Intratumoral heterogeneity of HER2 expression is common in gastric cancers and pose a challenge for identifying patients who would benefit from anti-HER2 therapy. The aim of this study is to compare HER2 expression in biopsy and resection specimens of gastric carcinoma by immunohistochemistry (IHC) and to find the ideal number of biopsy tumor fragments that can accurately predict HER2 overexpression in the corresponding surgically resected specimen. The HER2 IHC results of 702 paired biopsy and resection specimens of gastric cancer were compared.P = 0.0315 and P = 0.0052). ROC curve analysis and positive predictability showed that 4 fragments should be obtained to minimize the differences in HER2 scores between biopsy and resection specimen.The mean number of biopsy fragments among all cases was 4.3 (range 1–11). HER2 was positive in 130 (18.5%) endoscopic biopsies and in 102 (14.5%) gastrectomy specimens. Intratumoral heterogeneity of HER2 was found in 80 (61.5%) biopsies and 70 (68.6%) resection specimens. Out of the 70 surgical specimens with intratumoral heterogeneity, 24 (34.3%) of the corresponding biopsies were categorized as negative (positive conversion). In the 86 (12.3%) discrepant cases, negative conversion was observed in 57 (66.3%) cases and positive conversion in 29 (33.7%). The fragment numbers were significantly correlated with the discrepancy of results and positive predictability (In gastric carcinomas with discrepant HER2 results between biopsy and surgical resection specimens, intratumoral heterogeneity is common with most of them showing positive conversion. To predict HER2 status precisely, at least 4 biopsy fragments containing tumor cells are required. Since its first expression characteristics were described in gastric cancer in 1986, human epidermal growth receptor 2 (HER2) has become an established predictive biomarker in this disease , 4. Receet al [However, determining HER2 expression status in the practical setting presents a few challenges. One challenge is due to tissue sampling in biopsy specimens compared with the resection specimens. To ameliorate diagnostic accuracy and reduce discordance between biopsy and resection specimens, sufficient biopsy material is required. Several studies have compared HER2 results in paired biopsy and resection specimens and showed an overall concordance rate varying between 87 to 96% . Severalet al . have reet al  has propAnother challenge associated with interpreting HER2 status in gastric cancer is the protein's affinity for being heterogeneously expressed. High incidence of HER2 heterogeneity is found in up to 79.3% of HER2-positive gastric cancers within the same tumor  . IntratuOnly patients with primary gastric carcinoma who underwent both preoperative endoscopic biopsy and gastric cancer resection at Samsung Medical Center in Seoul, Korea from January 2013 to December 2014 were selected for this study. Patients who underwent preoperative chemotherapy and/or radiotherapy, or patients who were diagnosed with multiple gastric cancers were excluded. The clinicopathologic characteristics of 702 patients are described in Table A tumor fragment was defined as a piece of tissue containing 10 or more viable tumor cells in an endoscopic biopsy specimen as previously described .IHC for HER2 (PATHWAY HER-2/neu (4B5) rabbit monoclonal antibody, Ventana Medical Systems, Inc., Tucson, AZ) was performed in all cases with a BenchMark XT automated stainer . In operation specimens, we reviewed all hematoxylin and eosin stained tumor sections and selected a representative tumor block for IHC analysis. A gastrointestinal pathologist (KMK) evaluated the staining data with no previous knowledge of clinical or pathological parameters. HER2 IHC was scored according to the recently developed assessment guidelines for HER2-associated gastric cancers . In biopT-test was used to compare the number of total fragments between concordant (between biopsy and resection results) and discordant groups as well as between the biopsy positive and negative groups. Linear regression method was used to associate the difference in HER2 score between biopsy and resection specimen with the number of total fragments. A ROC curve was constructed to find a cutoff value for the number of total fragments. These analyses were conducted using SAS version 9.4  and R 3.1.1  by biostatisticians (CS and JSH).A two-sample n = 616, 87.7%) between biopsy and resection specimens, 86 (12.3%) paired specimens showed discrepant results. Of these discrepant cases, negative conversion (positive in biopsy and negative in resection) was found in 57 (66.3%) cases, and positive conversion was seen in 29 (33.7%) cases. After exclusion of equivocal 2+ cases, [The IHC results of 702 paired biopsy and gastrectomy specimens were analyzed . Actually, for six cases with HER2 3+ in biopsies but negative on operation specimen, number of biopsy fragments were 2 (n = 1), 3 (n = 2), 4 (n = 2) and 5 (n = 1) and all five cases with > 2 biopsy fragments showed intratumoral heterogeneity within biopsy specimens. In those 6 cases, we further performed IHC using all tumor blocks from operation specimen and found 2+ in four cases and 3+ in two cases with positive tumor cells ranging from 1% to 10% of total tumor volume. Moreover, cases with heterogeneity in either biopsy or operation specimens showed higher discrepant results compared to cases without heterogeneity  . Out of 102 HER2-positive operation specimens, 70 cases (68.6%) showed heterogeneity, in which 24 (34.3%) showed discrepancy (positive conversion). Heterogeneity found in biopsy specimens was significantly correlated with its surgical operation specimen (n = 159). This value was termed the “difference value”. For all paired specimens with a difference value of 0 (no difference between biopsy and resection HER2 status), the mean fragment count was 4.9 (n = 50). For the paired specimens with a difference value of 1, 2, and 3, the mean fragment count was 4.9 (n = 53), 4.3 (n = 47), and 2.9 (n = 9), respectively. Additionally, the number of biopsy fragments was significantly correlated with the difference value (P = 0.0096)  within the HER2-positive group was calculated  Figure . Hence, n = 63) in either biopsy or resection specimen were evaluated. HER2 equivocal (2+) cases were excluded to improve the precision of the calculation. Out of 63 cases, 8 cases showed positive conversion (negative in biopsy and positive in gastrectomy). The number of fragments in all positive conversion cases ranged from 1 to 5 . According to ROC analysis, the ideal cut-off value was 3 with an AUC of 0.8045 (Figure ‘Positive predictability’ was defined as the positive predictive value of HER2 in biopsy specimens. To assess the positive predictability in relation to the biopsy fragment numbers, fragment numbers within only the HER2 3+ positive group a n = 3 in eith5 Figure . TherefoAccurate prediction of HER2 status in endoscopic biopsies of gastric cancer is essential for accurate tumor characterization and also carries important therapeutic implications. However, the heterogeneity of HER2 overexpression in gastric cancer contributes to false-negative results in cases with limited biopsy material suggesting the necessity of extensive tissue sampling . To our Intratumoral heterogeneity is the major underlying cause of discrepant HER2 results between the biopsy and resection specimens –13, 15. In this study, the positive predictability of the biopsy specimen was also calculated. True positive cases were determined as HER2 3+ either in the biopsy or resection specimens. Out of 63 HER2 3+ cases, 12.7% of cases were negative in the biopsy specimen and positive in the resection specimen. Interestingly, all cases with positive conversion showed intratumoral heterogeneity in the surgical specimens confirming that heterogeneity is the major reason for the discordant results. In cases with positive conversion, the number of biopsy fragments ranged from 1 to 5. Endoscopic biopsies with six or more fragments showed 100% HER2 status correlation with the surgically resected specimen. In our statistical analyses, a minimum of 4 biopsy fragments was needed to accurately predict HER2 status in the resected specimens.In summary, due to significant intratumoral heterogeneity, the larger the number of endoscopic biopsy fragments available for HER2 IHC analysis in gastric cancer, the higher the correlation with HER2 status will be in the resection specimen. The discordance increased with smaller numbers of biopsy fragments. This warrants some caution in relying on HER2 IHC findings of endoscopic biopsy specimens alone to determine treatment regimens.Finally, we suggest obtaining at least 4 biopsy fragments containing cancer in endoscopic biopsy for accurate HER2 test and recommend to record tumor fragments number in HER2 IHC pathologic report.


Since 2003, all nursing staff at HUG follow a mandatory course in infection control in the context of an institutional program. As part of a quality improvement pilot program we implemented a knowledge assessment tool in 2 units of a 300-bed geriatric hospital.We aim to assess the nursing staff’s theoretical knowledge of infection control and its application in everyday practice.During the first period (01-04/2014) all nurses and nursing assistants in the 2 participating units were assessed for their theoretical knowledge of infection control during a 30-minute interview using a predefined questionnaire with 12 items. During the same period adherence to hand hygiene was measured based on the WHO framework. During a second period (4-6/2014) individual and group-level feedback about knowledge scores and hand hygiene adherence was provided. Individuals with suboptimal performance in either domain were targeted for individual training sessions. During a third period (6-12/2014) hand hygiene compliance and the implementation of infection control measures was audited.st phase increased from 59 to 98 %. 14 infection control measures were audited and all fulfilled the predefined criteria for adequacy.21 nurses and 13 nursing assistants were assessed during the 1st period. None remembered to have received training in infection control. 5 caregivers reached the maximum knowledge score and had hand hygiene adherence > 80%. 5 caregivers reached the maximum knowledge score but had a hand hygiene compliance < 50%. 1 caregiver reached low scores both with regard to knowledge and hand hygiene compliance. During the 3rd period overall hand hygiene compliance of 6 randomly selected caregivers having participated in the 1Despite a long institutional culture of patient safety and infection control only 15% of the nursing staff had very good theoretical knowledge of infection control and were able to implement this knowledge into good adherence to hand hygiene. This quality improvement pilot program made the whole team reflect on their practices and made it possible to identify caregivers in need of individualized training.None declared.


Cell motility is an actin dependent process requiring the formation and extension of lamellipodia or filopodia, and is absolutely essential for many cellular processes, especially for morphogenesis during development. During lamellipodial extension, actin dynamics involve switching between branch formation at the leading edge and proximal severing of existing actin filaments . Actin bWhat is known is that Arp2/3 dependent actin assembly and Cofilin dependent disassembly are coordinately regulated at the leading edge by Coronin 1B, which binds the Arp2/3 complex in a phosphorylation-dependent manner. When dephosphorylated, Coronin 1B inhibits the binding of the Arp2/3 complex to actin, but phosphorylation by protein kinase C at Ser2 reduces its association with the Arp2/3 complex, enabling actin branch formation [Cofilin is also regulated by phosphorylation but, in contrast to Coronin 1B, it is inactivated by phosphorylation, and becomes active through Slingshot phosphatase (SSH) dephosphorylation . The firOur recent study has revealed two new important players in cellular motility. We have found that WISp39 (Waf1 Cip1 stabilizing protein 39), an Hsp90 binding protein we had discovered , coordinUsing biochemical approaches to dissect the interactions of WISp39, phospho-Coronin 1B, SSH and Hsp90, we have demonstrated that WISp39 may be the scaffold that orchestrates the activity of key regulators of actin dynamics  to maintain lamellipodia protrusion and directed motility as shown in Figure We have previously demonstrated that WISp39 interacts with cyclin-dependent kinase inhibitor p21 and Hsp90 in a trimeric complex that stabilizes p21 against degradation . That stOur data support a key role for WISp39 in regulating actin dynamics to sustain directed cell motility. We suggest that WISp39 and its binding partner Hsp90 act as a crucial scaffold that integrates Coronin 1B, SSH and Arp2/3 complex at the leading edge.


Sox2 during blastocyst formation. First, we investigate the regulation of Sox2 patterning and show that SOX2 is restricted to ICM progenitors prior to blastocyst formation by members of the HIPPO pathway, independent of CDX2, the TE transcription factor that restricts Oct4 and Nanog to the ICM. Second, we investigate the requirement for Sox2 in cell fate specification during blastocyst formation. We show that neither maternal (M) nor zygotic (Z) Sox2 is required for blastocyst formation, nor for initial expression of the pluripotency genes Oct4 or Nanog in the ICM. Rather, Z Sox2 initially promotes development of the primitive endoderm (PE) non cell-autonomously via FGF4, and then later maintains expression of pluripotency genes in the ICM. The significance of these observations is that 1) ICM and TE genes are spatially patterned in parallel prior to blastocyst formation and 2) both the roles and regulation of Sox2 in the blastocyst are unique compared to other pluripotency factors such as Oct4 or Nanog.Pluripotent epiblast (EPI) cells, present in the inner cell mass (ICM) of the mouse blastocyst, are progenitors of both embryonic stem (ES) cells and the fetus. Discovering how pluripotency genes regulate cell fate decisions in the blastocyst provides a valuable way to understand how pluripotency is normally established. EPI cells are specified by two consecutive cell fate decisions. The first decision segregates ICM from trophectoderm (TE), an extraembryonic cell type. The second decision subdivides ICM into EPI and primitive endoderm (PE), another extraembryonic cell type. Here, we investigate the roles and regulation of the pluripotency gene  Pluripotent stem cells can give rise to any cell type in the body, making them an attractive tool for regenerative medicine. Pluripotent stem cells can be derived from the mammalian embryo at the blastocyst stage or they can be created from mature adult cells by reprogramming. During reprogramming, SOX2 helps establish pluripotency, but it is not clear how SOX2 establishes pluripotency in the blastocyst. We evaluated where SOX2 is present, how SOX2 is regulated, and where SOX2 is active during blastocyst formation. Our data show that the roles and the regulation of SOX2 are unique compared to other pluripotency/reprogramming factors, such as OCT4 and NANOG. SOX2 marks pluripotent cells earlier than do other factors, but does not regulate pluripotency until several days later. Rather, the earlier role of SOX2 is to help establish the yolk sac lineage, which is essential for gestation. Oct4, Nanog, and Sox2, during establishment of EPI cells in the blastocyst Sox2 in the blastocyst are unresolved. For example, several studies reported that Sox2 is restricted to the ICM by the blastocyst stage Sox2 expression in the blastocyst are unknown.To create and use pluripotent stem cells, it is essential to understand the origins of pluripotency during normal development. During mouse blastocyst formation, pluripotent epiblast (EPI) cells are established by two cell fate decisions that segregate pluripotent progenitors from extraembryonic tissues Sox2 expression is patterned, the functional roles of Sox2 in the blastocyst are not yet clear. ES cells cannot be derived from embryos lacking zygotic (Z) Sox2Sox2 is essential for pluripotency. In ES cells, Sox2 is required for the expression of pluripotency genes, such as Oct4 and Nanog, and for the repression of TE genes Sox2 might be required for initial expression of pluripotency genes and repression of TE genes in the ICM. However, the expression of pluripotency and TE genes in Sox2 Z null blastocysts has not yet been examined at the level of individual cells. Moreover, maternal (M) Sox2 is also thought to participate in blastocyst formation, which could partially compensate for loss of Z Sox2. RNAi knockdown of M and Z Sox2 in the zygote was reported to disrupt blastocyst formation Sox2 during development.In addition to the unresolved mechanism by which Sox2 mRNA is enriched in ICM progenitors starting at the 16-cell stage Sox2 is restricted to ICM progenitors. Using immunofluorescence and confocal microscopy, we observed that SOX2 is restricted to nuclei of ICM progenitors at the 16-cell stage and later  analysis of oocytes from fl/fl or delZp3Cre; Sox2 females  and ICM (OCT4) markers was normal in the absence of MZ Sox2  to E3.75  or reduced (1/5 Sox2 null embryos), expression of OCT4 was undetectable (1/5 Sox2 null embryos) or reduced (3/5 Sox2 null embryos), and expression of NANOG was also undetectable (1/1 Sox2 null embryos)  . These ome point . We concSox2 mRNA in ICM progenitors Sox2 in regulating cell fate does not become apparent until late blastocyst stage. It is possible that Sox2 is initially genetically redundant with other pluripotency factors, such as Oct4 or Nanog. Although phenotypes resulting from disruption of multiple pluripotency genes have not yet been reported in mice, there is evidence of genetic redundancy among zebrafish Oct/Sox/Nanog orthologues Fgf4 expression is reduced, but not eliminated, in the absence of either Sox2 or Oct4Fgf4, and possibly other targets, synergistically, as has been demonstrated in pluripotent stem cell lines Here we have examined the roles and regulation of SOX2 in the preimplantation embryo, with the goal of deepening our understanding of the origins of pluripotency during development. We showed that SOX2 is a unique, early marker of ICM progenitors, consistent with the reported early enrichment of Sox2 directly or indirectly. In ES cells, the YAP/TEAD complex been shown to bind upstream of Sox2 to promote its expression Sox2 in TE cells. It will be exciting to address this hypothesis in future studies in addition to examining whether position, polarity, and/or contact regulate Sox2 expression, as has been shown for Cdx2Sox2 and Cdx2 in the embryo. Finally, our observations are also consistent with LATS regulating the activity of an as-yet unidentified transcription factor that promotes expression of SOX2 in ICM progenitors. This hypothesis is also supported by qPCR evidence that Lats1/2 maintains expression of Sox2 in the blastocyst Our evidence suggests that SOX2 and CDX2 are patterned by HIPPO pathway components in parallel , but it Sox2 is activated de novo in the trophoblast lineage postimplantation, where it promotes trophoblast development Sox2 expression postimplantation as well. Investigation of the mechanisms by which Sox2 expression becomes activated in the extraembryonic ectoderm is an exciting opportunity to learn about the origins of trophoblast stem (TS) cells, which are derived from extraembryonic ectoderm Sox2 and Tead4Our study provides insight into the regulation of extraembryonic cell types during preimplantation development. In terms of the TE lineage, we showed that SOX2 is not detected in TE cells during preimplantation, nor is it required for their specification. These observations suggest that expression of While SOX2 does not activate TE gene expression, SOX2 also does not appear to repress TE gene expression in the ICM. This is in contrast to OCT4, which is known to repress expression of TE genes in the ICM and in ES cell lines Nanog null embryos has not yet been reported, in Gata6 null embryos, SOX2 is expressed in all ICM cells Gata6 and Nanog null embryos, with and without manipulations to the FGFR/MEK signaling pathway will help clarify the direct and indirect mechanisms regulating Sox2 expression in the ICM.Our analysis led us to explore the genetic regulation of PE specification as well. We have shown that in the ICM, SOX2 becomes expressed in a salt-and-pepper fashion, similar to NANOG. We have also shown that the salt-and-pepper distribution of SOX2 in the ICM depends on FGFR/MEK signaling, but it is not yet clear whether FGFR/MEK signaling regulates SOX2 expression directly, or whether FGFR/MEK signaling maintains cell fate, which in turn regulates SOX2 expression. Alternatively, NANOG or GATA6 could help regulate SOX2 expression in the ICM. While the expression pattern of SOX2 in Sox2 null embryos do not completely phenocopy Fgf4 null embryos, since NANOG was not upregulated in Sox2 null embryos as it is in Fgf4 null embryos Fgf4 in Sox2 null embryos are sufficient to repress NANOG, as we have shown for Oct4 null embryos Sox2 null embryos as has been observed in Fgf4 null embryos Sox2 null embryos is eventually able to restore expression of PE genes 93Knwtm1HsskTead4tm1.1AralCdx2del+Sox2) were generated by crossing mice carrying tm1.1LanSox2 with tm1(cre)Nagy129-AlplAll animal research was conducted in accordance with the guidelines of the University of California Santa Cruz Institutional Animal Care and Use Committee or by RIKEN CDB and Kumamoto University. The following alleles or transgenes were used in this study: 2. Microinjection of mRNA was performed as described As described previously Embryos were fixed, stained, imaged, and recovered for genotyping as previously described Sox2 MZ null embryos as previously described Sox2 alleles Chimeras were performed using Sox2 (GCGGAGTGGAAACTTTTGTCC and CGGGAAGCGTGTACTTATCCTT), Fgf4 (AGCAGGGGCAAGCTCTTC and GGGTACGCGTAGGATTCG), Oct4 (AGCTGCTGAAGCAGAAGAGG and AGATGGTGGTCTGGCTGAAC), and Actb (CTGAACCCTAAGGCCAACC and CCAGAGGCATACAGGGACAG).RNA isolation and single blastocyst qPCR was performed as previously described Figure S1Sox2, the anti-SOX2 antibody non-specifically detects a cytoplasmic pattern in TE cells, while the signal is specific to ICM at the blastocyst stage. B) SOX2 is detected in an increasing proportion of inside/ICM cells during formation of the blastocyst, with nearly 100% of ICM cells expressing SOX2 by E3.5. C) SOX2 does not colocalize with CDX2 in TE cells of the blastocyst, although SOX2 colocalizes with rare inside cells that express CDX2 , consistent with our prior observations that CDX2 is detected in rare inside cells at this stage Details of SOX2 expression pattern in the early embryo. A) In morulae lacking (EPS)Click here for additional data file.Figure S2Sox2 M null evaluated as a negative control), M Sox2 mRNA is not detectable (*) in individual wild type 2-cell embryos by qPCR , and M SOX2 is not detectable in nuclei of Sox2 Z null embryos at the 16-cell stage . B) At E4.5, neither CDX2 nor EOMES are detected in the ICM of Sox2 null embryos. Bar  = 20 µm.M Sox2 is not detectable in early embryos, and SOX2 does not repress TE fate in the ICM. A) M SOX2 protein is not detectable in wild type zygotes ((EPS)Click here for additional data file.Figure S3Sox2 acts upstream, and not downstream of Fgf4. A) qPCR analysis of single blastocysts at E3.75 shows that M Sox2 is not required for Fgf4 expression. B) Treatment of control (Sox2 M null or nonmutant) or Sox2 null (MZ or Z null) embryos for 42 h starting at E2.75 leads to complete upregulation of SOX17 and downregulation of NANOG throughout the ICM. C) Quantification of embryos shown in panel B . Bar  = 20 µm, p-value calculated by t-test; n.s.  = p>0.05; ANOVA performed for panel C.Z and not M (EPS)Click here for additional data file.Figure S4Sox2 null blastocysts. B) Average ICM cell number for time points examined in Sox2 null blastocysts at the equivalent of E4.25. D) Expression of DAB2 is eventually rescued in Sox2 null implantation-delayed blastocysts. The number of EPI cells is significantly reduced in Sox2 null implantation-delayed blastocysts, relative to wild type. By contrast, the number of PE cells is not significantly reduced in Sox2 null implantation-delayed blastocysts, relative to wild type, until the last time point examined. EPI and PE were defined based on SOX17 expression, since other EPI markers are not detectable in Sox2 null blastocysts at this stage. Bar  = 20 µm, p-value calculated by t-test, n.s.  = p>0.05.Evaluation of PE quality in late blastocysts. A) At E4.25, the average proportion of ICM cells in which GATA6 is detected is equivalent between control and (EPS)Click here for additional data file.Table S1Cell numbers detected in wild type embryos harvested at the indicated times (E3.0–E4.5).(DOCX)Click here for additional data file.


Multiple cell types including trophoblasts, osteoclasts and myoblasts require somatic cell fusion events as part of their physiological functions. In Drosophila Melanogaster the paralogus type 1 transmembrane receptors and members of the immunoglobulin superfamily Kin of Irre (Kirre) and roughest (Rst) regulate myoblast fusion during embryonic development. Present within the human genome are three homologs to Kirre termed Kin of Irre like (Kirrel) 1, 2 and 3. Currently it is unknown if Kirrel3 is expressed in adult human skeletal muscle.We investigated (using PCR and Western blot) Kirrel3 in adult human skeletal muscle samples taken at rest and after mild exercise induced muscle damage. Kirrel3 mRNA expression was verified by sequencing and protein presence via blotting with 2 different anti-Kirrel3 protein antibodies. Evidence for three alternatively spliced Kirrel3 mRNA transcripts in adult human skeletal muscle was obtained. Kirrel3 mRNA in adult human skeletal muscle was detected at low or moderate levels, or not at all. This sporadic expression suggests that Kirrel3 is expressed in a pulsatile manner. Several anti Kirrel3 immunoreactive proteins were detected in all adult human skeletal muscle samples analysed and results suggest the presence of different isoforms or posttranslational modification, or both.The results presented here demonstrate for the first time that there are at least 3 splice variants of Kirrel3 expressed in adult human skeletal muscle, two of which have never previously been identified in human muscle. Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance. These initial discoveries highlight the need for more molecular and functional studies to understand the role of Kirrel3 in human skeletal muscle. Somatic cell fusion events are critical for development of multicellular eukaryotic organisms. Postnatal growth and repair also frequently rely on somatic cell fusion events, especially in tissues such as skeletal muscle that contain multinucleated cells. However, the genes and molecular mechanisms underpinning this fundamental cellular process in humans are largely unknown. Multiple cell types including trophoblasts, osteoclasts and skeletal muscle require somatic cell fusion events in order to perform their physiological functions –3. The oIn humans, a single skeletal muscle fibre can contain thousands of nuclei . Each nuResearch findings from Drosophila have highlighted that key events in the muscle cell fusion process are cell-cell attraction, adhesion and subsequent actin nucleation at sites of cell-cell adhesion, the latter enabling membrane fusion –17. A wiPresent within the human genome are three Kirre homologs termed Kin of Irre like (Kirrel) 1 ,2 and 3 . This faOur primary aim was to assess whether Kirrel3 is present in uninjured and regenerating human skeletal muscle following mild damage-inducing exercises such as plyometric jumping and downhill running. Presence of Kirrel3 in the afore mentioned samples would raise questions about its function in skeletal muscle. Currently nothing is known about Kirrel3 in this tissue, or if it is indeed present in adult human skeletal muscle. While its presence alone would not confirm a role in the human muscle cell fusion process, it would provide initial support for further investigation. It is possible that it could mirror the role of Kirre, its Drosophila homolog, in regulating myoblast fusion events in adult human muscle.Our research findings demonstrate that at least three alternative splice variants of Kirrel3 are present in adult human skeletal muscle. Two of these splice variants have not been previously reported in the published literature. Detection of Kirrel3 mRNA in adult human skeletal muscle using standard PCR was sporadic with occasional detection in uninjured and regenerating skeletal muscle samples. Semi nested PCR increased the detection rate. Such sporadic detection, using standard PCR assessment, demonstrates that in adult human skeletal muscle Kirrel3 mRNA is present at very low levels. At the protein level, Kirrel3 immunoreactive proteins in uninjured and regenerating adult human skeletal muscle were observed. Further work is now required in order to ascertain the function of all Kirrel3 splice variants in human skeletal muscle and to provide more insight into stimuli promoting its expression and its subsequent post-transcriptional regulation.th exon 641 nucleotides 3′ to the missed spliced site in B. The 3′ untranslated regions of Kirrel3 A and B are 1079 and 370 nucleotides respectively.Analysis of the National Centre for Biotechnology (NCBI) gene database highlighted the presence of two human Kirrel3 reference sequences NM_032531.3 (hereafter referred to as Kirrel3 A) containing 3777 nucleotides and NM_001161707.1 (hereafter referred to as Kirrel3 B) containing 2534 nucleotides. Aligning Kirrel3 A and B mRNA transcripts to the human genome via DNA sequence present in the genomic contig NT_033899.8, demonstrated that Kirrel3 A has 17 exons while B has 14 exons , five extracellular Ig domains  and a transmembrane domain (AA 536-558 http://www.cbs.dtu.dk/services/TMHMM/). Five putative N-linked glycosylation sites  are predicted to be present in the extracellular domain of both Kirrel3 proteins. The intracellular domain of Kirrel3 A is predicted to contain an amphysisin SH3 binding domain (AA 759-773 http://scansite.mit.edu/). Also present at its C-terminal end is a Post Synaptic density protein 95, Drosophila discs Large tumour suppressor, zonula occludens (PDZ) binding domain corresponding to the amino acids THV [Kirrel3 A is the larger of the two Kirrel3 proteins with 778 amino acids (AA) while Kirrel3 B has 600 AA. The first 565 AA of Kirrel3 A and B are identical. Domains predicted within this region  demonstrated that they were all variants of Kirrel3  study by our group  were utiSkeletal muscle is made up of multinucleated cells (muscle fibres). The Drosophila homolog of Kirrel3, Kirre is involved in enabling muscle cell fusion events. Hence, Kirrel3 may be a putative muscle cell fusion regulator in humans. There is currently no published data available regarding Kirrel3 in adult human skeletal muscle. Therefore, the primary aim of this research was to assess for the presence of Kirrel3 in adult human skeletal muscle. By using PCR and transcript sequencing, we provide the first evidence that three alternatively spliced Kirrel3 mRNA transcripts are present in adult human skeletal muscle. Results from western blot analysis provide support for the presence of Kirrel3 protein in adult human skeletal muscle.The mRNA data highlight that in adult human skeletal muscle Kirrel3 mRNA expression levels are very low and that using standard PCR may result in false negatives. A nested PCR approach yielded a higher rate of Kirrel3 detection. It will be of interest for future studies to examine Kirrel3 mRNA expression in a model that is known to include a large amount of myogenesis such as can be seen in some muscle pathologies. Primary human myoblast cell cultures would provide a useful system to further investigate Kirrel3 and ascertain its importance to human muscle cell fusion. Murine proprioceptive neurons have been reported to express Kirrel3 , therefo-min [The physical process of transcribing Kirrel3 can be regarded as a specialised event because, compared to the vast majority of other genes within the human genome, Kirrel3 spans a very large genomic region of approximately 580kB. Such a long genome span is likely to impact on the rate of Kirrel3 mRNA transcript production. RNA polymerase II has been reported to be capable of transcribing large human genes at a rate of approximately 4 kb-min . This ra-min , while t-min .Results from our western blot experiments varied depending on the commercial antibody being used. Each antibody targeted different epitopes with one being extracellular and the other intracellular. The fact two immunoreactive proteins that migrated at approximately 70 and 75 kDa were detected by both antibodies, appears to support that these are Kirrel 3 proteins rather than breakdown products. However, the predicted molecular weight of Kirrel3A is 85 kDa therefore the Kirrel proteins within the 70 kDa range are likely to be one or two of the other Kirrel3 splice variants present in human skeletal muscle. It is possible that they represent one splice variant with the larger having undergone post-translational modification. Immunoprecipitation and mass spectrometry analysis will be useful in determining the identity of these immunoreactive proteins.The proteins detected at 50 and 55 kDa were detected only when using the Kirrel3 antibody targeting the intracellular domain. This antibody may be recognising partially degraded Kirrel3 proteins or these could be alternative truncated isoforms.For analysis of mRNA, our primers were directed towards the 3′ end region of Kirrel3 (intracellular coding region) and we can only speculate on whether or not alternative splicing may occur further downstream towards the 5′ end (extracellular coding region) that would produce even smaller Kirrel3 protein isoforms. If alternative splicing occurs at the 5′ end this could explain why the extracellular targeting antibody did not detect proteins of similar size as those detected by the intracellular targeting antibody. In future, we suggest that 5′ race experiments should be performed to provide useful information in evaluating this possibility.Our rationale for investigating Kirrel3 in human skeletal muscle was derived from research studies in Drosophila that demonstrated the involvement of two genes, Kirre and Rst, in the muscle cell fusion process during embryonic development . While o-/- murine myoblasts displayed reduced fusion capabilities during in vitro myogenesis [In vitro, the extracellular domain of Kirrel3 is capable of binding to the extracellular domain of nephrin, a type 1 transmembrane protein and member of the immunoglobulin superfamily . Nephrinogenesis .Kirrel3 is part of the Kirrel gene family that contains another two structurally similar members: Kirrel and Kirrel2 . In DrosThe splice variants, Kirrel3 A and B, that are present in adult human skeletal muscle and/or astrocytes are predicted to contain significantly different cytoplasmic domains. Kirrel3 A, but not Kirrel3 B, is predicted to contain a SH3 amphysisin binding domain and also a PDZ binding domain. Such differences are likely to result in the two isoforms having divergent functions. Amphysisin is a protein whose function is not completely understood, however, it is highly concentrated at nerve terminals . The C-eThe PDZ binding domain present in Kirrel3 A may confer Kirrel3 with the ability to alter the polarity of the cell in which it is expressed as the PDZ binding domain of human Kirrel3 is capable of binding to the cell polarity protein partitioning defective 3 (PARD3) . SatelliIn conclusion, the results presented here demonstrate for the first time that there are at least 3 splice variants of Kirrel3 present in adult human skeletal muscle, two of which have never previously been identified in human muscle. Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance. These initial discoveries highlight the need for more molecular and functional studies. Full length sequence information should be obtained on all Kirrel3 mRNA transcripts in order to predict the translated Kirrel3 proteins. Physiological studies should be done to confirm involvement in fusion, or not. Production of isoform specific anti Kirrel3 antibodies will enable identification of the cellular localisation of the different Kirrel3 isoforms. Obtaining such information will aid in our understanding of Kirrel3 and its function in human skeletal muscle.Healthy young men aged between 18-28 years of age volunteered to participate in one of two studies aiming to describe molecular and physiological responses to exercise-induced muscle damage. Participants were informed about the purpose and risks of the study in which they were to participate before signing an informed consent document. The experimental protocols were approved by the Committee for Human Research at Stellenbosch University and the studies were conducted according to the ethical guidelines and principles of the International Declaration of Helsinki.Participants were first taught how to perform the squat jump exercise. Subsequently their maximum squat jump height was measured. For the squat jump exercise intervention participants preformed 100 squat jumps at 90% of their maximum jump height. Jumps were divided into sets of 10 with 1 minute rest interval between each set. Prior to performing the 100 squat jumps participants exercised at a light to moderate intensity for 5 minutes on a treadmill.2max, 10% decline) on a motorized treadmill. They were allowed a 2 min standing rest between bouts and all the participants were able to complete all twelve bouts.The protocol used here was previously described . In brievastus lateralis muscle using a 5 mm trephine biopsy needle with assisted suction. Baseline biopsies were obtained from participants who had not engaged in any strenuous physical activity 7 days prior to the biopsy. Biopsies from participants who had performed the plyometric jumping protocol were obtained 4 and 24 hrs post completion of the protocol. Baseline and 24 hr biopsies were taken from the right leg while the 4 hr biopsy was taken from the left leg. Similarly, biopsies from participants who completed the downhill running protocol were taken at baseline and one and two days post downhill running in a consistent manner. Biopsies were frozen in liquid nitrogen cooled isopentane and stored at -80°C until use.Muscle biopsies were obtained from the 5 in 6 well plates (BD Bioscience) in growth media which contained 89% Dulbecco’s Modified Eagle Medium, 1% N-2 Supplement 100X, 10% Fetal Bovine serum . Once cells had reached 70% confluence they were lysed for RNA and protein isolation. Growth media was removed from wells before 200 ul of Tripure (Roche) was added per well of a 6 well plate and incubated at room temperature for 5 minutes with occasional gentle agitation. For protein isolation 150 ul of protein lysis buffer was added per well and incubated on ice for 5 minutes with occasional gentle agitation.Human astrocytes  were seeded at a density of 10Primer set 1 – Forward primer GCCGACTTCCAGACCATCTA, Reverse primer – TTTGAGGACCTCCAGCTGTT, Primer set 2 - Forward Primer CGCTATACGGTGGAGACCAT, Reverse Primer Same as primer set 1. Primer set 3 – Forward primer same as primer set 2, Reverse Primer – CGCTTTTCCCCCTATCTTTC. Amplicons were excised from the agarose gel, purified and sequenced at the central analytical facility at Stellenbosch University. For GAPDH primer set used was Forward primer – AATCCCATCACCATCTTCCA, Reverse primer - TGACAAAGTGGTCGTTGAGG.Muscle biopsies were sectioned on a cryostat (Leica Bio systems) at -20°C to obtain approximately 20 mg of tissue. Samples were then homogenised on ice in 1 ml of Tripure (Roche). RNA was isolated according to manufacturer’s guidelines and suspended in TE Buffer and stored at -20°C until use. For reverse transcription (RT) 1ug of RNA was DNAse treated according to manufacturers (Roche) guidelines. Subsequently the 1 ug of DNAse treated RNA was reverse transcribed using random hexamers in accordance with manufacturers (Roche – Transcriptor First strand cDNA synthesis kit) guidelines. For PCR a 2 ul aliquot of cDNA corresponding to 50 ng of RNA was used. Each PCR consisted of a total volume of 25 ul. Primers (Sigma Life Science) were used at a 1 uM concentration and remaining components of PCR followed manufacturers (Roche – Faststart PCR Master) recommended protocol. For semi nested PCR a 2 ul volume from the initial PCR was used in the second PCR instead of 2 ul of cDNA. Post PCR the 25 ul volume was mixed with 6 ul of loading buffer  and loaded onto a 1% agarose gel which contained sybr safe (Life Technologies) at 1X concentration and electrophoresed alongside a 100 bp ladder (Life Technologies). Gels were visualised on a Chemidoc MP (Biorad) that was supported with Image Lab software (Biorad). Kirrel3 Primer sets used were as follows (BCA) kit (Thermo Fischer Scientific) using Bovine serum albumin (Roche) as standards. 30 ug of protein lysate was mixed with 5× Lamelli buffer  to yield 1× and electrophoresed using the Mini Protean Tetra system (Biorad). Post electrophoresis proteins were transferred to a nitrocellulose membrane  via Trans Blot Turbo (Biorad) and membranes were stained with ponceau S to confirm transfer and equal protein loading. Subsequently membranes were washed 3 times for 5mins with 1× TBST  and then blocked for 1 hr at room temp in 1× TBST and 5% semi skimmed milk. The intracellular targeting primary rabbit anti human Kirrel 3 antibody (102960 Abcam Cambridge UK) and the extracellular targeting primary sheep anti Kirrel 3 antibody (AF4910 R and D systems Minneapolis USA) were incubated over night at room temp with gentle agitation in 1× TBST with 5% semi skimmed milk. For blocking peptide experiment intracellular targeting primary anti human Kirrel 3 antibody was incubated with blocking peptide at a ratio of 1:4 for 3 hrs at room temperature with gentle agitation. Post primary antibody incubation or antibody and blocking incubation membranes were washed 3 times for 5 mins and subsequently incubated either with goat anti rabbit secondary  at 1:15,000 or donkey anti sheep HRP conjugated secondary ( 6900 Abcam Cambridge UK) at 1:30,000 for 1 hr at room temp in 1x TBST and 5% milk. Membranes were subsequently washed 6X for 5mins followed by incubation with chemiluminescence detection reagents . Membranes were visualised on a Chemidoc MP (Biorad) which was supported with Image Lab software (Biorad).Muscle biopsies were sectioned on a cryostat (Leica Biosystems) at 12 μm to obtain ~20 mg of tissue. Samples were homogenised on ice in 700 ul of lysis buffer . Protein concentration was measured via a Bicinchoninic acid


Seed vigor is an important characteristic of seed quality. In this study, one rice population of recombinant inbred lines (RILs) was used to determine the genetic characteristics of seed vigor, including the germination potential, germination rate, germination index and time for 50% of germination, at 4 (early), 5 (middle) and 6 weeks (late) after heading in two years. A total of 24 additive and 9 epistatic quantitative trait loci (QTL) for seed vigor were identified using QTL Cartographer and QTLNetwork program respectively in 2012; while 32 simple sequence repeat (SSR) markers associated with seed vigor were detected using bulked segregant analysis (BSA) in 2013. The additive, epistatic and QTL × development interaction effects regulated the dry maturity developmental process to improve seed vigor in rice. The phenotypic variation explained by each additive, epistatic QTL and QTL × development interaction ranged from 5.86 to 40.67%, 4.64 to 11.28% and 0.01 to 1.17%, respectively. The QTLs were rarely co-localized among the different maturity stages; more QTLs were expressed at the early maturity stage followed by the late and middle stages. Twenty additive QTLs were stably expressed in two years which might play important roles in establishment of seed vigor in different environments. By comparing chromosomal positions of these stably expressed additive QTLs with those previously identified, the regions of QTL for seed vigor are likely to coincide with QTL for grain size, low temperature germinability and seed dormancy; while 5 additive QTL might represent novel genes. Using four selected RILs, three cross combinations of seed vigor for the development of RIL populations were predicted; 19 elite alleles could be pyramided by each combination. Oryza sativa L.) is one of the most important crops in the world. Recently, improving rice vigor become more important, because the direct seeding method has become increasingly important in many Asian countries due to its lower cost and its operational simplicity Seed vigor is an important characteristic of seed quality, reflecting potential seed germination, seedling growth, seed longevity, and tolerance to adversity Arabidopsis, the transcription factors, such as ABI3, FUS3, LEC2 and LEC1, have been identified as being master regulators of seed maturation Medicago trunculata and ArabidopsisSeed development is a crucial process in the lifecycle of plant, which can be divided into the two stages morphogenesis and maturation Seed vigor has been known as a complex quantitative trait which makes the genetic analysis of seed vigor very difficult According to the theory of developmental genetics, genes are expressed selectively at different growth stages 2∶10) derived from the cross of indica rice IR26 and japonica Jiucaiqing was employed to map the loci underlying four seed vigor traits, including germination potential, germination rate, germination index and time for 50% of germination. The QTLs with additive, epistatic and QTL × development interaction effects for seed vigor were conducted during three maturity stages in rice. Finally, the novel parental combinations for seed vigor were predicted in future rice breeding. The selected RILs and identified QTLs might be used to improve seed vigor by marker assisted selection approach.What happens in the dry maturity developmental process to improve rice vigor is still an open question. In this study, the objectives were to investigate the genetic control of developmental behavior of seed vigor in rice. One RIL population (FOryza sativa L.) varieties, Jiucaiqing (japonica) and IR26 (indica), and their 150 recombinant inbred lines (RILs) (F2∶10) were used in this study. The 25-day-old seedlings were transplanted into a paddy field at the Experimental Station of Nanjing Agricultural University  on June 20th, 2012 and also 2013. The plants were grown with 17 cm between plants within a row and 33 cm between rows. Filed management was carried out according to the local standard methods Two rice  was computed on the basis of the RIL population through analysis of variance using the formula: B2H  =  G2σ/(G2σ + e2/nσ), where G2σ is genetic variance, e2σ is error variance, and n is number of replicates.The experimental data were analyzed using the SPSS 19.0 software, and the phenotype of two parents was compared with Student's The seeds harvested in 2012 were used for identification of QTLs with additive, epistatic and QTL × development interaction effects for seed vigor in rice. The genetic linkage map based on 135 simple sequence repeat (SSR) markers at an average interval of 16.5 cM was constructed by Wang et al. 50 simultaneously and low vigor bulk with low GP, GR, GI and higher T50 simultaneously, were selected at each maturity stage respectively, and each bulk containing DNA from 10 individuals was used for BSA. A total of 168 SSR markers that revealed polymorphisms between Jiucaiqing and IR26 were used to determine the SSR markers associated with seed vigor.The seeds harvested in 2013 were used to test those QTLs detected in 2012 by the bulked segregant analysis (BSA) method. Two extreme phenotypic bulks, including the high vigor bulk with high GP, GR, GI and lower T50 at three maturity stages were selected. Then, the positive alleles of the stably expressed additive QTLs among the selected RILs were analyzed. Furthermore, the plant height and grain weight of the selected RILs were observed. Finally, the best three cross combinations were predicted to improve seed vigor in rice.The best cultivars with maximum phenotypic value and elite alleles might be used to design parental combinations for crop breeding 50) of Jiucaiqing and IR26 and their RIL population were investigated by using seeds harvested at 4 (early), 5 (middle) and 6 (late) weeks after heading, respectively , indicating a large amount of genetic variation in the population. The HB2 of GP, GR, GI and T50 was more than 94% at three maturity stages.The seed vigor , four for GR , five for GI , and five for T50 . The phenotypic variance explained by each QTL ranged from 6.95% to 30.08%. Two major QTLs qGR8.2 and qGI8 (r2>20%) were identified with a LOD score of 6.74 and 4.95 respectively, explaining 30.08% and 29.77% of the phenotypic variation, between RM6976 and RM6845 region on chromosome 8.There were sixteen additive QTLs identified at the early maturity stage , includiqGP3), GR (qGR7), GI (qGI7) and T50 (503.2qT), identified at the middle maturity stage , two for GR (qGR8.1 and qGR12), two for GI (qGI1.2 and qGI7) and one for T50 (501.2qT). Among these QTLs, the qGI1.2 was co-located with 501.2qT between RM6950 and RM5759 region on chromosome 1, and the qGI7 was co-located with qGR7 between RM8261 and RM5426 region on chromosome 7. The phenotypic variance explained by each QTL ranged from 9.93% to 40.67%. One major QTL qGR12 (r2>20%) was identified with a LOD score of 2.73, explaining 40.67% of the phenotypic variation, between RM1880 and RM20 region on chromosome 12.There were eight additive QTLs identified at the late maturity stage , includi50 respectively. The epistatic QTL was detected with only epistatic main effect, while no significant QTL × development interaction effect. The phenotypic variance explained by each epistatic QTL ranged from 4.64% to 11.28%, and the phenotypic variation explained by each QTL × development interaction ranged from 0.04% to 1.17%.A total of nine epistatic QTLs were identified in joint analysis of three-maturity phenotypic values in 2012 . Of them50. The additive QTL was detected with only additive main effect, while no significant QTL × development interaction effect. The phenotypic variance explained by each additive QTL ranged from 5.86% to 9.84%, and the phenotypic variation explained by each QTL × development interaction ranged from 0.01% to 1.08%.A total of five additive QTLs were identified in joint analysis of three-maturity phenotypic values in 2012 . Each onTo test those additive QTLs detected in 2012, the SSR markers associated with seed vigor were identified using BSA method in 2013. Two extreme phenotypic bulks, including the high and low vigor bulks, were selected at each maturity stage respectively . A total50 at three maturity stages respectively , which will greatly facilitate the detection of QTLs and QTL × development interactions. By comparison, the more additive QTLs were expressed at the early maturity developmental stages followed by the late maturity stage. We found that the expressions of QTL were differential at different maturity stages: the additive QTLs rarely co-localized among the different stages. Only the qGR1, qGI1.1 and 501.1qT on chromosome 1 coincided with qGL-1 for grain length qGI1.2 and 501.2qT on chromosome 1 was similar with the region of qGRL1.1 for grain dimension qSD1 for seed dormancy 503.1qT was on the similar location of qSD3.2 for seed dormancy qGR5 located on the same region of qSD-5 for seed dormancy qLTG-5-1 for seed low temperature germinability 507.1qT is near to Sdr4 for seed dormancy qGL7-2 for grain length qGI7, qGR7 and qGP7 on chromosome 7 was similar with the region of qSD7.1 for seed dormancy 507.2qT was on the similar location of qGL7 for grain length qGI8 and qGR8.2 located on the same region of qSD8 for seed dormancy on chromosome 8 qGI10 and qGR10 located on the same region of qLTG-10 for seed low temperature germinability qGI3, 503qT.2, qGP3, qGP4.1 and qGP5 which indicates that these additive QTLs might be novel genes. With the increase in the number of QTLs identified for seed vigor, the genetic control of seed vigor will be better understood.The additive QTLs which stably expressed over years might play important roles in seed vigor in different environments. Comparing the positions of these 20 stably expressed additive QTLs detected here with other QTLs reported previously, we found that the region of QTL Sdr4 has been cloned and identified as one of the major determinants of dormancy in rice, which affects the expression of several DELAY OF GERMINATION 1-LIKE (DOG1-LIKE) genes Sdr4 expression is positively regulated by OsVP1, a global regulator of seed maturation that is orthologous to maize viviparous 1 (VP1) and Arabidopsis ABI3qGI1.2 and 501.2qT identified in this study was similar with the location of OsVP1 on chromosome 1 OsVP1 during seed maturation. To date, only one a major rice QTL qLTG3-1 for low temperature germinability has been cloned qLTG3-1 is functionally associated with the vacuolation of the tissues covering the embryo, which results in the reduction of the mechanical resistance to coleoptile growth. In this study, one major QTLs qGR8.2 was co-located with qSD8 for seed dormancy qGR8.2 is now in progress to elucidate the molecular mechanism of seed vigor using near isogenic lines (NILs).The exploration of physiological and genetic mechanisms in seed vigor is a highlight of seed science Besides gene functional analysis, the genetic mechanisms of seed vigor can also be well revealed by functional mapping. Functional mapping integrates developmental principle of trait formation into a QTL framework, which can detect the specific QTLs that determine the developmental pattern of a complex trait In a hypothetical cross of two cultivars, the trait values of produced RILs can be predicted by the effects of all the detected loci. The best RIL with maximum value would represent the best cross. To improve seed vigor, all the elite alleles might be pyramided into one cultivar as far as possible. Thus, according to the information of phenotypic values and the allelic effects of the 20 stably expressed additive QTLs in two years, the best three cross combinations for the development of RIL populations were predicted in this study. A total of 19 elite alleles could be pyramided by each combination to improve seed vigor. These results showed that the selected RILs as new materials will be valuable in future rice breeding programs. The identified QTLs will be applicable to improve rice seed vigor by marker assisted selection.


Extensive description of severe asthma phenotypes has gained much attention in the latest years with the hope of achieving better management of this potentially problematic group of asthmatics. However, all previous studies have focused on patient cohorts, and not random population samples.To dissect the concept of severe asthma, and to determine whether clustered complaints or addressing individual complains, will give more coherent understanding of severity.From a large population based study in West Sweden, a subgroup of 744 people with active asthma was characterised with extensive clinical examinations and detailed questionnaires. In this cohort we defined different parameters of severity as follows –1) low lung function as FEV1% predicted below 70%, 2) multi-symptom asthma described day-time symptoms, 3) 2 or more night-time awakenings per month, 4) use of rescue medications 3 or more times per week, 5) any respiratory emergency visits and 6) use of oral steroids regularly or on exacerbations in the last year. We outlined particular subgroups of asthmatics who presented with different extend of overlap of these characteristics and looked into possible risk factors for the separate groups.The group of asthmatics who presented with at least one of severity features comprised of 374 people (50.3% of the active asthma group). The distribution for the individual complaints was day-time symptoms in 196 (36.3%), low lung function in 85 (11.4%), rescue medications in 130 (17.5%), night-time symptoms in 111 (14.9%), emergency visits in 78 (10.5%) and use of oral steroids in 66 (8.9%). When at least two complaints in a different combination were examined, the groups comprised of 2.1 -7.6% of the whole asthma population. The extent of the overlap between more than three complaints revealed tightly agglomerated groups representing 0.5 – 7.9% of all asthmatics. The risk factors that were outlined for the different groups were current smoking and having BMI > 30 with ORs ranging from 1.7 – 4.1 and 1.6 – 2.6, respectively.Further dissecting into the different severe asthma phenotypes in a random population could identify particular influencers for severity, and may more exactly stratify distinct groups that require extensive management.


ROP occurs in 95% of babies weighting less than 1000 g, all cases that had proceeded to the third stage will cause serious complications or complete blindness [Retrospective analysis of examination of 217 newborn of the risk group. . Due to the development of Respiratory Distress Syndrome all children needed respiratory therapy on the first week of living. The patients were divided into 3 groups according to the kind of respiratory therapy received. 1st group ( n-=86) received noninvasive respiratory therapy with Nosal Continuous Airway Pressure, 2nd group (n=69) – noninvasive therapy with Biphasic, and 3rd group (n=62) – invasive respiratory therapy with Artificial Respirating Unit.In 1st group we observed 0 cases of ROP, in 2nd - 3 cases were diagnosed, but all of them had a fast regression afterwards, in 3rd group – 20 children developed ROP, only 15 cases regressed and one child had to go through the laser coagulation of the retina vessels.Changing from invasive to noninvasive forms of respiratory therapy may lead to significant decrease of the risk of ROP.


To evaluate the efficacy of a 5 day short course of oral prednisolone when added with levocetirizine for management of acute urticaria.Prospective, randomized clinical trial was carried out in a teaching hospital.All patients were asked to evaluate the severity of pruritus on urticarial activity score (UAS).Patients were then given oral prednisolone 30 mg for 5 days and tablet levocetirizine 5 mg twice daily for 6 weeks and only levocetirizine tablet 5 mg twice daily for 6 weeks. Patients’ conditions were reassessed clinically with UAS calculated again 2 days later and again 5 days later.49 patients were enrolled; 24 patients received prednisolone with Levocetirizine and 25 received only Levocetirizine. The two groups had similar UAS at enrollment  , but at 2- and 5- day follow-up the prednisolone group had significantly lower UAS (1.4 and 0.2 versus 3.8 and 2.4 respectively) and greater clinical improvement in rash. Response did not correlate with age, sex, or identification of an allergen. No adverse effects were noted in levocetirizine group. In Prednisolone group two patients complained of gastritis.At the end of 6 weeks, 3 patients from the steroid group and 8 patients from the levocetirizine group continued to get urticarial wheals. The addition of a prednisolone short course improves the symptomatic and clinical response of acute urticaria to antihistamines. Patients with steroids improved more quickly and completely without major adverse effects.


Dual T1 mapping allows for a comprehensive assessment of myocardial tissue by combining detection of edema in the native scan and quantification of extracellular volume (ECV) after administration of Gadolinium. Recent studies proved the diagnostic value of T1 mapping in different pathologies; the aim of this study was, to evaluate the practicability and robustness of T1 mapping in assessing common pathologies in daily CMR practice.Since October 2012, we investigated 140 patients undergoing clinically indicated CMR examination by perfoming additional T1 mapping sequences which were not used for clinical diagnosis. We used a modified Short Modified Look Locker Inversion Recovery (ShMOLLI) sequence with 3 inversion pulses and a 4-(1)-3-(1)-2 readout pattern. The post Gadolinium scan for extra cellular volume calculation was performed 10 min. after administration of a bolus of 0.2 mmol/kg body weight gadopentetate dimeglumine. Diagnosis was based on clinical information and standard sequences for assessment of myocardial tissue comprising native T2 weighted dark blood turbospin echo (tse) sequences, pre and early post gadolinium T1 weighted tse sequences and inversion recovery spoiled gradient echo sequences for late enhancement. Assessment of T1 relaxation time and ECV values was based on 3 short axis views and 1 long axis view using the AHA/ACC 17-segment model.Native T1 relaxation time and ECV for the different pathologies are summarized in Table T1 mapping and ECV correlated well with myocardial alterations in commonly diagnosed cardiac disorders. It proved reliable and robust in daily clinical practice and allows for a good differentiation between normal findings and pcommon pathological CMR diagnoses. The combined use of native T1 and ECV quantification is promising approach for comprehensive assessment of the myocardium and may improve diagnostic accuracy of CMR in myocardial disease.None.


Plakophilin3 (PKP3) loss results in increased transformation in multiple cell lines in vitro and increased tumor formation in vivo. A microarray analysis performed in the PKP3 knockdown clones, identified an inflammation associated gene signature in cell lines derived from stratified epithelia as opposed to cell lines derived from simple epithelia. However, in contrast to the inflammation associated gene signature, the expression of MMP7 was increased upon PKP3 knockdown in all the cell lines tested. Using vector driven RNA interference, it was demonstrated that MMP7 was required for in-vitro cell migration and invasion and tumor formation in vivo. The increase in MMP7 levels was due to the increase in levels of the Phosphatase of Regenerating Liver3 (PRL3), which is observed upon PKP3 loss. The results suggest that MMP7 over-expression may be one of the mechanisms by which PKP3 loss leads to increased cell invasion and tumor formation. Matrilysin MMP7) is one of the smallest members of the MMP family and is a highly potent metallo-protease which can degrade casein, laminin, fibronectin, collagen III/IV/V/IX/X/XI, type I/II/IV/ V gelatins, elastin and proteoglycans , 2. MMP- is one oDesmosomes are cell-cell adhesion junctions present in both simple and stratified epithelial cells. Desmosomes anchor intermediate filaments in adjoining cells and thus play a central role in the formation of a tissue wide intermediate filament network, allowing cells to survive when they encounter mechanical stress during tissue repair , 20. ThrPlakophilin3 (PKP3) is the most widely expressed plakophilin family member and is ubiquitously present in all the layers of the stratified epithelia and simple epithelia except in hepatocytes . PKP3 foRecent results from our lab have shown that PKP3 loss leads to an increase in PRL3 (Phosphatase of regenerating liver-3) protein levels leading to the dephosphorylation of keratin8 (K8), which results in increased neoplastic progression and metastasis . To deteThe oligonucleotides used to generate the MMP7 shRNA constructs  were cloHCT116, HaCaT and FBM cells  were cultured as described previously , 24, 30.RNA isolated from the FBM derived vector control and PKP3 knockdown cells and HCT116 derived vector control and PKP3 knockdown cells were Cy3 labeled and processed for the Sureprint G3 Human GE 8x60k microarray by single color hybridization. The results obtained from the microarray were analyzed using the Agilent Feature Extraction software. Using normalized signal intensities  obtained from the microarray, the fold changes of genes altered in FBM derived PKP3 knockdown clone (shpkp3-2) has been compared to the vector control clone (vec). Similarly, the HCT116 derived derived PKP3 knockdown clone (shpkp3-2) has been compared to the vector control clone (vec). A functional classification of the differentially regulated genes was performed using GeneSpring GX 11.0 software and gene ontology browser. The significant pathway list for differentially regulated genes was obtained using the GeneSpring GX 11.0 and Biointerpreter software . The data for the FBM derived clones has been deposited in the NCBI GEO database (Accession number GSE61512), while the data for the HCT116 derived clones have been deposited in the NCBI GEO database (Accession number GSE64580). Functional classification of genes altered upon PKP3 loss was performed using the PANTHER Classification System software , 33.(-ΔΔCt) method [The forward and reverse oligonucleotides used in this study are shown in ) method . A changFor Western blots, the mouse monoclonal β actin antibody  was used at a dilution of 1:5000, the mouse monoclonal PKP3 antibody  at a dilution of 1:2000, mouse monoclonal MMP7 antibody  at a dilution of 1:100. Goat anti-mouse secondary antibody (Pierce) was used at a dilution of 1:2500. The blots were developed using Supersignal West Pico Cheminiluminescent Substrate (Pierce) according to the manufacturer’s instructions. Cells were lysed in 1X sample buffer as described  and prot2. Cells were observed by time lapse microscopy and images taken every 10 minutes for 20 hours using the AxioCamMRm Camera (Zeiss) with a 10X phase I objective. Axiovision software version 4.8 (Ziess) was used to measure cell migration. Three independent experiments were performed in triplicates for each clone. Invasion assays were performed as described [5 cells resuspended in 200μl of serum free media were added to the upper chambers and 400μl of serum containing media was added in the lower chamber. The inner side of the insert was pre-coated with 5μg of Matrigel (BD Biosciences). After 24 hours, cell culture inserts were then removed from the wells and the cells attached to the inner side of the insert were removed using cotton buds. The inserts with cells on the outer side of the membrane were fixed with 4% para-formaldehyde, stained with 1% crystal violet and mounted on slides using DPX mountant . Images were taken using Olympus SZ61 stereo microscope using a 10X objective lens. Three independent experiments were performed for each clone.Scratch wound healing assays were performed as described . The plaescribed , 36. Bri2500 cells of the HCT116 based plakophilin3 knockdown clones shpkp3-1 and shpkp3-2, the vector control clone (vec), the shpkp3-2 derived vector control and shpkp3-2 derived double knockdown clones were resuspended in 0.4% soft agarose as described . The celFoxn1nu/Crl) of 6–8 weeks old, provided by the ACTREC animal house facility, was used for the study. 1 x 106 cells of the HCT116 based shpkp3-2 derived vector control and double knockdown clones were resuspended in 100μl of PBS and injected sub-cutaneously in the dorsal flank of mice. Six mice were injected for each clone. Tumor formation was monitored at intervals of 2 to 3 days and tumor size was calculated weekly for 5 weeks using the formula (0.5x LV2) where L is the largest dimension and V its perpendicular dimension [3 was obtained 5th week post-injection for a mouse injected with shpkp3-2+vec.No surgical procedure was involved in the present study and therefore no anesthetic or analgesic was employed during these experiments. During injections, the animals were handled by trained, certified animal technicians and were injected by the in-house veterinarian with minimum distress to animals. Mice were sacrificed 5 weeks post injection. Animals were euthanized as per in-house Standard Operating Procedure (SOP) approved by the attending veterinarian (AV) of the ACTREC animal house facility. Carbon dioxide (CO2), an inhalant euthanasia agent recommended by the Committee for the Purpose of Control and Supervision of the Experiments on Animals (CPCSEA), Government of India, was used for euthanasia of mice. Euthanasia was performed under the supervision of the attending veterinarian and according to the American Veterinary Medical Association (AVMA) guidelines for the euthanasia of animals (2013 Edition). Briefly, a compressed CO2 cylinder was used as a source for carbon dioxide to control the inflow of gas, which was connected to a euthanasia chamber. The mice were kept in the chamber and an optimal flow rate was maintained to fill 20% of the chamber volume. After keeping the mice in the chamber, the CO2 cylinder supply valve was turned on to deliver the gas in the chamber so that animals were exposed to the gas slowly and steadily. After sufficient exposure like for 2 to 3 minutes, mice showed cessation of respiration and heart beats. The chamber was not prefilled with CO2 and was vented out post sacrifice and before the next animal was introduced into the chamber. Thereafter, the mice were removed from the chamber and a cervical dislocation performed to ensure that the mice were dead.BALB/c Nude mice , Ministry of Environment and Forest, Government of India. A controlled environment was provided to the animals with a temperature of 22±2°C and relative humidity maintained at 40–70%. A 12 hours day night cycle was maintained (7:00 to 19:00 day and 19:00 to 7:00 night). The animals were given autoclaved balanced diet prepared in house and sterile water. Individually ventilated Cage system  was used to house mice used in the experiments. These IVCs were provided with autoclaved corn cob as bedding for the mice. Animal euthanasia was done under the guidelines of AVMA as mentioned above using CPCSEA recommended euthanizing agent, carbon dioxide. The Institutional Animal Ethics Committee (IAEC) of the Advanced Centre for Treatment Research and Education in Cancer (ACTREC) approved all the protocols used in this report. The project number for the study is 16/2008 and was approved in November 2008.Previous studies from our laboratory have demonstrated that loss of PKP3 in HCT116, HaCaT and FBM cells leads to an increase in transformation in vitro and increased tumor formation and metastasis in vivo . To idenIn confirmation of previously reported results , 30, a rThe mRNA levels of the Matrix metalloprotease7 (MMP7) gene were found to be up-regulated upon PKP3 loss in all three cell types tested . To detePKP3 loss leads to an increase in cell migration as assayed by scratch wound healing assays . To detePKP3 loss leads to an increase in anchorage independent growth and an increase in tumor formation and metastasis in vivo . To detePrevious data from our laboratory has demonstrated that an elevation in the levels of Keratin 8 is observed in the HCT116 derived PKP3 knockdown clones and that this increase is due to an increase in the levels of the phosphatase of regenerating liver 3 (PRL-3) and inhibition of PRL-3 activity using a chemical inhibitor resulted in a decrease in cell migration . An inhiThe results in this report demonstrate that loss of PKP3 leads to an increase in the levels of MMP7 in three independently derived cell lines of different origins. The increase in MMP7 levels is required for cell migration and invasion in vitro and tumor formation in nude mice as loss of MMP7 in the PKP3 knockdown cells results in a reversal of these phenotypes. The increase in MMP7 mRNA is dependent on the expression of PRL-3, whose levels are elevated upon PKP3 loss . Thus, tMin mice leads to a decrease in tumor incidence. Our work has demonstrated that PKP3 loss leads to an increase in migration, invasion, tumor formation and metastasis and that these functions are dependent upon the increase in MMP7 expression upon PKP3 knockdown. To determine if a correlation exists between MMP7 and PKP3 levels across colon cancers at the transcript level, we analyzed the data sets in the Oncomine database (www.oncomine.org). When PKP3 levels in normal colon samples were compared with that in colon cancer, the levels were found to be unchanged, while MMP7 levels were higher in colon cancer samples. None of the databases present had actually validated the PKP3 levels using real time PCR or reverse transcriptase PCR. To determine if a correlation exists between MMP7 and PKP3 levels across colon cancers at the protein level, we examined the data deposited in the human protein atlas database (http://www.proteinatlas.org/). It was observed that a thorough immuno-histochemical analysis of PKP3 and MMP7 levels in a large dataset of colorectal cancer patients has not been performed to determine if any correlation exists between PKP3 and MMP7 levels. Surprisingly, MMP7 loss leads to an increase in colony formation in soft agar but a decrease in tumor formation in nude mice. These results suggest that the ability of MMP7 to induce the degradation of the extra-cellular matrix in vivo maybe essential for the ability of the PKP3 knockdown cells to form a tumor. Taken together these results suggest that the increase in MMP7 levels observed upon PKP3 loss is required for increased tumorigenesis in cells derived from the colon.MMP7 and membrane type-1 matrix metalloprotease (MT1-MMP) are metalloproteases which are exclusively produced by the epithelial cells of the colon while the rest of these matrix-metalloproteases such as MMP9, MMP11 (stromelysin 3) are produced by the stroma –43. MMP7PRL-3 levels are increased in colorectal cancers  and PRL-PKP3 knockout mice show symptoms of severe itching, intercellular edema, neutrophil infiltration, epidermal hyperplasia, hair loss and muscle wasting . BecauseThe results reported here lead to the generation of the following model. PKP3 loss leads to an increase in PRL-3 translation leading to an increase in K8 levels  or an inS1 Table(DOCX)Click here for additional data file.S2 TableCompiled data with complete dataset, differentially expressed genes and functional classification of altered genes observed upon PKP3 knockdown in FBM cell line.(XLSX)Click here for additional data file.S3 TableCompiled data with complete dataset, differentially expressed genes and functional classification of altered genes observed upon PKP3 knockdown in HCT116 cell line.(XLSX)Click here for additional data file.S1 Fighttp://www.pantherdb.org/about.jsp). S1B Fig, Cell type specific alterations in FBM derived PKP3 knockdown clones. Reverse transcriptase PCRs were performed using oligonucleotides specific for SAA4, EPPK1, MOBKL2b, MMP9, NR2F1, ΔNp63, IGFBP3, ARHGEF5 and GAPDH, in HaCaT, HCT116 and FBM derived PKP3 knockdown clones and the respective vector controls. Expression of GAPDH has been used for normalization. The PCR product size has been indicated in base pairs. S1C Fig, MMP7 loss leads to a decrease in migration. Phase contrast images of wound healing at 0 hours (start) and 20 hours (end of experiment) have been shown.S1A Fig, Functional classification of genes whose expression is altered >=2 folds in FBM derived clones and >=1.5 folds in HCT116 derived clones upon PKP3 knockdown. The mRNA expression profile of the FBM derived vector control (vec) was compared with that of the PKP3 knockdown clone (shpkp3-2). Similarly, mRNA expression profile of the HCT116 derived vector control (vec) was compared with that of the PKP3 knockdown clone (shpkp3-2). Differentially expressed genes which show more than two fold (for FBM) and more than 1.5 folds up-regulation or down-regulation in the PKP3 knockdown clones compared to the vector control clone were selected. The pie charts show functional classification of genes obtained from the microarray data analysis using the PANTHER Classification tool ((TIF)Click here for additional data file.S2 Fig6 cells of the vector control or the double knockdown clones were injected sub-cutaneously into nude mice and allowed to develop tumors. Representative images of mice have been shown. S2B Fig, LCN2 expression is not altered upon inhibition of PRL-3. The HCT116 derived PKP3 knockdown clones (shpkp3-1 and shpkp3-2) or the vector control (vec) were treated with either the vehicle control (DMSO) or 10 μM PRL-3 inhibitor-1 (PRL-3i) for 24 hours. The mRNA prepared from the treated cells was used as a substrate for reverse transcriptase followed by real time PCR reactions using oligonucleotides specific for LCN2. All expression was normalized to the levels of GAPDH. The fold change is graphed on the Y-axis and the clone name is on the X-axis. The standard errors are plotted and student’s t test was performed . Note that LCN2 levels are increased upon treatment with PRL-3 inhibitor. S2C Fig, MMP7 expression does not change upon inhibition of PRL3 activity in HaCaT derived clones. The HaCaT derived PKP3 knockdown clones (shpkp3-1 and shpkp3-2) or the vector control (vec) were treated with either the vehicle control (DMSO) or 10 μM PRL-3 inhibitor-1(PRL-3i) for 24 hours. The cell supernatants were collected and a100μg of acetone precipitated protein was resolved on 12% SDS PAGE gels followed by Western blotting with antibodies to MMP7. The same blot was stained with Ponceau to indicate equal loading.S2A Fig, Tumor formation is inhibited in the PKP3 knockdown cells upon MMP7 knockdown. 10(TIF)Click here for additional data file.


Blockade of the sphenopalatine ganglion with OnabotulinumtoxinA injections (SphenoBlock) represents a novel approach for treating intractable chronic cluster headache (iCCH).The aim of this pilot study was to explore the safety aspects and therapeutic potential of SphenoBlock.After signing written confirmed consent ten patients with iCCH were injected with 25 U (n=5) or 50 U (n=5) onabotulinumtoxinA towards the sphenopalatine ganglion in an observational study, approved by the Institutional Review Board, with 6 months follow-up. The procedure was performed with a novel image-guided technique. The primary endpoint was to evaluate safety of the procedure, but change in attack frequency from baseline to week 4, 12 and 24 was also registered.Data for the first 5 patients are presented. One patient experienced intermittent ipsilateral visual deficits lasting 4 weeks. Patient number 5 was a failed injection. Four patients were defined as frequency responders (>50% reduction from baseline) in week 4, 3 patients responded in week 12, and 2 patients in week 24 (Figure SphenoBlock in iCCH shows promising preliminary results and give reasons for cautious optimism for further studies on this low-cost alternative treatment of iCCH.


CUG repeats result in part from the inactivation of the muscleblind-like (MBNL) proteins. To test the role of MBNL3, we deleted Mbnl3 exon 2 (Mbnl3ΔE2) in mice and examined the onset of age-associated diseases over 4 to 13 months of age. Accelerated onset of glucose intolerance with elevated insulin levels, cardiac systole deficits, left ventricle hypertrophy, a predictor of a later onset of heart failure and the development of subcapsular and cortical cataracts is observed in Mbnl3ΔE2 mice. Retention of embryonic splice isoforms in adult organs, a prominent defect in DM1, is not observed in multiple RNAs including the Insulin Receptor (Insr), Cardiac Troponin T (Tnnt2), Lim Domain Binding 3 (Ldb3) RNAs in Mbnl3ΔE2 mice. Although rare DM1-like splice errors underlying the observed phenotypes cannot be excluded, our data in conjunction with the reported absence of alternative splice errors in embryonic muscles of a similar Mbnl3ΔE2 mouse by RNA-seq studies, suggest that mechanisms distinct from the adult retention of embryonic splice patterns may make important contributions to the onset of age-associated pathologies in DM1.Myotonic dystrophy type I (DM1) exhibits distinctive disease specific phenotypes and the accelerated onset of a spectrum of age-associated pathologies. In DM1, dominant effects of expanded  DMPK and immediately 5′ of SIX51214Myotonic Dystrophy type 1 (DM1) is a multi-system disorder resulting from the expansion of a CTG repeat sequence located in the 3′ untranslated region of DMPK RNAs encoding expanded CUG repeat sequences (CUGexp) play a central role in the development of key aspects of DM1 pathology678DMPK RNA encoding CUGexp with the MBNL family of proteins and allele specific silencing of SIX5 expression resulting from CTG tract expansion respectively, are sufficient to result in a subset of DM1 cardiac, skeletal muscle, ocular and gonad pathologies10111213141516171819Multiple lines of evidence demonstrate that dominant RNA effects resulting from the expression of mutant CUGexp to aberrantly sequester and functionally inactivate members of the muscleblind (MBNL) family of proteins10118212324Mbnl1Δ3/Δ3 muscle, heart and brain and in Mbnl2Δ2/Δ2 brain2628RNA dominant effects in DM1 have been hypothesized to stem in part from the ability of Clc-1 RNA splice defects has been shown to rescue myotonia in the HSALR DM1 mouse modelInsr) RNA has been implicated in the development of abnormal glucose tolerance in DM1262730Experiments directed at the examination of the causal role of RNA splice errors in the development of DM1 features are limited to myotonia, where reversion of Mbnl3, which is an X-linked gene, to develop Mbnl3ΔE2 mice38kD) and the retention of a truncated 27 kD Mbnl3 isoform (Mbnl327kD) translated from an ATG codon present in Mbnl3 exon 3, as previously described by us and Poulos and colleagues34Mbnl3ΔE2 mice demonstrate an accelerated onset of a subset of age-associated phenotypes observed in DM1, over a range of 4–13 months of age. Specifically, Mbnl338kD deficits trigger the early onset of abnormal glucose metabolism, elevated insulin levels, cardiac systole dysfunction that progresses to left ventricle hypertrophy, and a high incidence of subcapsular and cortical cataract formation. To test if DM1 specific splice errors contribute to the development of these phenotypes, we studied the splice patterns of twenty RNAs including the Insulin Receptor (Insr), Lim Binding Domain 3 (Ldb3) and Cardiac Troponin T (Tnnt2) RNAs in Mbnl3ΔE2 mice. DM1-like splice errors are not observed in these RNAs and reversion to the embryonic splice patterns is not observed for the Insr and Tnnt2 RNAs in Mbnl3ΔE2 skeletal muscle and heart. The modest splice error detected in the Ldb3 RNA in Mbnl3ΔE2 hearts does not recapitulate the embryonic splice pattern. Thus these data demonstrate Mbnl338kD deficits can cause the accelerated onset of age-associated DM1 pathologies and suggest that mechanisms distinct from splice alterations may contribute to the development of such DM1 phenotypes.In this study we tested the role of the third member of the muscleblind family, MBNL3, in DM1 etiology. We deleted exon 2 of Mbnl3ΔE2 mice on a 129sv background, in which exon 2 of the X-linked Mbnl3 gene was replaced by a Neomycin expression cassette12Mbnl3 mRNA expression in the adult soleus muscle and in the adult mouse heart, lens and brainTo test the potential effects of MBNL3 deficiency in the development of DM1 pathophysiology we developed Mbnl3+/+ and Mbnl3ΔE2 mice. Briefly, mice were fasted for 6 hours prior to testing and baseline blood sugar levels were obtained from a drop of tail blood. Subsequently, a bolus of sterile 5% dextrose in saline was injected IP at a dose of 1 g/kg at time zero and blood glucose levels were repeatedly tested for up to 3 hours following the injection. Area under the curve for the glucose tolerance test was calculated using an individual t-test at 4 months of age for male Mbnl3ΔE2 (n = 3) and Mbnl3+/+ (n = 4) mice. At 4 months of age, there was no statistically significant difference in blood glucose levels . However, a trend towards a shorter time for peak glucose levels to be achieved and elevated blood glucose levels 3 hours post injection was observed in Mbnl3ΔE2 mice when compared to Mbnl3+/+ controls  and Mbnl3ΔE2 mice (n = 7) (p < 0.001). Upon dextrose injection, peak glucose levels achieved were both significantly higher and were sustained at higher levels for a longer duration, with blood glucose levels ~57% higher in Mbnl3ΔE2 mice when compared to control animals, 3 hours post injection (p < 0.013) (Mbnl3+/+ and Mbnl3ΔE2 mice) adjusting for reads and time .At 7–9 months of age, baseline blood glucose levels were significantly different between male < 0.013) . At 7–9 Mbnl3ΔE2 mice was due to decreased insulin levels, we measured insulin levels in 8–10 month old Mbnl3+/+ and Mbnl3ΔE2 mice, at 0, 8, 15, 30 and 60 minutes following the intraperitonel injection of dextrose at a dose of 2 g/kg, subsequent to a 16 hour fast as previously describedMbnl3ΔE2 mice when compared to Mbnl3+/+ mice prior to dextrose injection and the incidence rate ratio (IRR) is significantly different between groups (Mbnl3+/+ and Mbnl3ΔE2 mice) adjusting for reads and time , demonstrating an 80% likelihood of higher insulin levels in the Mbnl3ΔE2 mouse group when compared to control mouse group .As aberrant splicing of exon 10a of the 7 months  and 11 m7 months  or by qPMbnl3+/+ and Mbnl3ΔE2 mice as a function of age. Left ventricular function and chamber dimensions were evaluated in male Mbnl3+/+ and Mbnl3ΔE2 mice by ultrasound echocardiography at 4 months  and 11 months  of age  and a consequent reduction in left ventricular function manifesting as diminished left ventricle percent fractional shortening , left ventricle ejection fraction  and velocity of circumferential fiber shortening  when compared to Mbnl3+/+ animals.Structure-function evaluation of the heart was carried out in male ) of age . These eMbnl3ΔE2 mice at four months of age occurs without differences in diastole chamber dimensions. In contrast, at 11 months of age, male Mbnl3ΔE2 mice exhibit a hypertrophic response, most likely in response to the cardiac systole deficits that manifest at 4 months of age. Specifically, significant increases in end-diastolic dimension , left ventricular mass , posterior wall thickness  and ventricular septal thickness  are observed in this age group. Finally, the E/A ventricular filling ratios are in the normal range suggesting no diastole dysfunction accompanying the hypertrophy. These age-related functional and morphological changes are shown in Left ventricle systole dysfunction in male As each sample set at 4 and 11 months of age are separate, typical corrections for multiple comparisons do not directly apply to these data. Given the small sample size, a statistically significant effect indicates very large effect sizes . Even with multiple comparison corrections, the most relevant outcomes of left ventricle percent fractional shortening at 4 months and left ventricular mass and posterior wall thickness at 11 months are significant.Mbnl3+/+ and Mbnl3ΔE2 mice at 4 and 11 months of age. The wave shapes and intervals were within the normal range for Mbnl3ΔE2 mice at these time points (Mbnl3ΔE2 mice (data not shown).Surface electrocardiograms were recorded in parallel from male e points . No arrhLdb3 and Tnnt2 mutations are known to cause cardiac dysfunction, dilated cardiomyopathy and cardiac hypertrophy37Tnnt2 isoforms in the adult heart is strongly associated with heart failure in humansTnnt2 in transgenic mice results in diminished cardiac efficiencyLdb3 and Tnnt2 in adult hearts, two splice errors previously described in DM133Mbnl3ΔE2 hearts. Ldb3 shows a modest but significant decrease in the inclusion of exon 11 in Mbnl3ΔE2 hearts when compared to Mbnl3+/+ hearts by RT-PCR at 7 months . This splice error is not reminiscent of the embryonic Ldb3 exon 11 splicing pattern . Consistent with the results from the skeletal muscle, Insr and Mbnl1 splice patterns are also not altered in Mbnl3ΔE2 hearts at either age .7 months  and by R pattern . The splMbnl3ΔE2 hearts, we analyzed splice events in Pdlim3/Alp, Trim55/Murf2, Mapt/Tau, Pdlim5, Sorbs1, Sorbs2, Fhod1, Spag9, Mbnl2, Myom1, Clta, Stx2, Csda, Sirt2, Atp2a1 and Atp11a RNAs, which show the aberrant retention of embryonic splice isoforms in adult Mbnl1ΔE2/ΔE2 heartsMbnl3+/+ and Mbnl3ΔE2 hearts and from E18 Mbnl3+/+ hearts. In 11 month Mbnl3ΔE2 hearts, none of splice events examined showed significant alterations when compared to 11 month old Mbnl3+/+ hearts  and Mbnl3ΔE2 (n = 8) lenses, demonstrated a single Grade-I subcapsular cataract in Mbnl3ΔE2 mice. No Mbnl3+/+ lenses studied at this age showed cataract formation , with ~60% showing advanced Grade-II and Grade-III cataracts as evaluated using LOCS II photographic grading standards. Cataracts in Mbnl3ΔE2 mice were primarily posterior subcapsular or cortical in origin . Electroretinography studies did not show significant retinal defects in Mbnl3ΔE2 mice at 13 months of age  is a multi-system disorder, which exhibits the accelerated onset of several age-associated disease phenotypes that include abnormal glucose metabolism, left ventricle systolic deficits, hypertrophy and heart failure, ocular cataracts that originate in the sub-capsular and cortical regions of the lens is a mul4232425262826272829303132hologies . SignifiMbnl3ΔE2 and Mbnl3+/+ mice. A trend towards elevated glucose levels was observed in Mbnl3ΔE2 mice at 4 months. At 7–9 months of age Mbnl3ΔE2 mice show aberrant glucose tolerance, with both higher peak glucose levels and sustained elevated glucose levels that did not return to baseline 180 minutes post injection. Overt diabetes was not observed at either age and baseline blood glucose levels were not significantly elevated in Mbnl3ΔE2 mice when compared to Mbnl3+/+ mice. In DM1 patients an increase in basal blood insulin levels is reported44Mbnl3ΔE2 mice. An increase in insulin levels was observed subsequent to dextrose injection in Mbnl3+/+ mice but not in Mbnl3ΔE2 mice. The lack of an increase in insulin levels subsequent to dextrose injection may be due to high, approximately a two fold elevation, in basal insulin levels in Mbnl3ΔE2 mice. Previous studies have strongly implicated elevated CUG-BP1 levels with aberrant glucose metabolism in DM1 patient cellsDmpk−/− animals that are maintained on a high fat dietEndocrine abnormalities occur frequently in DM1, with patients having an increased risk of developing diabetes142138kD deficits trigger cardiac systole deficits and ventricular hypertrophy, which can be an indicator of the later onset of heart failure. EKG abnormalities are not however observed in Mbnl3ΔE2 mice. In contrast, our previous studies have shown that deficits of Dmpk and Six5 result in conduction disorders but not echocardiographic abnormalities. Specifically, P-R prolongation, second and third degree heart block and QRS alterations are observed in Dmpk+/−, Dmpk−/− and Six5+/− mice respectively1516Mbnl1−/− loss results in arrhythmias, cardiac histopathology and sudden death and Lee et al. have further demonstrated that Mbnl1−/−/Mbnl2+/− mice show both arrhythmias, left ventricular hypertrophy with myocardial fibrosis25In DM1, sudden death is widely considered to result from cardiac arrhythmias and although progressive heart failure is less common, left ventricle systole dysfunction is associated with increased risk of overall mortality and sudden death14714748495047484950Mbnl3ΔE2 mice do not show significant ERG alterations. However these animals demonstrate an increase in ocular cataracts that are subcapsular and cortical in their origin. In our study subcapsular cataracts were first detected at 7 months of age in Mbnl3ΔE2 mice and by 12 months of age, 100% Mbnl3ΔE2 lenses examined showed subcapsular or cortical cataracts, with ~60% showing advanced Grade-II and Grade-III cataracts as evaluated using LOCS II photographic grading standards. This is in contrast to Mbnl3+/+ animals, where only one of eight lenses showed grade 1 subcapsular cataracts at 12 months of age. Previous studies have demonstrated that Mbnl1 loss results in subcapsular dust-like opacities and Six5 deficits increase the incidence of nuclear cataracts1718Six5 deficits to the development of DM1 ocular defects is unclear, because in mice Six5 loss results in nuclear cataracts, which are not prominent in DM1 patients. Taken together these data are consistent with Mbnl338kD and Mbnl1 deficits playing an important role in the development of the posterior subcapsular and cortical cataracts observed in DM1.DM1 patients develop an unusual form of ocular cataracts that are located primarily in the posterior subcapsular and cortical regions of the lens1425338kD deficits play a causal role in the development of a set of age-associated pathologies observed in DM1. Our results support the idea that DM1 pathology results from an aggregate of changes in DMPK, SIX5, CUG-BP1, MBNL1, MBNL2 and MBNL3 that occur downstream of the CTG repeat expansion. The potential synergistic interactions that result from these individual alterations and their relationship to the disease trajectory have however yet to be fully understood.Thus, results described in this study demonstrate that Mbnl3Mbnl3ΔE2 mice we examined splice site choice in RNAs that have been implicated in the development of DM1 pathology. Interestingly, a deficit of Mbnl338kD did not result in DM1-like splice errors in Insr and Tnnt2 in muscle and heart, two splice abnormalities, which can contribute to glucose intolerance and cardiac dysfunction and hypertrophy32373839Ldb3 exon 11 was however observed in Mbnl3ΔE2 cardiac muscle. This splice error is not reminiscent of the embryonic splice pattern, where inclusion of Ldb3 exon 11 is enhancedLbd3 splice defect observed in DM1 is an aggregate of two events, with Mbnl1 loss either overriding Mbnl3 effects or Mbnl1 loss producing larger changes than Mbnl3 loss. To more generally examine the role of Mbnl338kD depletion in the development of DM1 like splice errors we studied a range of splice events in the heart, skeletal muscle and lens of adult Mbnl3+/+ and Mbnl3ΔE2 mice and E18 Mbnl3+/+ embryos. No significant difference was observed for these splice events in adult Mbnl3+/+ and Mbnl3ΔE2 mice. These results are consistent with the observations of Poulos and colleagues who did not detect splice errors as a consequence of Mbnl338kD depletion in E18 forelimbs using RNA-seq analyses38kD target RNAs could underlie the phenotypes observed in Mbnl3ΔE2 mice34Mbnl3ΔE2 miceMbnl3ΔE2 mice. Deciphering the molecular basis of the accelerated onset of age associated pathologies in Mbnl3ΔE2 mice should provide important new insights into the molecular mechanisms that contribute to DM1.To test the molecular mechanism that underlies the onset of age-associated phenotypes in All experiments were performed in accordance with the institutional guidelines of both the University of Southern California (USC), and the University of California, Los Angeles (UCLA). The USC protocol was approved by the Institutional Animal Care and Use Committee at the University of Southern California, Los Angeles (Protocol number: 10347). The UCLA protocol (99-028) was approved by the UCLA Office of Animal Research Oversight.Left ventricular (LV) size, mass, wall thickness, ventricular and valve function and blood flow were assessed using methods similar to those previously described56Electrocardiograms were obtained for at least 15 minutes from each mouse under light isoflurane anesthesia by inserting two Pt needle electrodes  under the skin in the lead II configuration. The ECG data were amplified (Grass Technologies) and then digitized for analysis with HEM V4.2 software .All morphometry and histology experiments were carried out primarily as described by Jordan and colleagueset al.Glucose tolerance tests were performed according to a protocol modified from Andrikopoulos Mice were fasted in a clean cage with water but no food for 16 hours prior to collecting blood for baseline insulin level measurementsoC heat therapy pad in a Faraday cage with the visual axis of the eye to be measured directed vertically. A 27 g sub-dermal platinum needle electrode was placed in the lower  eyelid. Another identical (ground) electrode was placed in the  ear. The tip of a carbon-fiber wick electrode was placed in contact with the cornea and all three electrodes were connected to a pre-amplifier. A fiber optic cable was centered vertically over the eye to deliver steady background and/or flashes of light (Grass PS22 photo-stimulator powering a Grass Xenon flash unit) to within several millimeters of the corneal surface. Retinal electrical responses to flash stimuli were captured on a Nicolet Electrovisual Diagnostic System for analyses.For ophthalmic examination of the lens, mice were first lightly anesthetized with intra-peritoneal Ketamine HCl (100 mg/kg) and the pupils were dilated with a topical application of phenylephrine hydrochloride 2.5% and tropicamide 0.5% eye drops. Slit lamp examination of the anterior segment was performed using Haag-Streit B900 slit lamp  employing direct and retroillumination techniques. A digital fundus camera system  was used for red reflex and fundus color imaging to evaluate lens clarity. Images were analyzed based on Lens Opacities Classification System-IITotal RNA was prepared using Trizol  according to the manufacturer’s protocol. cDNA was synthesized from 5 μg of total RNA using the cDNA synthesis kit . cDNAs (150 ng) were used for splicing and qPCR studies with primers and PCR conditions described in How to cite this article: Choi, J. et al. Muscleblind-like 3 deficit results in a spectrum of age-associated pathologies observed in myotonic dystrophy. Sci. Rep.6, 30999; doi: 10.1038/srep30999 (2016).