Our findings are two-fold. First, we identified the phosphorylation events present in individual sMyBP-C variants at different states. Secondly, we quantified the relative abundance of each phosphorylation event, and of sMyBP-C phospho-species as a function of age and dystrophy, in the presence or absence of fatigue. Our results revealed both constitutive and differential phosphorylation of sMyBP-C. Moreover, we noted a 10–40% and a 25–35% reduction in the phosphorylation levels of select sites in old wild type and young or old mdx soleus muscles, respectively. On the contrary, we observed a 5–10% and a 20–25% increase in the phosphorylation levels of specific sites in young fatigued wild type and mdx soleus muscles, respectively. Overall, our studies showed that the phosphorylation pattern of sMyBP-C is differentially regulated following reversible (i.e. fatigue) and non-reversible (i.e. age and disease) (patho)physiological stressors.Myosin Binding Protein-C slow (sMyBP-C) is expressed in skeletal muscles where it plays structural and regulatory roles. The functions of sMyBP-C are modulated through alternative splicing and phosphorylation. Herein, we examined the phosphorylation profile of sMyBP-C in mouse slow-twitch soleus muscle isolated from fatigued or non-fatigued young (2-4-months old) and old (~14-months old) wild type and  MYBPC1 gene, the slow (s) skeletal isoform of MyBP-C is composed of seven immunoglobulin (Ig) and three fibronectin-III (Fn-III) domains. At the NH2-terminus of the protein, upstream of the first Ig domain, C1, is a sequence of ~50 amino acids enriched with proline (Pro) and alanine (Ala) residues, referred to as the Pro/Ala rich motif. Immediately following Ig C1, is a conserved linker region of ~100 amino acids, termed the M-motif. sMyBP-C differs from the cardiac and fast skeletal homologues, as it consists of a heterogeneous family of proteins, ranging in size from ~126 to ~131.5 kDa, resulting from extensive exon shuffling812Myosin Binding Protein-C (MyBP-C) comprises a family of accessory proteins in striated muscles that contributes to the assembly and stabilization of thick filaments and modulates the formation of actomyosin cross-bridges123456782-terminus of sMyBP-C is phosphorylated by both PKA and PKCThe NHmdx soleus muscles that were rested or subjected to an in vitro fatigue protocolmdx soleus muscles, we observed qualitative and quantitative differences in individual phosphorylation events or combinations thereof as a result of age, fatigue, and/or disease. Collectively, our studies are the first to demonstrate that the phosphorylation pattern of sMyBP-C is differentially regulated in response to age, fatigue, and disease in the slow-twitch soleus muscle.Using a panel of phospho-specific antibodies in combination with conventional and phosphate-affinity gel electrophoresis, we examined the phosphorylation profile of sMyBP-C in young (2–4 months old) and old (~14 months old) wild type and in vitro kinase assays using a series of phospho-ablated peptides along with our panel of phospho-specific antibodies (2-terminus of m-isoform3 (GST-sMyBP-C NH2 aa1-285) as it contains all known phosphorylation sites, including mSer-59 and mSer-204 present only in select variants , and 3. young (~4 months old) wild type and mdx mice exposed to an acute in vitro fatigue protocol. The lysates were analyzed by standard or phosphate-affinity SDS-PAGE immunoblotting. Given the presence of multiple sMyBP-C variants that have similar molecular weights (126–131.5 kDa)To study the phosphorylation profile of sMyBP-C mdx fatigued and un-fatigued samples, but significantly decrease (~20%) in the respective old mdx muscles , but not in fatigued young wild type tissue, compared to un-fatigued young wild type soleus . Moreove muscles .Evaluation of the phosphorylation status of sMyBP-C in young wild type soleus muscle using the α-pan-sMyBP-C antibody and phosphate-affinity SDS-PAGE revealed the presence of nine distinct species corresponding to differentially phosphorylated forms of the sMyBP-C variants . For easmdx soleus muscles demonstrated that they express the same major species as young wild type soleus muscle with the following exceptions: both young and old mdx soleus muscles contain an additional intermediate mobility species (purple dot) that is also present in aged wild type soleus muscle. However, young mdx soleus muscle lacks two high mobility species (orange and white dots), while aged mdx soleus muscle lacks one low mobility species (red dot) and one high mobility species (light pink dot) , and lacking five to seven species of intermediate and high mobility  . Interesmobility .2-terminus of sMyBP-C in soleus muscles obtained from the six animal groups described above. As previously, we consider the young wild type soleus muscle as the basis for qualitative and quantitative comparisons.Using our panel of phospho-specific antibodies  and stan2-terminus of human variants 1 and 2 (mdx (~4%), while dramatically decrease in un-fatigued young mdx (~33%) and old mdx (~14%) soleus muscles, compared to young wild type tissue  soleus muscles, compared to un-fatigued young wild type tissue (mdx) and dystrophic mdx (either young or old) soleus muscles contain similar levels to young wild type tissue, aged wild type tissue expresses reduced (~13%) levels of such species   .mdx soleus muscles, too, which also contain an additional intermediate mobility species (purple dot)  and one high mobility band (pink dot) (mdx soleus muscle expresses one low mobility band (yellow dot), two intermediate mobility bands (dark blue and purple dots), and one high mobility band (pink dot)  of differential abundance. Notably, select p-mSer62-containing species exhibit similar electrophoretic mobility with p-mSer59-containing forms. The five p-mSer62-containing species are detected in protein lysates prepared from old wild type and young ple dot) . In contink dot) . In contink dot) .mdx soleus muscles indicated a similar expression profile of p-mThr84-containing species compared to young wild type soleus muscle, with the only exception being the lack of a high mobility species (orange dot) in young mdx tissue , some of which exhibit similar electrophoretic mobility with p-mSer59- and p-mSer62-containing forms . The samx tissue . Interesectively . Moreovety forms .mdx soleus muscles express the same four p-mSer204-containing species, while young fatigued mdx soleus muscle expresses only one p-mSer204-containing form of intermediate mobility (dark green dot)  and three of intermediate mobility  . Examinaeen dot) .in vitro fatigue, aging, and chronic disease are conditions where contractility deficits have been linked to post-translational modifications of contractile proteins131415mdx mouse), aging in both the mdx and wild type background, as well as an acute in vitro fatigue protocol to reveal the dynamic range of phosphorylation events in sMyBP-C in slow-twitch muscle. Herein we present evidence of the complexity of MyBP-C phosphorylation in soleus muscle. Notably, the overall abundance of sMyBP-C phosphorylation is similar across the young soleus independent of genotype or fatigue; however, both age and dystrophy result in a modest, yet significant, reduction of sMyBP-C levels (Acute C levels . More imC levels . We therC levels  and phosC levels  of the iOf the nine distinct sMyBP-C forms expressed in young wild type soleus muscle (i.e. the base-line condition), a low mobility species physiological stimuli, such as age, stress, fatigue or disease. Alternatively, this may be the result of non-phosphorylated higher molecular weight sMyBP-C variants co-migrating with minimally phosphorylated lower molecular weight variants.The great majority of the identified sMyBP-C forms are phosphorylated at multiple, if not all known sites; however, phosphorylated species that contain only one (known) phosphorylation event are also present . The latmdx tissues (~16–25%), while its relative expression decreases dramatically in un-fatigued wild type and mdx samples (~0–9%). This is not true for the other low mobility species (yellow dot), which exhibits a ~22–30% relative abundance across the different soleus muscle groups. Moreover, the intermediate mobility species (light green dot) is of low relative abundance in all muscle groups (~0–6%), with the exception of the fatigued young wild type soleus muscle (~17%). On the contrary, the intermediate mobility species (dark green dot) shows a modest and similar relative abundance (7–12%) across the different soleus groups, with the exception of young fatigued mdx tissue where it is significantly increased (~25%). The remaining intermediate mobility species have varied expression among the different samples, ranging between 0–11% (turquoise dot) and 0–14% (dark blue dot). Notably, both of these species are absent in young fatigued wild type and mdx tissues. Lastly, the five high mobility species also exhibit differential expression and abundance across the different soleus muscles. The two extremes are represented by the species marked with the pink and white dots; while the former is present in all six soleus groups with a relative abundance ranging between 8–25%, the latter is only present in aged wild type tissue where it exhibits a relatively low abundance (~2%). The other three high mobility species  are expressed in select soleus groups and their relative expression varies between 0–13%, 0–8% and 0–16%, respectively.Similar to young wild type soleus muscle, the relative abundance of individual immunoreactive bands varies significantly within each of the remaining soleus muscle groups, as well . As suchin vitro fatigue is a reversible physiological stress. We observe considerable differences in the phosphorylation profile of sMyBP-C between these reversible and non-reversible conditions; an exciting result as it likely indicates regulatory activity. Specifically, exertion of fatigue results in a significant increase in the overall phosphorylation levels of residues mSer59 and mThr84 physiological states, while d mThr84 . Similard mThr84 . This isin vitro findings reveal intra- and inter-dependent relationships between the phosphorylation events that take place in the NH2-terminus of sMyBP-C. As such, individual phosphorylation events within the Pro/Ala rich motif exhibit varied dependence on the presence of additional phosphorylation events within the same motif  was produced as a GST-fusion protein as described inRecombinant protein corresponding to the NHIn vitro kinase assays were performed as described in112, 2 mM ATP and 1 mM DTT, and the reaction mixture was incubated at 20 °C for 25 min. The assay was terminated by addition of SDS sample buffer and separated by standard SDS-PAGE, transferred to nitrocellulose, and probed with each of the phospho-specific antibodies (listed below).SEW 3′, 5′ EWSLGESPAGC 3′, 5′ CANSQLSTLFVEK 3′, and 5′ RSAFKRSGEGQED 3′ containing phosphorylated mSer59, mSer62, mThr84, and mSer204 (underlined) were used to immunize rabbits for production of polyclonal antibodies . Phospho-specific antibodies were obtained through two rounds of affinity purification, following the manufacturer’s protocols. Briefly, in the first step, immune serum was purified against the phosphorylated form of the respective antigenic peptide coupled to sepharose beads. In the second step, the eluted antibodies were further purified against the non-phosphorylated form of the respective peptide, also coupled to sepharose beads. The antibodies contained in the flow-through fraction from the second affinity column were used as affinity purified phospho-specific antibodies at 750 ng/ml in subsequent immunoblotting experiments.Mouse antibodies to pan-sMyBP-C  and Hsp90 , which served as loading control, were used according to the manufacturers’ instructions. In addition, custom polyclonal antibodies to each of the four previously identified phospho-sites of sMyBP-C  skeletal isoform of MyBP-C, fMyBP-C, including the Pro/Ala rich motif, Ig C1, and the M-motif , was also produced as a GST-fusion protein and tested to ensure the specificity of the generated phospho-antibodies. Following affinity purification, the recombinant slow and fast MyBP-C NH2-terminal proteins and control GST-protein were either left untreated or treated with PKA and/or PKC, separated by SDS-PAGE, transferred to nitrocellulose, and probed with each of the phospho-specific antibodies , and flash frozen in liquid nitrogen. For each experiment, four to seven (n = 4–7) different soleus muscles were used per animal group.All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Maryland, School of Medicine. Freshly isolated soleus muscles were collected from young (~2 months) and old (~14 months) adult male wild type C57BL/6Scsn/J and dystrophic mdx mice was performed using in vitro methods, as previously described12mdx animals, three (n = 3) soleus muscle pairs  were used.Fatigue of the soleus muscle from young (~4 months) wild type and TM  and a 10% w/v solution of acrylamideLysates from mouse soleus muscles were prepared as previously reported10Quantification of the relative content of total sMyBP-C and of the relative expression of each phosphorylated residue among and within samples, respectively, was performed in standard SDS-PAGE blots by ImageJ software . Percent (%) expression was normalized to the levels of Hsp90, which served as loading control, and subsequently to the levels of sMyBP-C present in the young wild type un-fatigued sample, which served as the basis for all our quantifications. Experiments were repeated three times, using three different muscle samples. Significance was calculated via student’s t-test (p < 0.01). In addition, we used Image J software to quantify the relative abundance of specific immuno-reactive bands within a sample. To this end, we used the quantified relative content of total sMyBP-C for each sample as a representation of total sMyBP-C protein within that sample. The relative abundance of each immuno-reactive band recognized by the pan-sMyBP-C antibody in the phosphate affinity SDS-PAGE blots was calculated as a percentage of the total protein within that sample. Lastly, we used the phosphate affinity SDS-PAGE western blots probed with each of the phospho-specific antibodies to determine the residues that are phosphorylated within a specific immuno-reactive band detected with the pan-sMyBP-C antibody.How to cite this article: Ackermann, M. A. et al. The Phosphorylation Profile of Myosin Binding Protein-C Slow is Dynamically Regulated in Slow-Twitch Muscles in Health and Disease. Sci. Rep.5, 12637; doi: 10.1038/srep12637 (2015).


The worldwide prevalence of obesity has reached pandemic proportions, and with it have come other associated metabolic diseases, such as insulin resistance and type 2 diabetes (T2D). Intensive research has identified the frequent coexistence of obesity with a state of inflammation in metabolic tissues such as adipose tissue and liver . MultiplIn general, a majority of cellular double-stranded RNA (dsRNA)-binding proteins, such as dsRNAdependent protein kinase (PKR) and retinoic acid-inducible gene-I (RIG-I)-like receptor family, have been known to exclusively recognize exogenous dsRNA and induce inflammatory responses . HoweverPKR, a pathogen-sensing protein, is activated by excess nutrients and its aberrant activity plays a key role in the induction of inflammatory responses, insulin resistance, and abnormal glucose metabolism in obesity . In addiob/ob mice (a leptin-deficient obese mouse model) resulted in improved glucose tolerance, accompanied by significant reduction of JNK activity and eIF2α phosphorylation [More recently, we have identified TRBP, a trans-activation response (TAR) element-binding protein, as a physiologically-crucial component involved in obesity-induced PKR activation and metabolic dysfunction . TRBP isrylation . These fOur study suggests that metabolic inflammation results, at least in part, from deranged dsRNA processing/signaling, which adversely regulates systemic glucose metabolism in obesity. Given that TRBP is a dsRNAbinding protein involved in multiple cellular events, TRBP may be the signaling node that senses metabolic stress levels through the recognition of dsRNA networks, and links to miRNA outputs and PKR-mediated inflammatory signaling networks under the stress conditions. Identifying the molecular basis by which changes of endogenous dsRNA dynamics trigger chronic inflammation would pave the way for developing novel therapeutic strategies for not only obesity-induced metabolic diseases but also other chronic inflammatory diseases.


QuestionA 60-year-old man, known with Fabry disease, secondary left ventricular hypertrophy, and moderate aortic valve stenosis had a check-up in the outpatient clinic. An electrocardiogram (ECG) at rest Fig.  showed sWhat is your diagnosis, and what does it tell you about the possible pre-excitation?AnswerYou will find the answer elsewhere in this issue.


NTR and sortilin, and in the absence of NGF, TrkA recruits p75NTR as a hired killer to induce neuroblast apoptosis. This variety of potential pro- and anti-apoptotic outcomes is required to eliminate neuroblast excess yet maintain sufficient neuroblast numbers for development of the fully functional SNS.1Spatial and temporal changes in the expression of nerve growth factor (NGF) and its receptor tropomyosin-related kinase A (TrkA) are important in regulating cell fate choice as neuroblast delineate from the neural crest, migrate throughout the developing embryo, differentiate along the sympatho-adrenal lineage and eventually give rise to the sympathetic nervous system (SNS). Within this context, the ligation of active NGF by TrkA protects neuroblasts against apoptosis and promotes differentiation; pro-form NGF, in the absence of TrkA, promotes neuroblast apoptosis via the low-affinity NGF receptor p752 The characterisation of the mechanisms through which NBs resist apoptosis is critical for the future development of novel pro-apoptotic therapeutic strategies for use in this tumour type.Neuroblastomas (NBs) are devastating extra-cranial paediatric tumours that originate from neuroblasts of neural crest origin transformed at different stages along the sympatho-adrenal lineage. Not surprisingly and consistent with this origin, NBs are comprised of cells that are blocked in the particular differentiation state at the time of transformation and selected for resistance to apoptosis by a combination of oncogene activation, oncosuppressor inactivation and physiological apoptosis protection mechanism(s) conserved at the time of transformation, and exhibit different patterns of neurotrophin and neurotrophin receptor expression.3 The identification of tumour necrosis factorα (TNFα) as a pro-apoptotic cytokine paved the way for characterisation of the extrinsic apoptosis pathway and led to identification of the TNFα, Fas, TNF-related apoptotis-inducing ligand (TRAIL) and Apo3L pro-apoptotic cytokine family. Pro-apoptotic cytokines induce apoptosis through the extrinsic and/or intrinsic pathways by activating cognate TNFR1, FasL, FasR, DR3, DR4 and DR5 death receptors. Among this cytokine family, TRAIL is considered to be a promising chemotherapeutic agent because of its selective pro-apoptotic action on tumour, but not non-transformed cells.6 However, the potential therapeutic application of TRAIL in NB has been hampered by observations of TRAIL resistance in NB models, which has been attributed to caspase-8 silencing, altered decoy and functional TRAIL receptor expression, expression of the cellular FLICE-like inhibitory protein (cFLIP) inhibitory analogue of caspase-8, protective IP3K/Akt/mTor signalling or protective RIP/nuclear factor kappa binding (NF-κB) signalling. Accordingly, re-sensitisation of TRAIL-resistant NB cells to TRAIL-induced apoptosis has been reported for agents that increase the expression of caspase-8, ectopic overexpression of functional TRAIL receptors, the inhibition of cFLIP expression and agents that promote a pro-apoptotic expression pattern of TRAIL-induced apoptosis components.10Apoptosis can be activated via extrinsic and/or intrinsic pathways.Cell Death Discovery publication,11 we describe a novel NGF-mediated mechanism for sensitising TrkA-expressing SH-SY5Y NB cells to TRAIL-induced apoptosis that abrogates anchorage-independent tumorigenic growth in vitro. In our study, the TRAIL-resistant phenotype of SH-SY5Y NB cells (In our recent NB cells  was founNB cells  did not c, and reducing cytosolic levels of X-linked inhibitor of apoptosis, and was inhibited by ectopic overexpression of Bcl-xL, confirming that apoptosis was TrkA tyrosine kinase dependent, caspase dependent and mediated through the intrinsic mitochondrial pathway. Furthermore, NGF sensitisation of TrkA-expressing SH-SY5Y NB cells to TRAIL-induced apoptosis was temporary, limited to the first 6 h of NGF treatment and was eventually inhibited by increased Mcl-1 expression, but could be further optimised by siRNA Mcl-1 knockdown and by NF-κB inhibitors.NGF sensitisation of TrkA-expressing SH-SY5Y NB cells to TRAIL-induced apoptosis was abrogated by TrkA tyrosine kinase and caspase inhibitors, associated with permeabilisation of the outer mitochondrial membrane, increasing cytosolic levels of mitochondrial Omi/Htr2a and cytochrome Cell death Discovery study,11 not only adds to the potential ways in which NGF interaction with TrkA regulates the cell fate choice of neuroblasts, but may also extend to other TrkA-expressing cell types, other neurotrophin/neurotrophin receptor interactions, and potentially to other members of the TNF family and cognate receptors. We are currently investigating these possibilities. This mechanism may also help to explain the association between expression of fully spliced TrkA receptors, better prognosis and spontaneous remission in NB.1 Finally, our observations provide a novel potential pro-apoptotic therapeutic strategy, based on ‘painless' forms of NGF12 or alternative TrkA agonists, TRAIL or alternative TRAIL receptor agonists and inhibitors of NF-κB and/or Mcl-1, for future development in the treatment of favourable and unfavourable TrkA-expressing NBs, characterised for the expression of the TRAIL apoptosis pathway components and Bcl-2 family members, and cFLIP-dependent TRAIL resistance.The discovery of this novel pro-apoptotic TRAIL-mediated immunological dimension to NGF-TrkA function, described in our


Specifically, mRNA and protein levels of HERV-K (HML-2) are up-regulated in the blood plasma or serum of breast cancer patients. Here, we collected blood samples of 49 breast cancer patients and analyzed mRNA expressions of various HERVs env genes including HERV-R, HERV-H, HERV-K, and HERV-P by real-time PCR. The expression of env genes were significantly increased in the blood of primary breast cancer patients but were decreased in patients undergoing chemotherapy to a similar level with benign patients. When we compared the group currently undergoing chemotherapy and those patients undergoing chemotherapy simultaneously with radiotherapy, HERVs env genes were reduced more in the chemotherapy only group, suggesting that chemotherapy is more effective in reducing HERV env gene expression than is radiotherapy. Among chemotherapy groups, HERV env gene expression was the lowest in the taxotere- or taxol-treated group, suggesting that taxotere and taxol can reduce HERVs env expression. These data suggest the potential to use HERVs env genes as a diagnosis marker for primary breast cancer, and further studies are needed to identify the mechanism and physiological significance of the reduction of HERV env gene expression during chemotherapy.Human endogenous retroviruses (HERV)  Retroviruses, a form of mobile genetic elements, have important roles in both disease and primate evolution. Endogenous retroviruses (ERVs) was pathogenic, as well as being beneficial to a host in some cases. Furthermore, retroviruses also have played a key role in primate evolution that resulted in the incorporation of these elements into the human genome . Human Eenv mRNAs, including HERV-R (ERV3-1) and HERV-E, which are expressed in these same ovarian cancer tissues. Anti-HERV antibodies including anti-ERV3 (30%), anti-HERV-E (40%) and anti-HERV-K (HML-2) (55%) were also detected in patients with ovarian cancer but not in normal female controls [env gene, was found to be significantly expressed in the majority of melanoma cells and other cancers [Recent studies have suggested a strong relationship between HERVs and cancer based on the mRNA expression profile of HERVs in normal and cancer tissues ,14,15,16controls . Furthercontrols ,19,20,21 cancers ,23.env protein in both adult human organs and in tumors using a tissue microarray and also compared the expression of HERV-R (ERV3-1) between normal and tumor tissues to study the relationship between HERV-R (ERV3-1) and tumor formation. HERV-R (ERV3-1) was highly expressed in normal organs and was significantly increased in various tumors, including lung adenocarcinoma, renal cell carcinoma, papillary carcinoma, hepatocellular carcinoma, and adenocarcinoma in the gastrointestinal tract [env gene mRNA expressions in the blood of 49 breast cancer patients to identify blood HERV env gene expression and to examine the potential use of HERVs as a breast cancer detection marker.We previously analyzed the HERV-R (ERV3-1) al tract . Here, wenv genes expression of various HERVs including HERV-R (ERV3-1), HERV-H, HERV-K (HML-2), and HERV-P were analyzed by real-time PCR.We collected blood samples of 10 healthy normal and 47 breast cancer patients containing benign tumors (Benign), primary breast cancers without chemotherapy (Primary no chemo), primary breast cancers undergoing chemotherapy (Primary chemo), and recurred and metastasized cancers. Recurred and metastasized cancers were divided into two groups: before chemotherapy (Meta prechemo) and undergoing chemotherapy (Meta chemo). However, Meta prechemo patients were treated with chemotherapy when they had primary tumors. The env was significantly increased in the blood of Primary no chemo samples but reduced in patients undergoing or previously treated with chemotherapy to similar level with normal or benign patients  compared to normal or benign group. HERV-K (HML-2) env expression was slightly reduced in the Primary chemo group but still showed a significant increase compared to normal or benign group. When we compared cancers with and without chemotherapy, we found that HERV-K (HML-2) expression, similar to HERV-R (ERV3-1), was reduced in groups with chemotherapy  env, including an increase in Primary no chemo group, and decrease in chemotherapy groups  and cancer cells . In addition, our previous reports showed HERV-R (ERV3-1) was highly expressed in certain tumors [env gene in human tissues, the env fragments were detected in the mRNA of several tissues  and various cancer cells  by RT-PCR analysis [env genes of HERV-R, HERV-H, HERV-K, and HERV-P indicated that high levels of expression were detected in various cancer tissues compared to the adjacent normal tissues [The relationship between HERV-derived protein and breast cancer was first identified through the finding of the expression of HERV-K-MEL t cancer . In addit cancer . That stt cancer . Other st cancer ,20. HERVt cancer . An RT-Pn tumors . In caseanalysis . Transcranalysis . Quantitenv mRNAs including HERV-R (ERV3-1), HERV-H, HERV-K (HML-2), and HERV-P in whole blood samples from breast cancer patients and compared the mRNA expression patterns of each HERV env gene. The expressions of HERV-K (HML-2) and other HERV env genes were significantly increased in the blood of breast cancer patients and decreased in the patient groups undergoing chemotherapy. These data agree with previous studies and also suggest other HERV candidates as diagnostic markers or therapeutic targets for breast cancer.Here, we first analyzed various HERV env gene reduction by chemotherapy. Among chemotherapy groups including chemotherapy and radiotherapy group and a chemotherapy only group, the expressions of all HERV env genes were the lowest in the chemotherapy only group, indicating that radiotherapy has no effect or increases the expression of HERV env genes. This data agrees with our previous report showing that radiation induces HERV-R (ERV3-1) env expression by epigenetic modulation [env genes. Contreras-Galindo et al. found virus-like particles in the plasma of lymphoma patients [env expression comes from these viral particles. However, it is also possible that this expression comes from the contamination of debris nucleic acids from lymphocytes, circulating tumor cells, or exosomes. In this study, we collected whole blood to include whole possibilities including virus, lymphocytes, circulating tumor cells, or exosomes but excluded the possibility of genomic DNA contamination by treating DNase before constructing cDNA from purified total RNA. It is also an important issue whether the decreased expression of HERV env genes correlates with remission or simply just the chemotherapy treatment. The Meta Pre Chemo and Meta Chemo group consisted mainly of patients having recurred breast cancers, which is much more malignant than primary tumor. These groups had treated or have been treating with chemotherapy and showed the significant low level of all types of HERV env genes comparing with No Chemo group, suggesting the possibility that chemotherapy reduces the expression of HER env genes , 10 primary breast cancers without chemotherapy (Primary no chemo), 20 primary breast cancers undergoing chemotherapy (Primary chemo), 5 recurred and metastasized cancers before chemotherapy (Meta prechemo), and 5 metastasized cancers undergoing chemotherapy (Meta chemo). All primary and metastasized tumors were invasive ductal carcinoma. Both the collection and analysis of all samples were approved by the Institutional Review Board of Kosin University Gospel Hospital. Informed consent was obtained from each patient enrolled in the study .env gene RNA from particles in plasma or serum, lymphocytes, and circulating tumors. We treated blood samples with RBC lysis buffer  to remove red blood cells and added Trizol reagent  to purify total RNA according to the manufacturer’s guidelines. All purified RNA from all samples was treated with DNase before constructing cDNA to remove genomic DNA contamination. One microgram of total RNA was subjected to reverse transcription using the AffinityScrip™ Multiple Temperature cDNA synthesis kit . Relative quantitative real-time PCR was performed in a Applied Biosystems 7500 Fast Real-Time PCR System  using the SYBR® Green PCR Master Mix  according to the manufacturer’s instructions with the primer sets we used in previous study [We purified total RNA from whole blood which contains all possible HERV us study  and descus study .env genes in breast cancer patients are presented relative to the benign patients group as control with the calculated mean values and 95% confidence intervals. Statistical significance of the differences between groups was determined using a two-tailed Student’s t-test. p values less than 0.05 were considered statistically significant.All PCR reactions were coupled to melting-curve analysis to confirm the amplification specificity . Non-temenv show the same pattern of regulation as HERV-K (HML-2) and may be potent diagnostic markers and therapeutic targets for breast cancer patients.In conclusion, various HERV genes including HERV-R (ERV3-1), HERV-H, and HERV-P


Kaposi's sarcoma (KS) is a common malignancy in untreated AIDS patients that is caused by the infection of Kaposi's sarcoma-associated herpesvirus (KSHV). KS lesions are characterized by spindle-shaped endothelial cells with infiltration of inflammatory cells and neo-angiogenesis. KSHV commonly establishes default latent infection, but lytic replications occur in a small fraction of cells and play critical roles in KS pathogenesis by serving as a reservoir of infectious virion for persistent infection and inducing continuous paracrine factors for tumor progression. KSHV lytic infection triggers a variety of cellular machinery for replication and remodels cellular transcriptome. Distinct transcriptional patterns are exhibited in the immediate early (IE) and late stages of lytic replication. Previous studies have revealed that multiple pathways activate IE gene expression during lytic infection and reactivation from latency. However, the mechanism of late lytic gene expression regulation has remained largely undefined for a long time. Recently, studies have revealed that both host and viral factors, ORF45-prolonged c-Fos and TATA box binding protein (TBP)-like protein ORF24, are required for late lytic transcription , 2.Our previous observations demonstrated that KSHV tegument protein ORF45 interacts with p90 ribosomal S6 protein kinase (RSK) and induces reciprocal ERK-RSK activation through the formation of ORF45-ERK-RSK multiprotein complexes that protect active ERK-RSK from dephosphorylation, consequently activating a sustained ERK-MAPK cascade that is essential for lytic replication , 4. DefiAs the sensor of sustained ERK-RSK activation, prolonged c-Fos activation accelerates late lytic transcription, whereas transient ERK-RSK activation-mediated rapid c-Fos activation induces IE gene expression. Two types of c-Fos exhibit the distinct promoter-binding profile in the KSHV DNA genome  and therRhadinovirus) and that prolonged c-Fos regulates their lytic progression. However, the similar RSK-binding region is not found in ORF45 homologues of γ1-herpesvirus (Lymphocryptovirus), such as EBV BKRF4, suggesting this mechanism does not occur in other families of herpesviruses and that there are other conserved mechanisms for the lytic progression of these herpesviruses.γ2-herpesviruses encode conserved ORF45 homologues that share a RSK-binding region , suggestOur studies have shown that KSHV genes are divided into c-Fos-dependent and c-Fos-independent clusters that are activated by c-Fos directly or by secondary transactivation of RTA or other c-Fos targets, respectively. The expression of c-Fos-independent clusters still requires sustained ERK-RSK activation, suggesting that other transcription factors or cooperative viral and cellular partners are involved in KSHV viral transcription. One study has revealed that the transcription factor Elk-1 is activated by ERK-RSK signaling of the ORF45 homologue of γ2-herpesvirus . FurtherWe have revealed the role of the ORF45-ERK-RSK cascade in viral lytic progression, while the functions in oncogenic transformation and host-virus interaction remain largely unknown. Prolonged c-Fos activation may attenuate inflammatory responses, which play important roles in tumorigenesis and the progression of KSHV-associated diseases. Furthermore, sustained ERK-RSK activation may be involved in the conversion of cell morphology and transformation. Given that RSK and other MAPK inhibitors significantly suppress KSHV lytic replication, it is interesting to evaluate the therapeutic role of ERK-RSK signaling in KSHV pathogenesis. Additionally, targeting sustained ERK-RSK activation is a promising therapeutic approach for KSHV-related diseases.In conclusion, our studies revealed that KSHV ORF45 induces sustained ERK-MAPK activation during late lytic replication. Consequently, c-Fos accumulation and transactivation are prolonged to accelerate lytic transcription during the late stage of KSHV lytic life cycle. Rapid and prolonged c-Fos activation mediates lytic transcription during the IE and late stage of KSHV lytic replication, respectively, indicating that c-Fos timing determines KSHV lytic progression. The prolonged c-Fos activation may represent a common regulation for late lytic transcription and replication of γ2-herpesviruses.


Tyrosine kinase inhibitors of epidermal growth factor receptor (EGFR-TKIs) are standard treatments for advanced non-small-cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations. Nowadays, tumor tissues acquired by surgery or biopsy are the routine materials for EGFR mutation analysis. However, the accessibility of tumor tissues is not always satisfactory in advanced NSCLC. Moreover, a high proportion of NSCLC patients will eventually develop resistance to EGFR-TKIs. Invasive procedures, such as surgery or biopsy, are impractical to be performed repeatedly to assess the evolution of EGFR-TKI resistance. Thus, exploring some convenient and less invasive techniques to monitor EGFR-TKI treatment is urgently needed. Circulating cell-free tumor DNA (ctDNA) has a high degree of specificity to detect EGFR mutations in NSCLC. Besides, ctDNA is capable of monitoring the disease progression during EGFR-TKI treatment. Certain serum microRNAs that correlate with EGFR signaling pathway, such as miR-21 and miR-10b, have been demonstrated to be helpful in evaluating the efficiency of EGFR-TKI therapeutics. A commercialized serum-based proteomic test, named VeriStrat test, has shown an outstanding ability to predict the clinical outcome of NSCLC patients receiving EGFR-TKIs. Analysis of EGFR mutations in circulating tumor cells (CTCs) is feasible, and CTCs represent a promising material to predict EGFR-TKI-treatment efficacy and resistance. These evidences suggested that non-invasive techniques based on serum or plasma samples had a great potential for monitoring EGFR-TKI treatment in NSCLC. In this review, we summarized these non-invasive approaches and considered their possible applications in EGFR-TKI-treatment monitoring. Lung cancer is the most commonly diagnosed cancer as well as the leading cause of cancer-related deaths . Non-smaNowadays, EGFR gene mutations are the standard predictive biomarkers for selecting NSCLC patients to receive EGFR-TKI treatment. The point mutation in exon 21 (L858R) or deletion in exon 19 predicts good response to EGFR-TKIs , while tNon-invasive approaches, usually based on plasma or serum samples, showed great potential for monitoring EGFR-TKI treatment in recent years. Circulating cell-free tumor DNA (ctDNA), which is released from tumor cells to circulating blood, can be applied to detect EGFR mutations in NSCLC patients –23. MoreIn summary, serum ctDNA, miRNAs, proteomic biomarkers, and CTCs can be obtained non-invasively, making them ideal substitutes for tumor tissues Fig. . In addiCancer-associated genetic alterations, such as point mutations, deletions, copy number variations, and methylation patterns, can be detected in ctDNA , 36. In When ctDNA is used to analyze gene mutations in NSCLC patients, one vital issue is whether the genetic variation within blood was consistent with tumor tissues. Table p = 0.038) [Regarding the clinical application of ctDNA, another important issue is whether ctDNA is capable of predicting EGFR-TKI-treatment efficacy. Indeed, several studies have analyzed the association between pre-treatment ctDNA EGFR mutation status and clinical outcomes. Goto et al.  evaluate= 0.038) . Recentl= 0.038) . In thisIn NSCLC, despite an initial response, resistance to EGFR-TKIs will occur and thereafter tumor progresses. For this reason, identification of the molecular mechanisms of EGFR-TKI resistance is needed. Several mechanisms responsible for EGFR-TKI acquired resistance have been described, including the second T790M mutation of EGFR, amplification of MET or HER2, and mutations of PIK3CA or BRAF , 52. TheMicroRNAs (miRNAs) play critical roles in tumor development, including tumor initiation, invasion, and metastasis –56. SeveShen et al. performed a study to identify the miRNA panel in the serum of NSCLC patients with different EGFR mutation status . They reAnother study, which aimed to investigate the role of miR-21 in the acquired resistance to EGFR-TKIs, showed that miR-21 can mediate EGFR-TKI resistance by down-regulating PTEN and PDCD4 and activating the PI3K/Akt pathway . BesidesThe above available evidences suggested that serum miR-21 and miR-10b were helpful for monitoring EGFR-TKI treatment. Future data may update these results and validate the practical utility of circulating miRNAs in NSCLC.Similar to other high-throughput technologies, proteomics are able to detect multiple proteins that are expressed as a cause or consequence of ongoing diseases . Proteomp = 0.000) for OS and 0.49  for PFS [Taguchi and his colleagues reported a mass spectrometry serum test which was able to identify a subset of NSCLC patients with better outcomes after EGFR-TKI therapy in 2007 . Then th for PFS . Further for PFS . Their rp = 0.022). Chemotherapy is likely to result in improved outcomes for patients with a VeriStrat “poor” status. This trial complemented the results of previous retrospective studies and helped to identify the subgroup of NSCLC patients who may not benefit from EGFR-TKIs.The PROSE trial, the first prospective, randomized proteomic stratified phase III study, was designed to evaluate the predictive value of the VeriStrat test on the outcome in advanced NSCLC patients treated with either EGFR-TKIs or standard chemotherapy . InformaIn this emerging field, the number of studies is relatively limited but growing. Another phase III study (EMPHASIS) was opened for advanced squamous cell NSCLC . Patients with relapsed squamous cell lung cancer are assigned to two groups (VeriStrat “good” and VeriStrat “poor”) and then randomized between EGFR-TKI (erlotinib) and chemotherapy (docetaxel). One of its goals is to explore the predictive and prognostic ability of the VeriStrat test signature. In addition, a clinical trial that aims to measure whether VeriStrat could be a biomarker of benefit from treatment with standard chemotherapy regimens in first-line NSCLC patients is currently recruiting participants . These ongoing trials may prospectively validate the predictive role of this serum-based proteomic test in NSCLC patients.Circulating tumor cells (CTCs), which are disseminated from tumor tissues, have the potential to complement tumor tissues for tumor characterization and cancer-treatment monitoring . VariousThe EGFR gene mutation profiles of CTCs in NSCLC have been analyzed by several studies. Recently, a study investigated the EGFR mutations in CTCs by coupling the CellSearch System with next-generation sequencing (NGS) . In 37 aAs mentioned above, the dynamic changes in ctDNA-based EGFR status are able to predict the benefit of EGFR-TKI treatment . SimilarDNA extracted from CTCs, like other sources of DNA, has been shown to be an effective material for the detection of the T790M mutation of EGFR in NSCLC. Maheswaran et al. performed an EGFR mutation analysis on CTCs in NSCLC patients treated with EGFR-TKIs . Hence, Non-invasive approaches, usually based on serum or plasma samples, are showing great potential to monitor EGFR-TKI treatment in NSCLC Fig. . ctDNA h


Initial data suggest that objective anti-tumor response rates may be higher with anti-PD-1 agents compared with ipilimumab and the safety profile may be more tolerable. This review explores the development and next steps for PD-1 pathway inhibitors, including discussion of their novel mechanism of action and clinical data to-date, with a focus on melanoma.Checkpoint inhibitors are revolutionizing treatment options and expectations for patients with melanoma. Ipilimumab, a monoclonal antibody against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), was the first approved checkpoint inhibitor. Emerging long-term data indicate that approximately 20% of ipilimumab-treated patients achieve long-term survival. The first programmed death 1 (PD-1) inhibitor, pembrolizumab, was recently approved by the United States Food and Drug Administration for the treatment of melanoma; nivolumab was previously approved in Japan. PD-1 inhibitors are also poised to become standard of care treatment for other cancers, including non-small cell lung cancer, renal cell carcinoma and Hodgkin's lymphoma. Immunotherapy using checkpoint inhibition is a different treatment approach to chemotherapy and targeted agents: instead of directly acting on the tumor to induce tumor cell death, checkpoint inhibitors enhance or  V600E/KBRAF mutation, are associated with high response rates (~20–80%), prolonged progression-free survival (PFS) (5–9 months), and improved overall survival (OS) . A phase III study is evaluating nivolumab alone or with ipilimumab versus ipilimumab in patients with previously untreated advanced melanoma; the primary endpoint is OS [NCT01844505]. Another phase II study has a similar design, without the nivolumab alone group, and has ORR as the primary read-out [NCT01927419]. Other combinations of a CTLA-4-targeted agent plus a PD-1 pathway inhibitor are also being evaluated in phase I trials, including ipilimumab plus pembrolizumab or MPDL3280A, and tremelimumab plus MEDI4736 [Several fully-accrued trials are further investigating ipilimumab plus nivolumab in patients with advanced melanoma, and an expanded access program is now available . A phaseMEDI4736 .Detailed treatment algorithms and recommendations are available for the approved agent ipilimumab and nivolumab  toxicity profile. As such, treatment of patients with combinations or sequential approaches will require the close attention of clinicians for the development of immune-related toxicities. The understanding that immunologic AEs are caused by uncontrolled off-target immune responses, and therefore frequently require active treatment with steroids, is critical for clinicians to effectively manage patients receiving these therapies.


Talimogene laherparepvec, an investigational oncolytic immunotherapy, is a modified herpes simplex virus type-1 (HSV-1) designed to selectively replicate in tumors and to initiate a systemic immune response to target cancer cells. Intralesional administration of talimogene laherparepvec is intended to result in oncolysis within injected tumors, with lytic cell destruction promoting the local release of progeny virus and tumor derived antigens. GM-CSF, a product of the viral transgene, is also produced locally such that it can recruit and stimulate antigen presenting cells to further enhance systemic anti-tumor immune response.In a retrospective analysis of a Phase III melanoma trial investigators found that about two-thirds of the lesions injected with talimogene laherparepvec shrank 50% or more. Among uninjected tumors similar responses were seen in one third of lesions of the skin and lymph nodes and about a sixth of uninjected visceral lesions providing an indication that the treatment is triggering systemic immune effect.mGM-CSF) was employed in syngeneic tumor models (A20 and CT26).To further dissect the mechanism of action underlying innate and adaptive immunity following viral administration, a talimogene laherparepvec expressing murine GM-CSF  lesion and marked size increases in injected tumor draining lymph nodes. Flow cytometric profling, gene expression analysis, and IHC revealed infiltration of T cells, innate effector cells and antigen presenting cells in tumors and draining lymph nodes. Innate immune responses in the injected tumor are characterized by the induction of Interferon response gene signatures and result in the systemic release of proinflammatory cytokines. These effects are, at least in part, dependent on STING activity, with in vitro functional assays supporting direct agonism of this pathway by the virus. Systemic anti-tumor responses appear to be T cell driven: T cells expressed higher levels of activation markers as early as 2 days post-treatment in injected tumors, with evidence of T cell activation in contralateral lesion by day 7. To elucidate anti-viral and anti-tumor T cell responses, ex vivo re-challenge assays were performed using splenocyte- and draining lymph node-derived T cells following treatment with OncoVEXmGM-CSF; viral treatment increased systemic anti-tumor immunity and showed induction of anti-viral responses.When administered intratumorally (3 doses of 5x10^6 PFU) OncoVEXin vivo observations suggest that intratumoral injection of OncoVEXmGM-CSF activates multiple immune-mediated mechanisms of action, leading to T cell activation and induction of anti-tumor immunity in mice.In conclusion, in vitro and


The mammalian neocortex contains many distinct inhibitory neuronal populations to balance excitatory neurotransmission. A correct excitation/inhibition equilibrium is crucial for normal brain development, functioning, and controlling lifelong cortical plasticity. Knowledge about how the inhibitory network contributes to brain plasticity however remains incomplete. Somatostatin- (SST-) interneurons constitute a large neocortical subpopulation of interneurons, next to parvalbumin- (PV-) and vasoactive intestinal peptide- (VIP-) interneurons. Unlike the extensively studied PV-interneurons, acknowledged as key components in guiding ocular dominance plasticity, the contribution of SST-interneurons is less understood. Nevertheless, SST-interneurons are ideally situated within cortical networks to integrate unimodal or cross-modal sensory information processing and therefore likely to be important mediators of experience-dependent plasticity. The lack of knowledge on SST-interneurons partially relates to the wide variety of distinct subpopulations present in the sensory neocortex. This review informs on those SST-subpopulations hitherto described based on anatomical, molecular, or electrophysiological characteristics and whose functional roles can be attributed based on specific cortical wiring patterns. A possible role for these subpopulations in experience-dependent plasticity will be discussed, emphasizing on learning-induced plasticity and on unimodal and cross-modal plasticity upon sensory loss. This knowledge will ultimately contribute to guide brain plasticity into well-defined directions to restore sensory function and promote lifelong learning. Nevertheless, SST-interneurons are promising candidates in the context of mediating experience-dependent cortical plasticity through inhibition of distal dendrites of pyramidal neurons where intracortical inputs converge. As such they are ideally positioned to control plasticity of excitatory inputs that synapse onto the same dendritic location. At the same time, they strongly innervate PV-interneurons and are as such well-positioned to regulate inhibition instructive to brain plasticity [The dynamic and delicate interplay of excitatory and inhibitory neurons allows the brain to process, adapt, and respond to environmental cues coming in through the sensory systems. In the healthy brain, inhibitory transmission in general prevents runaway excitation and sharpens the response properties of excitatory neurons , 2. By p neurons . A vast  neurons ). In conasticity –28. A coasticity , includiasticity , which wInhibitory neurons use gamma-amino butyric acid (GABA) as their main inhibitory neurotransmitter and are mostly local-circuit interneurons. Their axonal arborization is typically restricted to the neocortex and does not project into the white matter . Nonethe3aR, and neuronal nitric oxide synthase (nNOS). Sets of these molecular markers partially overlap to different extents in distinct subtypes [3aR-interneurons make up three nonoverlapping classes, together forming approximately 100% of the interneurons in mouse cortex [3aR-interneurons  can be largely subdivided into VIP- and non-VIP-expressing interneurons [3aR-interneurons are mostly small neurogliaform cells with a large number of short, radiating dendrites forming spherical structures and have highly branched, thin axons [In general, most attempts to classify interneurons are based on the expression of different molecular markers, morphological and electrophysiological properties , 9. Recesubtypes , but thee cortex , 42–44. e cortex . A well-e cortex . 5HT3aR-rneurons , 47–49. rneurons . Bi-tuftrneurons . Non-VIPin axons . Neuroglin axons , 45 Further subdividing interneurons based solely on molecular markers has its limitations as distinct functional subclasses can express the same calcium-binding proteins or neuropeptides, and this may be functionally not very informative. On the other hand, many studies currently take advantage of the availability of many transgenic mouse lines in which molecular interneuron subclasses can be reliably and reproducibly targeted across experiments and laboratories in order to elucidate their functional roles in cortical processing , 53. As It has been clear for some time that the cortical SST-inhibitory cell population is not homogeneous. Several distinct SST-populations, in addition to Martinotti cells, have already been described in the mouse cortex, and more are likely to follow –57. Thesμm [μm [κ-opioid and/or SST-receptor-expressing pyramidal neurons present in layers V-VI in a long-lasting manner [Martinotti cells, described for the first time in 1889 by Martinotti , constitμm . A charaμm [μm . Within μm [μm , 63, 64.μm [μm , as wellμm [μm . Physiolμm [μm . This isμm [μm –68. Neveμm [μm . Mostly,μm [μm . Moleculμm [μm , PV, andμm [μm , 60. Parμm [μm , reinforμm [μm . In summg manner . FinallyStudies that have looked into the characteristics of Martinotti cells have focused largely on morphological properties to identify the proper subset of interneurons, such as the presence of layer I-targeting axon collaterals . HoweverX98-neurons are found in layers V and VI, GIN-neurons in layers II/III and to a lesser extent in layer V. Both are mainly layer I dendrite-targeting neurons and colocalize with CB, like Martinotti cells , 72, andOn the other hand, X94-neurons were shown to be present in layers IV and V. The layer IV and V SST-interneurons show no similarity with Martinotti cells based on electrophysiological or morphological properties and thus most likely belong to a distinct SST-subpopulation. Almost no axon branches to layer I were found; instead they remain local within layer IV, where they inhibit other fast-spiking interneurons such as PV-interneurons  or, in tA closer look into the characteristics of GFP-interneurons in the GIN-line revealed the presence of SST-interneurons lacking the typical Martinotti cell morphology and behavior, suggesting a larger heterogeneity in SST-subclasses than originally presumed , 56. In Another study revealed at least three distinct clusters of SST-subtypes in the GIN-strain. Here an unsupervised cluster analysis combined electrophysiological and morphological features and as such verified the presence of Martinotti cell-properties in 50% of GFP-interneurons with extensively branching layer I-targeting axon collaterals , yet the largest subset of these projecting cells is SST-positive (91%) and colocalizes with nNOS and NPY. They have been found mainly in layer VI and the white matter, but also layer II/III, and connect cortical areas across the areal boundaries  Figure . Recentlh nAChRs . Lypd6 wh nAChRs . It was h nAChRs .β1 and Caβ4 , HCN3, Caα1G , HCN2, Kv3.2, Kv4.2, and Kv1.1 are also found in Martinotti cells [Previous studies predicting electrical properties based on single-cell gene expression profiles found that ion channels are clustered around specific calcium-binding proteins, characteristic for particular interneuronal subpopulations. These ion channel clusters likely account for the unique electrophysiological properties present in different interneuron subclasses, and moreover also within the SST-interneuron populations. Several ion channels such as the voltage-gated potassium channels Kv2.1, Kv3.3, Kv4.2, and Kv3.1 and the hyperpolarization activated sodium/potassium channel HCN4, for example, are found in Martinotti cells, but not in fast-spiking interneurons or CB-negative SST-interneurons . In additi cells . For theti cells  and the ti cells  in the n3aR-) interneurons, a consensual connectivity scheme has been proposed in which PV-interneurons somatically inhibit themselves. SST-interneurons do not inhibit each other, yet they do inhibit PV-interneurons. Lastly, VIP-interneurons strongly inhibit SST-interneurons, and to a lesser extent PV-interneurons [The discharge properties of individual interneurons ultimately depend on the network they are embedded in as interneurons and excitatory neurons are reciprocally connected . Inhibitrneurons , 81–83. In the following section, the input and output relationships of SST-subpopulations will be further reviewed as the heterogeneity in SST-interneuron populations in combination with lamina-specific distributions of their pre- and postsynaptically connected neurons suggests distinct functions in cortical processing. Several studies will be highlighted that look particularly into SST-subtypes, in agreement with the abovementioned subdivision of SST-interneurons.A receptor-mediated synaptic input [2+-spikes are generated, and prolonged regeneration of these spikes can elicit high frequency bursting in the soma of the pyramidal neurons in layer V [2+-spikes [2+-influx is an important factor in modifying synaptic transmission at glutamatergic synapses [The best studied SST-interneurons, the Martinotti cells, have the highest connection probability with pyramidal cells, and the lowest with fast-spiking interneurons , 63, 73.ic input , 86. Wit layer V . When th+-spikes , 88–90. synapses , Martinosynapses . In the synapses , suggestsynapses , 95. It synapses .B-receptors on their glutamatergic boutons [B-mediated synaptic suppression can be rapidly and reversibly activated in a state-dependent manner. As such SST-interneurons can gate synaptic plasticity by relieving tonic inhibition from excitatory synapses when the interneurons become suppressed during sensory activity [Martinotti cells present in layers II/III and V share much of the same properties, yet the laminar distribution of their presynaptic neurons suggests an additional, layer-specific level of control over these Martinotti cells. A study from Gentet et al.  investig boutons . GABAB-mactivity .In addition to targeting layer I pyramidal dendrites, another study in rats reported layer II/III CB+ SST-interneurons targeting the axon initial segment of layer II/III pyramidal neurons, where action potentials are generated. This property has been previously attributed solely to Chandelier cells . HoweverSST-interneuron mediated inhibition on PV-interneurons in both layers II/III and V  could esSeveral studies have linked layer IV and V X94-neurons to a PV-interneuron targeting circuit; however these X94-neurons have so far only been reported to target PV-interneurons within the thalamorecipient layer IV. The difference between layer IV and V X94-neurons can be found in their input sources, be it cortical (for layer IV X94-neurons) or thalamocortical (for layer V X94-neurons) , 74.Layer IV X94-Cells. The X94-SST-subpopulation present in layer IV innervates only neurons within this layer in contrast to layer I-targeting Martinotti cells. The main input sources originate within the cortex, in contrast to the layer IV fast-spiking PV-interneurons that mainly receive thalamic input [ic input , 105. Thic input .Layer V X94-Cells. Layer V X94-cells have been reported to be involved in feedforward inhibitory mechanisms onto layer V pyramidal neurons. Generally, fast-spiking PV-interneurons have mainly been associated with thalamic feedforward inhibition [hibition , yet thihibition .The faster kinetics reported in part of the GIN-labeled subpopulations  allows aThe function of inhibitory long-range corticocortical projecting SST-neurons is only poorly understood and poses more difficulties to investigate due to their very low abundance. However, an interesting potential function is the communication and synchronization of activity within and between cortical areas through regulating rhythmic oscillations . As suchIn addition to chemical synapses, SST-interneurons, like VIP- and PV-interneurons –113, areEven though gap junctions constitute a simpler form of signal transmission reminiscent of invertebrates, they are very important also during mammalian development when chemical synapses are still being established (for a review see ). HoweveAnother line of evidence adding to the importance of interneurons and SST-interneurons in particular in cortical processes underlying mechanisms for attention, learning and memory, and cortical plasticity is the fact that SST-interneurons can be effectively depolarized by ACh via muscarinic  and nicoIn summary, SST-interneurons maintain the excitation/inhibition balance within cortical networks by virtue of their central position in the connectivity schemes with both excitatory and inhibitory neurons. They mediate both feedback and feedforward inhibition to filter through relevant signals during active behavior and attention; they contribute to the switch from somatic to dendritic inhibition, opening windows of opportunity for integration mechanisms, and are involved in mediating rhythmic oscillations within the cortex. As such, the wide range of inputs and outputs of different SST-populations makes them quite diverse yet pivotal players at all levels of cortical network processing.It has long been appreciated that inhibition plays a key role in mediating different aspects of experience-dependent synaptic modifications in the brain to optimally process and respond to the world around us , 18, 130In this section several studies will be highlighted each putting forward SST-interneurons as interesting candidates in mediating cortical experience-dependent and learning-induced plasticity by virtue of their distinct laminar distribution and intrinsic and network properties.A receptor α1 subunit in postsynaptic terminals, and increased frequency of spontaneous inhibitory postsynaptic currents in the trained cortical areas following associative learning [Learning-induced plastic reorganization of cortical activity involves modifications and strengthening of excitatory synaptic inputs onto pyramidal neurons . Such chlearning –137. Addlearning , somatoslearning , or motolearning .In motor cortex, for example, SST-interneurons regulate the spatiotemporal specificity of learning-induced structural plasticity of excitatory synapses and thus acquisition of motor skills. Chen et al.  observedAn interesting mechanistic link through which SST-interneurons may be engaged in learning-induced plastic processes is through cholinergic signaling. The cholinergic system itself is already linked to learning processes and its associated cortical plasticity, as boosting the cholinergic system and stimulating the basal forebrain from which the cholinergic neurons project to the cortex, has an effect on learning and cortical information processing –146. A lCholinergic receptors so far have been described in a subpopulation of SST-interneurons, more specifically layer II/III Martinotti cells and layer IV X94-SST-interneurons. Indeed, the study of Cybulska-Klosowicz et al.  suggesteEarly in life, brain plasticity is enhanced during well-defined windows of brain development, or critical periods (CP), when experience-driven activity strongly modifies both structurally and functionally the neuronal basis originally laid out by a genetic blueprint , 150. Du2+-spikes, learning, and plasticity in general. As ODP requires structural rewiring of excitatory synapses onto dendritic spines of pyramidal neurons, SST-interneuron-mediated inhibition may be ideally localized within the cortical network to contribute to these processes. One study initiated the characterization of how SST-interneurons, and more specifically Martinotti cells in the GIN-mouse line, functionally mature during development and whether this can be correlated with the CP for ODP [The role of PV-interneurons in this phenomenon of ODP has been the focus of many studies, as it has been long held that their functional maturation is crucial and even exclusive in opening the CP. PV-interneurons presumably also play an important role in closing it, although other mechanisms, such as epigenetic regulation of transcription or the maturation of the extracellular matrix to structurally stabilize the neural circuits, are also ascribed to CP closure . Neverth for ODP . The autBuilding further on this link between the maturation profile of SST-interneurons and ODP, another study transplanted GABAergic precursors for PV- and SST-interneurons from the medial ganglionic eminence (MGE) into cortical regions near V1b in 7-day-old mice. Thirty-three to 35 days after the transplantation, when the transplanted cells were at an equivalent age of postnatal days 26–28 , this new wave of PV- and SST-interneuron maturation was found to induce a second window for ODP following short-term monocular deprivation, as measured by changes in the ocular dominance index. Remarkably, transplants depleted of PV-precursors but still containing SST-precursors were capable of inducing plasticity similarly robust to transplants that only contained PV-precursors but that were depleted in SST-precursors. Furthermore, transplants depleted in both cell populations could not induce ODP, indicating a mediating role for both PV- and SST-interneurons but not other MGE-derived cell populations. This study showed for the first time that, in addition to PV-interneurons, also SST-interneurons and more importantly their maturation profiles are crucial mediators for starting up windows of plasticity during development . DespiteCritical periods of enhanced sensitivity are closed or severely constrained in adulthood; still certain manipulations that reduce the inhibitory level in V1b to a juvenile or CP-like condition of immature inhibition can cause the reopening of critical periods, allowing CP-like plasticity to occur well into adulthood –166. Thi2+-currents through these ion channels [Again, a link with cholinergic neuromodulation can be made as VIP-interneurons, as SST-interneurons receive direct nicotinic cholinergic inputs, which modulate the cortical state and sensory responses. As such, the VIP-SST disinhibitory circuit is well positioned to be a target for manipulation of experience-driven plasticity by this additional level of neuromodulation. It remains to be determined however how the cholinergic system converging on VIP-interneurons can be regulated. Recently, such a regulatory system has been found specifically in PV- and SST-interneurons, but not in VIP-interneurons. PV- and SST-interneurons both express Lynx family members of nAChR modulators that regulate the cholinergic signaling on these interneuron cell types. Specifically in PV-interneurons, Lynx1 functions as a cholinergic brake and has been observed to restrict adult ODP. Deletion of Lynx1 resulted in an enhanced nAChR signaling and successfully restored adult ODP . In infrchannels , 76. It Additional evidence for an instructive role for SST-interneurons in adult ODP can be found in the structural plasticity of inhibitory synapses in ODP. Indeed it was found that adult monocular deprivation results in a loss of inhibitory synapses on distal apical dendrites of layer II/III pyramidal neurons , again hExtensive sensory loss results in a compensatory response of the spared senses, not only within the same modality , but also between other spared modalities . This cross-modal plasticity especially occurs in multimodal areas, but, for example, also in monocularly driven visual areas, in contrast to ODP, which is mainly restricted to V1b , 177. InIn early deprived mammals, the plastic reorganization usually encompasses cortical, but also subcortical structural rewiring. A famous example consists of studies done on the congenitally blind mole rat, in which it was shown that the thalamocortical visual pathway is invaded by auditory input . Also inIn another study in hamsters enucleated at birth, a reduction was seen in the number of CB-interneurons in layer V of V1, together with PV-interneurons, which additionally showed an increase in layer IV. It could be that the affected primary visual cortex adopts the GABAergic features of the auditory cortex through cross-modal rewiring . The autIn addition to reorganization within the deprived cortical area, loss of sensory function also leads to structural and functional alterations in the other intact modalities. In this context, one study looked into the barrel cortex of young mice that were olfaction-deprived starting from postnatal day 12. The authors described that olfactory deprivation recruits more GABAergic neurons in the barrel cortex. Specifically, using the GIN-mouse strain, they observed an increase in the number, the fine processes, and the encoding capacity for action potentials in SST-interneurons in the barrel cortex 1 week after olfactory deprivation . It rema per se unimodal, but are interconnected by direct reciprocal cross-modal corticocortical connections [Cross-modal plasticity is not necessarily restricted to critical periods , but cannections –213. As nections , 214. Itnections , 216 as nections , 217–221nections . Layer Inections .Cross-modal modulation of neuronal responsiveness is mostly suppressive, which is indicative for an important role for inhibition in communication between different (early) sensory modalities –226. IndAlso in adult cross-modal plasticity both the takeover of the deprived cortical area and hypersensitivity of the spared modalities are observed . Indeed,These studies indicate an interesting potential role for SST-interneurons in both the takeover of the deprived area by intact modalities and the hyperexcitability seen in the intact sensory areas, in both young and adult mammals. In any case, further cell-type specific studies are required to understand a possible contribution of SST-interneurons to these mechanisms.In summary, SST-interneurons make up a highly diverse group of interneurons that establish cortical, state-dependent inhibition at multiple levels and timescales in the cortical column. They are involved in both feedback and feedforward inhibitory and disinhibitory circuits and can as such integrate different streams of sensory information. Because of these properties, SST-interneurons are gaining more attention in plasticity research and indeed several lines of evidence suggest a central role for multiple SST-interneuron subtypes in regulating both learning- and sensory deprivation-induced plasticity phenomena, both during development and in adulthood. Particularly in layer II/III, where bottom-up and top-town sensory inputs integrate, SST-interneurons, and perhaps the VIP-SST-disinhibitory circuits, come forward as pivotal players in several plasticity mechanisms, suggesting interesting substrates for future research.The use of transgenic mouse lines has proven to be an invaluable tool to characterize the distinct SST-interneuron subsets known today –56, and


Non-heart-beating donation, in the situation where a patient cannot be diagnosed as dead using brainstem criteria, may be an alternative to overcome shortage of donors. Preclinical studies showed that functional recovery of hearts after circulatory determination of death (DCDD) were similar to that of hearts from brain-dead donor. However, there are several experimental methods of simulating DCDD conditions such as exsanguination and hypoxic cardiac arrest both followed by warm ischemia.We aimed to compare two non-heart beating models in rat heart transplantation in which grafts were retrieved under exsanguination- or apnea-induced non-heart-beating conditions.Donor hearts were either left in situ for 10 min after cardiac arrest, which was induced by rapid exsanguination  or subjected to hypoxic cardiac arrest followed by 10 min of warm ischemia . Additionally, a control group, in which heart grafts were retrieved from heart beating donors, was used (n = 6). Then, hearts were perfused with a cold preservation solution (Custodiol), explanted, stored at 4°C in Custodiol for 1h and heterotopically transplanted. We evaluated left-ventricular graft function and assessed protein expression 1.5h after transplantation.After transplantation, significantly decreased systolic function  was observed in both exsanguinations- and apnea-groups compared to controls. Additionally, apnea-group showed further impaired diastolic function when compared with the exsanguination-group . Protein levels of caspase-3, tumor necrosis factor alpha, cyclooxygenase-2, nuclear factor KappaB, inducible nitric oxide synthase, cytochrome-c and SERCA-2 (assessed by Western Blot) did not differ between the groups.Our results showed that both models appear to be useful for investigating functional and molecular characterization of potential use of hearts after DCDD.


Recently, long non-coding RNAs (lncRNAs) were found to be implicated in cancer progression. However, the contributions of lncRNAs to Hepatitis C virus-related hepatocellular carcinoma (HCC) remain largely unknown. Here, we characterized lncRNA expression in 73 tissue samples from several different developmental stages of HCV-related hepatocarcinogenesis by repurposing microarray data sets. We found that the expression of 7 lncRNAs in preneoplastic lesions and HCC was significantly different. Among these significantly differently expressed lncRNAs, the lncRNA LINC01419 transcripts were expressed at higher levels in early stage HCC compared to dysplasia and as compared with early stage HCC, lncRNA AK021443 level increase in advanced stage HCC while lncRNA AF070632 level decrease in advanced stage HCC. Using quantitative real-time reverse-transcription PCR, we validated that LINC01419 was significantly overexpressed in HBV-related and HCV-related HCC when compared with matched non-tumor liver tissues. Moreover, functional predictions suggested that LINC01419 and AK021443 regulate cell cycle genes, whereas AF070632 is associated with cofactor binding, oxidation-reduction and carboxylic acid catabolic process. These findings provide the first large-scale survey of lncRNAs associated with the development of hepatocarcinogenesis and may offer new diagnostic biomarkers and therapeutic targets for HCV-related HCC. Hepatocellular carcinoma (HCC) is one of the most frequently diagnosed cancers in the world and is the second leading cause of cancer-related deaths worldwide . DespitelncRNAs are defined as nonprotein-coding RNAs that are greater than 200 nucleotides in length . MultiplIn this study, we examined lncRNA expression in 73 tissue samples, including healthy liver tissue, cirrhotic tissue, dysplastic nodules, and HCC samples. The cirrhotic tissue, dysplastic nodules, and HCC samples were obtained from patients with HCV infections. Our data show that several lncRNAs could serve as biomarkers for the detection or prognosis of HCV-related HCC. Furthermore, we predicted the functions of the lncRNAs by investigating coexpression networks of the differentially expressed lncRNAs and protein-coding genes.p value < 0.05, fold change >2). Then, we performed a gene ontology (GO) analysis to enrich for gene sets based on differentially expressed protein-coding genes. We observed that genes that were up-regulated in HCC samples mainly participated in mitosis and the cell cycle , we re-annotated the entire collection of probes using the ncFANs utility. As a result, we identified probes for 17,600 protein-coding genes and 2,863 lncRNAs in these published microarrays. Next, we reanalyzed the expression patterns of the protein-coding genes across 5 groups of patient samples: control, cirrhosis, dysplasia, early HCC and advanced HCC. Because HCC always occurs in cirrhotic livers, we compared the gene profiles associated with the preneoplastic lesion stage (cirrhosis and dysplasia stages) with those of HCC samples to identify the critical molecular events associated with HCC progression. Our results indicated that 585 protein-coding genes  and 7 ln33plus2.0cle Fig. , whereascle Fig. .Next, we evaluated the changes in expression of the 7 lncRNAs during the five stages of HCC  using one-way ANOVA (paired with an F test). We noted that LINC01419 was characterized by a significant increase in transcript expression from dysplasia to early HCC Fig. . The lncBecause early detection biomarkers are critical for effective HCC care, we chose to further validate LINC01419, whose expression increased sharply from dysplasia to early HCC. We analyzed LINC01419 expression using quantitative real-time polymerase chain reaction (qRT-PCR) in 15 pairs of HCV-related HCC samples, 55 pairs of HBV-related HCC samples and their corresponding adjacent non-tumor liver tissues Fig. . These rTo investigate the role of lncRNAs in HCC pathogenesis, we used the gene expression profiles of 75 patient samples to construct a coding-non-coding co-expression network. The resulting network includes 220 lncRNA genes and 3,924 coding genes with 105,508 coding-coding edges, 7,114 noncoding-coding edges and 324 noncoding-noncoding edges (online resource). Next, we predicted the functions of the lncRNAs using two different methods, a module-based analysis and a hub-based analysis. First, the co-expression network was parsed into forty module-based subnetworks using an MCL algorithm. Twenty of these modules had at least one enriched GO term. Next, we obtained 154 lncRNA-centered subnetworks with at least one enriched GO term by parsing the co-expression network into different hub-based subnetworks.The functions of the 3 deregulated lncRNAs associated with the initiation or progression of HCC were predicted using the module-based method or the hub-based method. Our results suggest that LINC01419 and AK021443 were classified into the same module with the other 36 lncRNAs and 637 protein-coding genes. Because the co-expressed module could be regarded as a functional unit, LINC01419 and AK021443 may have the similar function with those 637 protein coding genes. Thus, by performing Go analysis on those protein-coding genes, we conclude that the functions of LINC01419 and AK021443 were mostly involved with cell cycle progression Fig. . The funIn the present study, we reanalyzed a large microarray dataset to investigate the pathogenesis of HCV-related HCC. There are three main advantages of using this approach. First, with the exception often healthy liver tissue samples, the samples used in this microarray dataset were derived from patients with hepatitis C virus infection, which is the leading cause of HCC in the United States and Europe . This stIncreasing evidence has suggested that lncRNAs serve critical functions . SeveralThe cell cycle controls cell division, which is critical to cancer. Cyclin-dependent kinases (CDKs), which are key regulators of the cell cycle, have been investigated as potential therapeutic targets for cancer . Cell cyTaken together, our results provide a comprehensive understanding of lncRNAs in HCV-related HCC and a basis for identifying specific biomarkers or therapeutic targets for HCC.The microarray data used in this study were generated using the Affymetrix platform HG-U133A Plus 2.0. We downloaded these publicly available data sets from the Gene Expression Omnibus (GEO) database (accession number: GSE6764). The microarray data assessed the gene expression profiles of 75 samples consisting of 10 normal liver tissue samples, 13 cirrhotic liver tissue samples, 17 dysplastic nodules, and 35 HCCs. The preneoplastic lesions (cirrhosis and dysplasia) and HCC samples were obtained from 38 patients infected with HCV. Patients with other etiologies, such as HBV infection, were excluded from our analysis.35 HCC samples were categorized using the four following criteria: (i) according to tumor size, where well-differentiated tumors <2 cm in diameter with no vascular invasion and satellites were categorized as very early stage HCC (8 cases); (ii) according to tumor differentiation, where microscopic tumors measuring <2 cm in diameter, well-to moderately differentiated tumors measuring 2–5 cm in diameter without vascular invasion or satellites and tumors with 2–3 well-differentiated nodules measuring <3 cm in diameter were categorized as early stage HCC (10 cases); (iii) according to tumor number, where poorly differentiated tumors measuring >2 cm in diameter with microvascular invasion and satellites or tumors measuring >5 cm in diameter were categorized as advanced stage HCC (7 cases); (iv) according to vascular invasion and satellites, where tumors with macrovascular invasion or diffuse liver involvement were categorized as very advanced stage HCC (10 cases) [Sixty-five frozen HCC tissues and matched adjacent normal liver tissues were randomly obtained from sixty-five patients who underwent hepatectomy at Peking Union Medical College Hospital . This patient cohort consisted of fifty-five patients who were infected with HBV and fifteen patients who were infected with HCV. We used these samples to perform quantitative real-time polymerase chain reaction (qRT-PCR) analysis.t-test, one-way ANOVA (paired with an F test) and Benjamini Hochberg (BH) FDR correction were performed using the Limma statistical package. Genes with a fold-change >2 and BH FDR-adjusted p-values < 0.05 were considered differentially expressed.Non-coding RNA Function Annotation Server (ncFANs) can be used to quantify the levels of messenger RNAs and long non-coding RNAs depending on the re-annotation of Affymetrix microarray probes . We subm® Green Master mix (Applied Biosystems) and a StepOne™ Real-Time PCR System . The relative expression levels of the lncRNAs were calculated using the comparative Ct method. GAPDH gene expression was included as an internal control. The primers for LINC01419were (forward) 5′-GAAACTCCGAACACATCTG-3′ and (reverse) 5′-TTCTCCTGCTGGTTGATT-3′; for GAPDH, (forward) 5′-TCAAGGCTGAGAACGGGAAG-3′ and (reverse) 5′-GTGAAGACGCCAGTGGACT-3′.Total RNA was isolated using TRIzol reagent (Invitrogen). First-strand cDNA was synthesized using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to instructions suggested by the manufacturer. Real-time PCR was performed using Power SYBRp-value significance threshold of < 0.01. Finally, we used the Enrichment Map plugin [The DAVID Bioinformatics Tool  was usedp plugin  for Cytop plugin  to visuath percentile. A Pearson's correlation coefficient or a Spearman Rank correlation coefficient was used to estimate the relationship of each gene pair, including coding-coding, coding-lncRNA and lncRNA-lncRNA gene pairs. The p-values of the correlation coefficients for all gene pairs were calculated using Fisher's asymptotic test and then were adjusted using Bonferronimultiple testing correction. We chose co-expression gene pairs with p-values < 0.01 to construct the coding-non-coding co-expression network. Based on the coding-non-coding co-expression network, we used module-based and hub-based methods embedded in ncFANs to predict the function of the lncRNAs. In the module based method, modules of co-expressed genes in the coding-non-coding co-expression network were identified using the Markov cluster algorithm (MCL). The protein coding genes in the same module as the lncRNAs were further analyzed using gene ontology analysis to predict the functions of the lncRNAs in this module. In the hub-based method, the co-expression network was parsed to identify subnetworks comprising a central lncRNA and its neighboring protein-coding genes. The functional enrichment of these connected protein-coding genes was considered the function of the corresponding lncRNA. Only function enrichments with p-values < 0.01 were retained.Gene expression data for 73 tissue samples was submitted to ncFANs to generate a coding-non-coding co-expression network. To build the co-expression network, we chose genes (both protein-coding genes and lncRNA genes) with expression differences that ranked in the top 75


Chest-X-ray is recommended for routine use in patients with suspected pneumonia, but its use in emergency settings is limited. In this study, the diagnostic performance of a new method for quantitative analysis of lung ultrasonography was compared with bedside chest X-ray and visual lung ultrasonography for detection of community-acquired pneumonia, using thoracic computed tomography as a gold standard.Thirty-two spontaneously breathing patients with suspected community-acquired pneumonia undergoing computed tomography examination were consecutively enrolled. Each hemi-thorax was evaluated for the presence or absence of abnormalities by chest X-ray and quantitative or visual ultrasonography.Quantitative ultrasonography showed higher sensitivity (93%), specificity (95%), and diagnostic accuracy (94%) than chest X-ray , or visual ultrasonography , or their combination .Quantitative lung ultrasonography was considerably more accurate than either chest X-ray or visual ultrasonography in the diagnosis of community-acquired pneumonia and it may represent a useful first-line approach for confirmation of clinical diagnosis in emergency settings.


A significant portion of patients with ST-elevation myocardial infarction (STEMI) display multivessel coronary artery disease (MVD). However, data on the association of MVD and reperfusion success are scarce. Thus, we thought to analyse the impact of MVD on infarct size, microvascular obstruction (MO), and myocardial salvage index (MSI) assessed by cardiac magnetic resonance imaging (CMR) in a large unselected cohort of patients with STEMI reperfused by primary percutaneous coronary intervention (PCI).STEMI patients reperfused by primary PCI (n=1074) within 12 hours after symptom onset underwent CMR 3 days after the index event (interquartile range [IQR] 2-4). Infarct size and MO were measured 15 min after gadolinium injection. T2-weighted and contrast-enhanced CMR were then used to calculate MSI. Severity of coronary artery disease was graded as single-vessel disease compared to MVD. Further, a detailed set of clinical, angiographic, and electrocardiographic parameters was recorded. The primary endpoint was defined as a composite of death, non-fatal myocardial reinfarction, and congestive heart failure (MACE). Clinical follow-up was conducted after 12 months.MVD was present in 48.5% (n=521) of all patients. Patients with MVD were older  and more often diabetics  in comparison to those with single-vessel disease. Angiographic reperfusion success defined as TIMI-flow III post-PCI , and ST-segment resolution  were similar between both groups.Patients with MVD displayed no significant differences in infarct size  and extent of MO , as well as MSI  in comparison to patients with single-vessel disease.Finally, the presence of MVD was significantly associated with the time-dependent occurrence of MACE (log-rank comparison p=0.004).MVD is not associated with impaired reperfusion success assessed by CMR. The adverse clinical outcome of patients with MVD might thus rather be explained by more advanced coronary artery disease itself and unfavourable baseline characteristics.None.


EGFR exons 2-7 results in a novel glycine at the junction and yields a tumor-specific antigen with demonstrated immunogenicity in mice and humans. Using a live-attenuated Listeria monocytogenes- based vaccine expressing a 21-AA neo-peptide from EGFRvIII (LmEGFRvIII), we demonstrated that prophylactic vaccination protects against subsequent challenge with an EGFRvIII-expressing squamous cell carcinoma (SCCVII-EGFRvIII). Similarly, therapeutic vaccination three days post-implantation prevented outgrowth of EGFRvIII-expressing tumors but not parental EGFRvIII-negative tumors. Conversely, we found that LmEGFRvIII vaccination was insufficient to clear large established SCCVII-EGFRvIII tumors despite eliciting large numbers of polyfunctional EGFRvIII-specific CD8+ T cells. We hypothesized that localized inflammation elicited by radiation therapy could recruit EGFRvIII-specific CD8+ T cells into the tumor. We demonstrated that while neither LmEGFRvIII vaccination nor radiation therapy alone were able to control the EGFRvIII-expressing tumors, the combination of LmEGFRvIII and radiation therapy led to tumor regression and durable cures. We are currently exploring the potential mechanisms for their synergy including the role of T cell recruitment, survival and epitope spreading.EGFRvIII is a constitutively active and tumor-specific deletional mutant of EGFR found in multiple tumor types including glioblastoma multiforme, and has been reported in head and neck squamous cell carcinomas (HNSCC). The deletion of


Relation of antiplatelet therapy (APT) discontinuation with the risk of serious cardiovascular events has not been fully addressed yet. This study is aimed to evaluate the risk of ischemic event after APT discontinuation based on long-term APT status of large cohort. In the CREDO-Kyoto Registry Cohort-2 enrolling 15939 consecutive patients undergoing first coronary revascularization, 10470 patients underwent percutaneous coronary intervention either with bare-metal stents (BMS) only (N=5392) or sirolimus-eluting stents (SES) only (N=5078). Proportions of patients taking dual-APT were 67.3% versus 33.4% at 1-year, and 48.7% versus 24.3% at 5-year in the SES and BMS strata, respectively. We evaluated daily APT status  and linked the adverse events to the APT status just 1-day before the events. No-APT as compared with dual- or single-APT was associated with significantly higher risk for stent thrombosis (ST) beyond 1-month after SES implantation , while higher risk of no-APT for ST was evident only until 6-month after BMS implantation . No-APT as compared with dual- or single-APT was also associated with significantly higher risk for spontaneous myocardial infarction (MI) and stroke regardless of the types of stents implanted. Single-APT as compared with dual-APT was not associated with higher risk for serious adverse events, except for the marginally higher risk for ST in the SES stratum. In conclusion, discontinuation of both aspirin and thienopyridines was associated with increased risk for serious cardiovascular events including ST, spontaneous MI and stroke beyond 1-month after coronary stenting. Early reports suggested that premature discontinuation of dual antiplatelet therapy (APT) was the most potent predictor of stent thrombosis (ST) after drug-eluting stent (DES) implantation –6. HowevThe Coronary REvascularization Demonstrating Outcome study in Kyoto (CREDO-Kyoto) percutaneous coronary intervention (PCI)/coronary artery bypass grafting (CABG) registry cohort-2 is a physician-initiated non-company sponsored multicenter registry enrolling consecutive patients undergoing first coronary revascularization procedures at 26 centers in Japan from January 2005 to December 2007 . The desThe research protocol  was apprThe outcome measures for the current analysis were Academic Research Consortium definite ST, spontaneous MI, and stroke . SpontanFollow-up data on clinical events together with APT status were collected by the experienced clinical research coordinators in the independent clinical research organization , we estimated the incidence rate during those time intervals. We expressed the incidence rate as the number of events per 100 person-years . CumulatCategorical variables were expressed as number and percentages. Continuous variables were expressed as mean +/- SD or median and interquartile range. In the comparison of incidence rates among each APT status, we used the likelihood-ratio chi-square test.To identify prognostically important factors other than daily APT status, we constructed cox regression models for each endpoint including 39 variables used in our previous report . Based oThe duration from APT discontinuation to the onset of adverse event was compared between the single-APT and no-APT groups, using Wilcoxon signed-rank test.Analysis was made by using JMP 10.0. , and Excel 2010 . P value <0.05 was considered as statistically significant.Baseline characteristics were previously described . In brieThe proportion of patients receiving dual-APT was higher in the SES stratum than in the BMS stratum  . The ratThe proportion of no-APT patients was small, but increased during follow-up in both SES and BMS strata  .In the SES stratum, the incidence rates for ST tended to be higher in no-APT patients as compared with dual- or single-APT patients in all the time intervals between 31 days and 1460 days. Cumulative incidence rate for ST beyond 1-month was significantly higher in no-APT patients as compared to patients with dual- or single-APT . TherefoIn the BMS stratum, higher risk for ST in no-APT patients was evident only in the interval of 31–180 days. Incidence rates of ST were very low regardless of APT status in all the time intervals between 181 days and 2190 days. Cumulative incidence rate of ST beyond 6-month in no-APT patients was not significantly different from that in dual- or single APT patients .In both the SES and BMS strata, incidence rates for ST were not significantly different between dual-APT and single-APT in any time intervals beyond 1-month. However, single-APT as compared with dual-APT after SES implantation, but not after BMS implantation, was associated with higher cumulative incidence rate for ST beyond 1-month Figs  and 5. TNo-APT as compared with dual- or single-APT was associated with higher risk for spontaneous MI, while single-APT as compared with dual-APT was not associated with higher risk for spontaneous MI regardless of the timing after stent implantation and regardless of the stent types implanted Figs  and 7. TNo-APT as compared with dual- or single-APT was also associated with higher risk for stroke, while single-APT as compared with dual-APT was not associated with higher risk for stroke regardless of the timing after stent implantation and regardless of the stent types implanted Figs  and 9. TIn the current analysis, APT status just 1-day before the event might not be causally related to the event, if APT status had changed a few days before the event. Therefore, we evaluated APT status during 30 days before the event in 73 patients with definite ST in the SES stratum . In 2 paThe main findings of the current novel analysis linking serious cardiovascular events after coronary stent implantation to APT status just 1-day before the events are as follows; (1) Discontinuation of both aspirin and thienopyridines was associated with significantly higher risk for ST up to 4-year after SES implantation, while higher risk for ST after discontinuation of both aspirin and thienopyridines was evident only until 6-month after BMS implantation; (2) Discontinuation of both aspirin and thienopyridines was also associated with significantly higher risk for spontaneous MI and stroke; (3) Discontinuation of either thienopyridine or aspirin only as compared with dual-APT was not associated with higher risk for serious cardiovascular events beyond 1-month after coronary stent implantation, except for the marginally higher risk for ST after SES implantation.Antiplatelet agents consists of several classes including, (1) Cyclooxygenase-1 (COX-1) inhibitors like aspirin, (2) P2Y12 (Adenosine diphosphate [ADP] receptor) inhibitors like thienopyridines represented by Clopidogrel and Ticlopidine, (3) Phosphodiesterase inhibitors like Cilostazol, (4) Glycoprotein IIB/IIIA inhibitors or others. Dual-APT mainly with aspirin and thienopyridines is the cornerstone therapy to prevent ST or other ischemic event within 30 days after coronary stent implantation , 26. HowPrevious reports evaluating the influence of APT discontinuation on ST could be classified into the following 5 types according to their methodologies: 1) randomized controlled trials comparing single-APT with dual-APT at various timings after coronary stent implantation –15, 2)  2, 5, 8, randomizIn the current analysis, we linked serious cardiovascular events after coronary stent implantation to APT status just 1-day before the events. The biggest advantage of this methodology was its ability to evaluate the APT status at the time of cardiovascular events. The timing of APT discontinuation after stent implantation was also taken into consideration. Discontinuation of aspirin and thienopyridines was separately analyzed. Furthermore, we evaluated the influence of APT discontinuation on serious cardiovascular events other than ST including spontaneous MI and stroke.It was noteworthy that nearly half of the SES patients still continued dual-APT even at 5-year after coronary stent implantation. Many of these patients seemed to have continued prolonged dual-APT mainly due to concerns on very late ST of SES, because the rate of dual-APT continuation in the SES stratum remained high even after exclusion of those patients with other indications of prolonged dual-APT such as prior stroke and peripheral vascular disease. In several recent reports with second-generation DES, nearly 90% of patients continued dual-APT at 1-year , 28, sugAnother important observation in the current study was that discontinuation of both aspirin and thienopyridines was associated with markedly increased risk for ST. The finding was consistent with our previous report from the j-Cypher registry . The curThis study has several limitations. First, we cannot deny the possibility of recall bias when the information on APT status was obtained from the patients and/or their relatives. Second, we did not assess the actual compliance and adherence to APT. Third, PARIS registry reported that cardiac events after cessation of dual-APT depend on the clinical reasons for cessation and early risk for events due to disruption (cessation of APT due to bleeding or non-compliance) is substantial irrespective of stent type . We did Discontinuation of both aspirin and thienopyridines was associated with increased risk for serious cardiovascular events including ST, spontaneous MI and stroke beyond 1-month after coronary stenting.S1 Appendix(DOCX)Click here for additional data file.S1 Protocol(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 FigAPT = antiplatelet therapy, BMS = bare-metal stents, DAPT = dual-APT, PCI = percutaneous coronary intervention, and SES = sirolimus-eluting stents.(TIF)Click here for additional data file.S2 FigValues are expressed as median (interquartile range) days. APT = antiplatelet therapy, BMS = bare-metal stents, SAPT = single-APT, SES = sirolimus-eluting stents, and ST = stent thrombosis. As for the definitions of the duration, see (TIF)Click here for additional data file.S3 FigValues are expressed as median (interquartile range) days. APT = antiplatelet therapy, BMS = bare-metal stents, MI = myocardial infarction, SAPT = single-APT, and SES = sirolimus-eluting stents. As for the definitions of the duration, see (TIF)Click here for additional data file.S4 FigValues are expressed as median (interquartile range) days. APT = antiplatelet therapy, BMS = bare-metal stents, SAPT = single-APT, and SES = sirolimus-eluting stents. As for the definitions of the duration, see (TIF)Click here for additional data file.