Basaloid squamous cell carcinoma (BSCC) is a rare variant of squamous cell carcinoma. The occurrence of BSCC in the nasal cavity is extremely rare. In the present study, two cases of BSCC occurring in the maxillary sinus are reported and the clinicopathological features and immunohistochemical characteristics of this rare tumor are discussed. Two patients, aged 85 (case 1) and 60 years (case 2), presented with nasal tumors and persistent nasal obstruction. In each case, the biopsy or resected specimen of the maxillary sinus tumor revealed an infiltrative proliferation of solid epithelial nests composed of basaloid cells exhibiting hyperchromatic nuclei without conspicuous nucleoli and scant cytoplasm. Mitotic figures were frequently observed and spherical hyalinized materials were present within the tumor nests. Immunohistochemically, the tumor cells exhibited diffuse positive immunoreactivity for p63 and perinuclear dot-like positivity for vimentin, leading to a final diagnosis of BSCC of the maxillary sinus. Furthermore, it was demonstrated for the first time in the two cases that cathepsin K, a cysteine protease with marked collagenolytic and elastolytic activities, was expressed in a diffuse manner. One patient (case 2) succumbed to multiple metastases, while the other (case 1) remains alive with the disease. In conclusion, it was demonstrated that cathepsin K was immunopositive in two cases of BSCC of the maxillary sinus and that it may be involved in tumor invasion by this highly aggressive carcinoma. BSCC wad larynx . Since td larynx . The occd larynx –9. In thAn 85-year-old Japanese female patient presented with a nasal tumor and right-sided exophthalmos. Computed tomography (CT) and magnetic resonance imaging (MRI) scans revealed a right maxillary sinus tumor invading into the right ethmoid sinus, orbit and dura mater . A biopsA 60-year-old Japanese male patient presented with an approximate three-month history of persistent nasal obstruction. CT revealed a right-sided maxillary sinus tumor with destruction of the surrounding bone tissues and dural invasion. A biopsy specimen from the maxillary sinus tumor revealed a poorly-differentiated carcinoma. Surgical resection of the maxillary sinus tumor was subsequently performed. Following surgery, the patient received chemotherapy. The post-operative course was uneventful, although local recurrence occurred one and a half years subsequent to the surgery. This was followed by the development of multiple liver and lung metastases to which the patient succumbed.μm thick sections, then deparaffinized and rehydrated. Each section was stained with hematoxylin and eosin, then used for immunostaining. Immunohistochemical analyses were performed using an autostainer  according to the manufacturer’s instructions. The following primary antibodies were used: mouse monoclonal antibody against α-smooth muscle actin , mouse monoclonal antibody against cathepsin K , mouse monoclonal antibody against cytokeratin , mouse monoclonal antibody against high molecular weight cytokeratin , mouse monoclonal antibody against epithelial membrane antigen , mouse monoclonal antibody against p63  and mouse monoclonal antibody against vimentin .The formalin-fixed, paraffin-embedded tissue blocks of the maxillary sinus specimens were cut into 3-The biopsy specimen from the maxillary sinus tumor exhibited an infiltrative proliferation of solid epithelial nests composed of basaloid cells and the surface epithelium was eroded . These bThe surgically resected specimen of the maxillary sinus showed an infiltrative proliferation of solid epithelial nests composed of the basaloid cells that had scant cytoplasm and hyperchromatic nuclei without conspicuous nucleoli . SphericIn the present study, two cases of BSCC of the maxillary sinus are described. The clinicopathological features of the 26 previously reported cases of BSCC of the nasal cavity, as well as the 2 present cases, are shown in The main histopathological differential diagnostic consideration for BSCC is adenoid cystic carcinoma (particularly the solid variant) since these tumors are also composed of basaloid cells and may have areas with a cribriform growth pattern . The maiTo further identify the markers of this devastating disease, the expression of cathepsin K, a cysteine protease with marked collagenolytic and elastolytic activities, was investigated. Cathepsin K cleaves multiple sites within the triple helix of collagen types I and III, as well as at extracellular regions, whereas other proteases are more limited in their proteolytic activities. Cathepsin K was first demonstrated to play a significant role in osteoclast-mediated bone resorption , and it


Biotinidase was identified in secretome analysis of thyroid cancer cell lines using proteomics. The goal of the current study was to analyze the expression of biotinidase in thyroid cancer tissues and fine needle aspiration (FNA) samples to evaluate its diagnostic and prognostic potential in thyroid cancer. Immunohistochemical analysis of biotinidase was carried out in 129 papillary thyroid cancer . Comparison of aggressive and non-aggressive PTC showed decrease in overall biotinidase expression in the former (p = 0.001). Loss of overall biotinidase expression was associated with poor disease free survival  = 3.1). We examined the effect of subcellular compartmentalization of nuclear and cytoplasmic biotinidase on patient survival. Decreased nuclear expression of biotinidase was observed in PTC as compared to benign tissues (p<0.001). Upon stratification within PTC, nuclear expression was reduced in aggressive as compared to non-aggressive tumors (p<0.001). Kaplan-Meier survival analysis showed significant association of loss of nuclear biotinidase expression with reduced disease free survival . Cytoplasmic biotinidase expression was reduced in aggressive thyroid cancers in comparison with non-aggressive tumors  = 0.29) which was evident by its significant association with advanced T stage , nodal metastasis , advanced TNM stage  and extrathyroidal extension . However, in multivariate analysis extrathyroidal extension emerged as the most significant prognostic marker for aggressive thyroid carcinomas . In conclusion, loss of overall biotinidase expression is a novel marker for thyroid cancer aggressiveness. Thyroid cancer is the most common malignant endocrine tumor and accounts for >90% of cancers of the endocrine glands, with an estimated annual incidence of 122,803 cases worldwide At present, fine-needle aspiration (FNA) is the most commonly used pre-operative technique for diagnosis of thyroid nodules >1 cm in size. However, even the use of ultrasound-guided FNA is often beset with inconclusive biopsy results  In search of new cancer biomarkers for this malignancy, we analyzed the secretome from thyroid cancer cell lines to identify cancer-relevant secreted proteins that can serve as potential biomarkers This study was approved by the Research Ethics Board of Mount Sinai Hospital, Toronto, Ontario, Canada. Archived formalin-fixed paraffin-embedded tissue blocks from the Mount Sinai Hospital Tumor Bank were retrieved, reviewed by the pathologist (CM), and cut to provide 5 µm-thick sections for immunohistochemical staining. Diagnoses were derived from histopathological analyses and clinical examination. Benign cases included multinodular goiters, hyperplastic nodules, and follicular adenomas, whereas all non-aggressive and aggressive tumors examined here were well-differentiated papillary thyroid carcinomas which included the following variants: classic, follicular, oncocytic, diffuse sclerosing, and tall cell. Defining features of tumor aggressiveness were TNM stage IV classification, distant metastasis, perineural invasion, and disease recurrence or persistence. Additional consideration was given to the following potential risk factors of aggressiveness including: TNM stage III, vascular invasion, extrathyroidal extension, lymph node metastasis, and non-classical papillary thyroid cancer variant type .Based on these criteria, the 163 tissue samples examined in this study were categorized as 34 benign , 81 non-aggressive , and 48 aggressive  tumors.Formalin-fixed paraffin-embedded cell blocks from fine-needle aspiration preparations from 43 patients were similarly obtained from the Mount Sinai Hospital Tumor Bank and each sample diagnosis was confirmed by histological examination of the respective thyroidectomy specimen (CM). Accordingly, the FNA samples were categorized as 23 benign, 13 non-aggressive, and 7 aggressive cases.The patient follow up data were retrieved from the clinical database to correlate the protein expression in tumors with clinical outcome for evaluation of the prognostic relevance of biotinidase. The patients were followed up for a maximum period of 19.5 years.Slides were immunostained as described previously Immunopositive staining was evaluated in five areas of the tissue sections as described Of the 129 thyroid cancer cases, follow-up data were available for 116 (90%) patients, while 13 patients (10%) were lost to follow-up. Thyroid cancer patients were monitored for a maximum period of 19.5 years . Recurrence with or without metastases was observed in 19 of 116 (16.4%) patients monitored during the follow-up. Ninety seven patients who did not show recurrence were alive until the end of the follow-up period. Disease-free survival was expressed as the number of months from the date of surgery to recurrence or till the last possible follow up in case of patients who are disease free.The immunohistochemical data were subjected to statistical analysis using SPSS 17.0 software . Scatter plots were used to determine the distribution of cytoplasmic, nuclear and overall biotinidase expression in benign, non-aggressive and aggressive thyroid cancers. Sensitivity and specificity were calculated and quantified using receiver operating characteristic (ROC) analysis. Based on the optimal sensitivity and specificity as revealed by ROC analysis, cut-offs were generated for biotinidase protein expression. For overall biotinidase expression, a cut-off value of ≥3.6 was defined as positive immunoreactivity for statistical analysis. A cut-off value of ≥2 and ≥4 was defined as positive criterion for individual nuclear and cytoplasmic biotinidase immunopositivity respectively for detailed statistical examination. The relationships between biotinidase expression and clinicopathological parameters were tested using Chi-Square and Fischer’s exact test. Two-sided p values were calculated and p<0.05 was considered to be significant. Prognostic significance of overall, nuclear and cytoplasmic biotinidase expression was assessed by Kaplan-Meier survival and multivariate Cox-proportional Hazards regression analysis.Of the 34 benign tissues analyzed, 33 tissues (97.1%) showed overall as well as nuclear accumulation of biotinidase protein . CytoplaThe reduction in overall biotinidase expression significantly correlated with advanced T stage , nodal metastasis , Stage III+IV tumors , and extrathyroidal extension . Furthermore, loss of cytoplasmic and nuclear biotinidase individually also showed significant association with advanced T stage , nodal metastasis , stage III+IV tumors , and extrathyroidal extension  that can not only help distinguish benign thyroid from malignant tumors but also have the ability to discriminate between aggressive and non-aggressive thyroid cancers. The findings of our study suggest that biotinidase satisfies both these criteria. In addition, our study demonstrates biotinidase levels are decreased in aggressive thyroid carcinomas and suggests its potential to serve as a marker for tumor aggressiveness. This novel use of biotinidase underscores its potential to serve as a tool to identify aggressive thyroid cancers in early stages for more focused therapy. Recently Kang FNA is an essential tool for the management of thyroid nodules and FNA of all thyroid nodules >1 cm has been recommended Classically age, gender, tumor stage, extrathyroidal extension (spread outside the thyroid capsule), nodal status, histology type, histology grade all have prognostic significance and were observed to be associated with biotinidase expression in our study. To determine independent prognostic significance for biotinidase, all these conventional markers of poor prognosis for thyroid cancer were incorporated into a multivariate model and the additional significance of biotinidase was assessed. However, in multivariate analysis extrathyroidal extension and nodal status emerged more significant than biotinidase as markers for poor prognosis . Given the association of biotinidase with conventional markers of poor prognosis, biotinidase did not have independent prognostic significance in this study cohort. Nevertheless, our findings are of significance in view of the limited studies on biotinidase in human cancers; importantly these few reports corroborate and support our observations.Drosophila melanogasterThe role biotinidase plays in cancer aggressiveness remains to be elucidated. A probable hypothesis would center on the role of biotin as a co-factor for a plethora of enzymes responsible for chromatin structure and stability. Biotinidase cleaves biocytin thereby making free biotin readily available. Loss of this enzyme would subsequently cause a biotin deficient state which would in turn affect histone biotinylation in chromatin remodeling. It is known that biotinylation of K12 in histone H4 is important for repair of DNA and heterochromatin structures as well as repression of genes and transposons to maintain genomic stability and reduce cancer risk in human cells and BRAF(V600E) is considered a negative prognostic marker in PTC and might have been a confounding prognostic factor in our analysis. One of the limitations of our study is the non-availability of BRAF(V600E) mutation data in our cohort of thyroid cancer patients. The majority of PTCs are initiated by genetic events involving mutation of BRAF or RAS and translocations producing RET/PTC oncogenes


Bisphenol A (BPA) is a monomer found in commonly used consumer plastic goods. Although much attention in recent years has been placed on BPA's impact as an endocrine disruptor, it also appears to activate many immune pathways involved in both autoimmune disease development and autoimmune reactivity provocation. The current scientific literature is void of research papers linking BPA directly to human or animal onset of autoimmunity. This paper explores the impact of BPA on immune reactivity and the potential roles these mechanisms may have on the development or provocation of autoimmune diseases. Potential mechanisms by which BPA may be a contributing risk factor to autoimmune disease development and progression include its impact on hyperprolactinemia, estrogenic immune signaling, cytochrome P450 enzyme disruption, immune signal transduction pathway alteration, cytokine polarization, aryl hydrocarbon activation of Th-17 receptors, molecular mimicry, macrophage activation, lipopolysaccharide activation, and immunoglobulin pathophysiology. In this paper a review of these known autoimmune triggering mechanisms will be correlated with BPA exposure, thereby suggesting that BPA has a role in the pathogenesis of autoimmunity. Bisphenol A  is a monomer used in the manufacture of polycarbonate plastics. BPA is used in diverse forms of plastic products in the food and electronic industries and in various types of commonly used consumer goods, such as plastic containers, utensils, toys, water bottles, and fax paper. BPA has been shown to leach out of products, and high levels of the monomer have been identified in human and animal samples . The extBPA has been studied extensively as an endocrine disruptor, and numerous papers have shown how BPA may impact perinatal, childhood, and adult health . BPA hasBPA also has been shown to have potential adverse neurological effects, especially with respect to fetal brain development and promotion of neurodegenerative diseases . Mice moThis paper explores the worldwide exposure to BPA and its potential role in the growing epidemic of autoimmune disease. Although no human or animal studies have been published linking BPA to the onset of autoimmune disease, the potential seems very high due to the physiological influences of BPA and current immunological models regarding loss of self-tolerance and autoimmunity. In addition to known immune mechanisms promoted by BPA that overlap with autoimmune generation, some early evidence also indicates that BPA may contribute to mechanisms that promote autoimmune expression and progression .The hepatic biotransformation of BPA depends on phase I oxidation/reduction involving glutathione and phase II glucuronidation, glutathione, and sulfate conjugation . HealthyA growing body of evidence shows increased toxic loads deplete hepatic tolerance, which leads to over activation of the innate and adaptive immune response and the development of autoimmune disease . Higher BPA disruption of cytochrome P450 enzymes may be a potential mechanism for autoimmune pathophysiology. The cytochrome P450 (CYP) monooxygenases play a crucial role in the liver and various other tissues and are involved with oxidation of organic substances and the bioactivation of drugs and xenoestrogens . CYP actImpairment of hepatic biotransformation of CYP expression may lead to ROS pathophysiology of autoimmunity. ROS have the ability to induce autoreactive molecules that may be involved with both the onset and the exacerbation of autoimmunity . CYP enzIn a study of mice offspring, BPA exposure to 15 and 300 mg/L of drinking water induced cytochrome CYP17 downregulation leading to potential proteomic alterations in immune function . These mAlthough the peptide hormone prolactin is known primarily for its role in lactation, it also plays a critical role in modulating immune and inflammatory responses through various immune signaling pathways . ProlactA review of hyperprolactinemia and autoimmunity has found increased prolactin levels associated with production of anti-DNA antibodies, islet cell antibodies, thyroglobulin antibodies, thyroid peroxidase antibodies, adrenocortical antibodies, and transglutaminase antibodies with individuals suffering from systemic lupus erythematosus (SLE), diabetes mellitus type 1, Hashimoto's disease, Addison's disease, and celiac disease . Prolact In vitro and in vivo studies have found that BPA mimics estradiol and induces hyperprolactinemia [BPA is an endocrine disruptor with powerful effects on the pituitary lactotroph cells, which are estrogen responsive and promote prolactin release.ctinemia . TherefoA link between BPA exposure and increased prolactin levels was found in women workers in occupational settings within one year. They demonstrated marked prolactin level increases and a multivariate analysis found BPA exposure was an independent risk factor for increased serum prolactin levels .The correlation between hyperprolactinemia and autoimmune disease promotion has been reported in the literature in multiple papers during the past 20 years . AdditioIn addition to the impact of BPA on prolactin release through its estrogenic influence on pituitary cells, BPA also appears to directly affect immune cell signaling pathways and thus immune responses . BPA is Various estrogen-promoted mechanisms have been found to trigger autoimmune reactivity. The reticulum transmembrane protein UNC93B1, which is essential for trafficking toll-like receptors (TLRs) from endoplasmic reticulum and is found to play a role in autoimmunity, has been shown to be upregulated by estrogenic signaling . Estroge in vivo [Many of these immune-stimulating responses that perpetuate chronic inflammation and autoimmunity may also be potentiated by the estrogenic activity of BPA . BPA sti in vivo . BPA tre in vivo . Overall in vivo . These BBPA has hapten and estrogenic activity, both of which play roles in activating hyperactive immune responses that may occur in autoimmune pathophysiology.BPA exposure leads to aquatic animal hemocyte immune dysfunction, potentially increasing its role in induced autoimmunity through immune dysregulation. BPA injected into mussels leads to significant lysosomal membrane destabilization and a dramatic decrease in phosphorylation of the stress-activated p38 mitogen-activated protein kinases (MAPKs) and CREB-like transcription factor (cAMP-responsive element-binding protein) in mussels . These rIn addition to turning on gene expression of autoreactive T cells, alterations in these MAPK and STAT signaling pathways lead to chronic activation of antigen-presenting cells (APCs), loss of regulatory T cells (CD4+CD25+), apoptosis of APCs, and inhibition of innate and adaptive immunity wind-up found in the pathogenesis of autoimmunity . The sigCytokines have been shown to play a key role in the pathogenesis of autoimmune disease. The shift of cytokines into Th-1/Th-2 dominance and the IL-17/IL-23 (Th-17) axis has been shown to play pivotal roles in the model of autoimmunity and the breakdown of self-tolerance . BPA hasThe impact of BPA on naïve immune systems using T cell receptor transgenic mice followed by measurement of cytokine responses to antigens suggest that BPA can augment Th-1 reactions when administered orally in low doses (1.5 mg to 1.8 mg/kg weight) in water. Specifically BPA increased antigen-specific interferon gamma production leading to exaggerated T cell activation and polar Th-1 and Th-2 shifts . These mAnimal studies have also shown that BPA exposure promotes cytokine inflammatory shifts associated with potential autoimmune development. BPA administered to mice in drinking water produced significant shifts of lymphocytes subpopulations. The production of inflammatory Th-1 type cytokines (IFN-gamma) was induced while Th-2 cytokine (IL-4) was suppressed with BPA treatment, promoting the transcription of IRF-1. The mRNA expression of GATA-3 was inhibited in BPA-treated groups in dosages of 0.015, 1.5, and 30 mg/mL for 4 weeks . These rA study comparing the effect of BPA exposure on cytokine activity in adulthood and prenatally demonstrated that in adulthood exposure to BPA significantly promoted antigen-stimulated production of IL-4, IL-10, and IL-13, but not IFN-gamma. However, mice exposed prenatally to BPA showed increased production of not only IL-4 but also IFN-gamma. The percentages of T regulatory function (CD4+CD25+) were decreased in both groups exposed to BPA . Loss ofThe delicate interplay between Th-1, Th-2, and Th-17 expression appear to be a key factor in autoimmune pathophysiology. Evidence indicates that BPA may induce polarity in this delicate balance and trigger inflammatory reactions, potentially leading to loss of self-tolerance as noted in subsequent paragraphs. The impact of BPA on the pathogenesis of abnormal cytokine shifts most likely occurs from complex web-like reactions. BPA's role as both a hapten and estrogenic endocrine disruptor appears to promote multiple interwoven pathways involved in adverse cytokine shifts that may play a role in autoimmune pathogenesis .Bacterial translocation of lipopolysaccharides (LPS) has the ability to activate oxidative and nitrosamine stress pathways associated with the inflammatory responses and pathophysiology of autoimmune responses . BPA dirAdditionally, decreased activation of LPS-induced inflammatory reactions has also demonstrated a reduction in inflammatory sequelae of autoimmune cytokine and chemokine expression. Specifically, mice injected with BPA exhibited increased endotoxin-induced macrophage activation, suggesting that BPA may potentiate infectious autoimmune inflammatory reactions via enhanced tumor necrosis factor and nitric oxide reactivity . TherefoAntigen-presenting cells such as dendritic cells and macrophages appear to play a potential role with BPA and autoimmune reactivity. Dendritic cells (DCs) are important antigen-presenting cells that play a critical role in adaptive immunity due to their ability to activate naïve T cells, which, when overzealous, could promote autoimmune activity . DCs proMacrophage modulation of nitric oxide release is also critical for the regulation of apoptosis and differentiation of T cells that may lead to progression of autoimmune disease . AdditioIncreased immunoglobulin reactivity from endocrine disruptors such as BPA may raise concerns about immune hyperactivity associated with autoimmune immunopathology. The activation of immunoglobulins has a potential to promote inflammatory or anti-inflammatory activities through the activation of regulatory B (Breg) cells. Recent research in mice has shown that when B cell expression shifts into IL-10 production, there are suppressive effects on inflammatory responses. However, promotion of IgE-producing B cells plays a direct role in promoting inflammatory responses and the development of immune upregulation associated with most underlying inflammatory conditions, such as allergies and autoimmunity .+ T cells and antigen-specific IgE levels in sera via the stimulation of Ca2+/calcineurin-dependent nuclear factor of activated T cells binding sites (NF-AT) [Recent research has shown that BPA has a direct impact on increasing immunoglobulin expression into the inflammatory IgE response, thereby potentially promoting an inflammatory cascade in autoimmunity. Specifically, exposure to BPA was shown to increase IL-4 production in CD4 (NF-AT) . These i (NF-AT) . The pot in vitro and in vivo in murine models of SLE. The study researchers suggested that BPA exacerbates preexisting autoimmune diseases such as SLE and that continued exposure to endocrine disruptors may potentiate the incidence and severity of autoimmune diseases [In a murine model for SLE, animals implanted with BPA specifically demonstrated B cell activation and promotion of autoimmune disease such as lupus nephritis. BPA implantation enhanced autoantibody production by B1 cells bothdiseases .Evidence of BPA on expressing B cell activity towards inflammatory expression and autoimmune development may partly explain the complex immune web reactions of this endocrine disruptor. Although inflammatory immunoglobulin reactivity may have a role to play in autoimmune expression, it is most likely part of a larger complex immune reaction that is linked to this very reactive endocrine disruptor.BPA binds to host protein, potentially creating a new epitope for immune reactivity. BPA binds to protein disulfide isomerase (PDI), also known as BPA-binding protein , a multiBPA and triiodothyronine (T3) possess such a degree of molecular structure similarity that BPA may act as an antagonist compound on T3 receptor sites . When coAryl hydrocarbon receptors (AhR) are involved with regulating immune responses and the development of TH-17 cells, which are key effector T cells in a variety of human autoimmune diseases.  ExposureWith the growing epidemic of autoimmune disease worldwide and the extensive use of consumer goods containing BPA, we must examine the risk of BPA as a potential triggering compound in autoimmune disease. Although no specific evidence has linked human or animal autoimmune disease development to BPA exposure, many of the mechanisms known to exist in autoimmune pathophysiology also appear to exist with immune reactivity from BPA exposure . Further


Patients with ST-elevation myocardial infarction (STEMI) treated with fibrinolysis are increasingly, and ever earlier, referred for routine coronary angiography and where feasible, undergo percutaneous coronary intervention (PCI). We sought to examine the randomized clinical trials (RCTs) on which this approach is based.We systematically searched EMBASE, Medline, and references of relevant studies. All contemporary RCTs (published since 1995) that compared systematic invasive management of STEMI patients after fibrinolysis with standard care were included. Relevant study design and clinical outcome data were extracted.Nine RCTs that randomized a total of 3320 patients were identified. All suggested a benefit from routine early invasive management. They were individually reviewed but important design variations precluded a formal quantitative meta-analysis. Importantly, several trials did not compare a routine practice of invasive management after fibrinolysis with a more selective 'ischemia-guided' approach but rather compared an early versus later routine invasive strategy. In the other studies, recourse to subsequent invasive management in the usual care group varied widely. Comparison of the effectiveness of a routine invasive approach to usual care was also limited by asymmetric use of a second anti-platelet agent, differing enzyme definitions of reinfarction occurring spontaneously versus as a complication of PCI, a preponderance of the 'soft' outcome of recurrent ischemia in the combined primary endpoint, and an interpretative bias when invasive procedures on follow-up were tallied as an endpoint without considering initial invasive procedures performed in the routine invasive arm.Due to important methodological limitations, definitive RCT evidence in favor of routine invasive management following fibrinolysis in patients with STEMI is presently lacking. A 66-year-old man presented at 4 AM at a community hospital with chest pain of 90 min duration. His vital signs were stable. The ECG showed ST-segment elevation of 3-5 mm in inferior leads and precordial leads V5-V6 and ST-segment depression of 3-4 mm in leads V1-V3. He received aspirin, clopidogrel, intravenous morphine and bolus fibrinolytic therapy 2 h after the start of his symptoms followed by low molecular weight heparin. Over the next hour, the pain subsided significantly and an ECG showed over 50% resolution of ST-elevation. He was awakened at 8 AM for an ECG that showed Q-waves and 1 mm ST-elevation and T-wave inversion in inferior leads and T-wave inversion only in V5-V6. Should the patient be transferred to the nearest tertiary cardiac center 550 km away for cardiac catheterization and revascularization or progressively mobilized and risk stratified with an ECG stress test a few days later?The treating clinicians believed the patient should be transferred to the tertiary cardiac center for coronary angiography and revascularization if feasible. However, it was Friday past noon and a snowstorm had developed that was sustained over the weekend. The patient continued to do well and was ambulatory Sunday. On Monday morning, the new attendant staff felt that a satisfactory exercise test could obviate the need for invasive management. The patient performed well on this test and was discharged on aspirin, a statin and a beta-blocker plus clopidogrel for 3 months. His life was uneventful a year later.Fibrinolysis remains a cornerstone for the treatment of ST elevation myocardial infarction (STEMI) when primary percutaneous coronary intervention (PCI) is not readily available. However, important clinical questions arise with fibrinolysis. Should a 'pharmacoinvasive approach', that is, fibrinolysis followed by routine coronary angiography with PCI when anatomically feasible, be performed in patients with STEMI or is an equivalent clinical benefit achieved with a selective, ischemia-guided approach to coronary angiography and revascularization? And if a pharmacoinvasive approach is superior, when after fibrinolysis should patients undergo invasive intervention? And is the magnitude of benefit compelling enough to justify systematic recourse to the limited and costly resources of tertiary cardiac care?Older studies (≥ 15 years ago) showed no benefit of systematic invasive cardiac management of patient with STEMI after fibrinolysis. HoweverThis review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses, or PRISMA statement.We systematically searched EMBASE and Medline to identify all RCTs published in English that compared systematic invasive management of STEMI patients after fibrinolysis with prevailing 'standard' or 'usual care'. A detailed description of our literature search strategy, conducted in June 2010, and corresponding key words is found in Additional file We included RCTs that compared fibrinolysis followed by standard or usual care with fibrinolysis plus routine invasive management, performed at any time following fibrinolysis. We included studies that differed only in the timing of invasive management after fibrinolysis because later invasive management was their standard of care and because these studies have been invoked in support of a routine pharmacoinvasive approach. We excluded 'rescue PCI' studies (where emergency PCI was performed because fibrinolysis was assessed to have failed)-13 and 'We extracted data regarding study design, including sample size, duration of follow-up, and use of angiography, PCI, and clopidogrel (or ticlopidine) in each treatment group, and clinical outcomes at 30 days, 6 months, and 1 year. Where necessary, relative risks, 95% confidence intervals (CI), and p-values were calculated from presented count data. Data were extracted in duplicate, with disagreements resolved by consensus.The disparate protocols and heterogeneous comparative groups of the included trials precluded a formal meta-analysis. The limited available data also prevented the exploration of sources of heterogeneity via meta-analytic tools such as meta-regression. We have therefore opted to systematically review each individual trial.Out literature search identified 3,788 potentially relevant publications Figure . Of thesThe characteristics of the trials examined and their outcomes are summarized in Tables In this trial, 99 patients with STEMI were randomized in non-PCI hospitals to streptokinase alone and 100 patients were randomized to streptokinase followed by systematic transfer for immediate PCI. RevascuIn this study, 197 STEMI patients were treated with reteplase and randomized to 1 of 2 approaches: immediate stenting  or elective stenting after 2 weeks. Thirty-The GRACIA-1 study randomized 500 STEMI patients to a routine invasive strategy within 24 hours of fibrinolysis or to an ischemia-guided 'conservative' approach. The hypIn this study, 170 STEMI patients treated with fibrinolysis were randomized to routine immediate PCI or fibrinolysis alone with rescue or deferred PCI as clinically indicated. CardiacThis RCT compared immediate versus delayed PCI in 164 STEMI patients treated with fibrinolysis  plus abciximab. The comThis trial randomized 304 patients with STEMI to fibrinolysis alone (n = 100), fibrinolysis plus transfer for PCI within 24 hours (n = 104), or primary PCI (n = 100). In-hospThis study randomized 600 patients, aged ≤75 years, to a pharmacoinvasive approach or standard ischemia-guided management (including rescue PCI) after receiving half-dose reteplase and abciximab. In the In this study, 528 patients were randomized to PCI performed within 4 hours after fibrinolysis (early pharmacoinvasive group) and 511 patients were randomized to 'standard' treatment . The useThis RCT compared a strategy of immediate transfer for PCI with an ischemia-guided approach after fibrinolysis  in 266 patients situated too far away for timely primary PCI. In the This complete and detailed review of the RCTs that evaluated routine invasive management against a comparator strategy following fibrinolysis in patients with STEMI has found important methodological limitations to performing a meta-analysis of the available evidence. Importantly, the comparator to routine invasive management has varied widely from systematic but deferred invasive management to large differences in the proportion of patients in the comparator arm who underwent invasive management. Additionally, several potential biases were identified that might confound the findings of the individual RCTs. Thus, this systematic review concludes that definitive RCT evidence in favor of routine invasive management following fibrinolysis in patients with STEMI is presently lacking.Although primary PCI has tended to become the favored reperfusion therapy wherever it is readily available, a substantial proportion of patients with STEMI still receive fibrinolysis. Since the widespread introduction of fibrinolysis 3 decades ago, a recurring clinical question has been: Should all patients be subsequently routinely referred for invasive management ? Or, following fibrinolysis, should invasive management be more selective or 'ischemia-guided', based on higher risk characteristics such as recurrent or refractory ischemia and non-invasive risk stratification such as an exercise test or myocardial imaging suggesting the presence of a significant ischemic substrate? A secondary question for clinicians favoring routine invasive intervention after fibrinolysis has been its timing: earlier or later? The issue at stake is important both clinically and from the perspective of allocation of costly technological infrastructure. Should treatment of STEMI systematically engage the resources of tertiary cardiac care or can clinicians exercise sufficient clinical acumen to selectively reserve invasive management to identifiable high-risk patients without prejudice to overall patient welfare? Guidelines have attempted to address these questions but the strength of evidence on which these recommendations are based has not been critically reviewed.In the more contemporary era, fueled by post-hoc analysis of clinical trials,30 and iHowever, given the diverse and non-standardized study interventions, routine invasive versus a fluctuating standard care approach (with invasive rates varying from 7% to 67%) or simply an early versus deferred universal invasive strategy (in which there was no contrast in intervention rates only in timing), we believe a quantitative meta-analysis is inappropriate. We have therefore chosen instead to qualitatively review the RCTs individually. Other difficulties in determining if these RCTs establish a causal benefit of a pharmacoinvasive approach include: use of a sub-optimal fibrinolytic agent; confounConsequently, while these RCTs are regularly cited to support a pharmacoinvasive approach, this systematic review suggests that these potential biases and heterogeneity in the evidence makes definitive conclusions hazardous. Importantly, meta-analysis with a summary effect size and corresponding 95% CI represents only the random error and not the systematic errors associated with these potential biases.The RCTs have evaluated routine versus selective invasive management with a wide variation in the latter, or have compared only the timing of a pharmacoinvasive approach. Because of this evidence base and because clinicians increasingly favor the pharmacoinvasive approach, the optimal rate of coronary angiography and revascularization following fibrinolysis in STEMI patients remains unclear. This question assumes that clinical judgment can be sufficiently reliable that the patients who will benefit from invasive management will be accurately identified and unnecessary procedures can be avoided in those who will not derive benefit. Observations from registries and clinical studies do suggest the presence of a threshold effect-39. Abovzeitgeist of acute coronary care. Yet our critical and systematic review of available data from RCTs suggests that evidence to support this 'pharmacoinvasive' approach versus a truly selective ischemia-guided approach is more byzantine than conclusive. We believe guidelines should reflect this uncertainty rather than endorse the prevailing penchant[Ever earlier routine coronary angiography and anatomy-driven PCI in STEMI patients treated with fibrinolysis is being strongly promoted by reviews, meta-analyses, supporting editorials, opinion-leaders, and guidelines-34,41-45 penchant. While aThe authors declare that they have no competing interests.PB conceived the project and is the principal author of the manuscript. KBF made substantial contributions to analysis and interpretation of data and critical revision of the manuscript. JB made substantial contributions to interpretation of data and critical revision of the manuscript. All authors have given final approval of the version to be published and take public responsibility for its content.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/11/34/prepubAppendix. Literature Search StrategyClick here for file


Recent studies have provided evidence for sensory-motor adaptive changes and action goal coding of visually guided manual action in premotor and posterior parietal cortices. To extend these results to orofacial actions, devoid of auditory and visual feedback, we used a repetition suppression paradigm while measuring neural activity with functional magnetic resonance imaging during repeated intransitive and silent lip, jaw and tongue movements. In the motor domain, this paradigm refers to decreased activity in specific neural populations due to repeated motor acts and has been proposed to reflect sensory-motor adaptation. Orofacial movements activated a set of largely overlapping, common brain areas forming a core neural network classically involved in orofacial motor control. Crucially, suppressed neural responses during repeated orofacial actions were specifically observed in the left ventral premotor cortex, the intraparietal sulcus, the inferior parietal lobule and the superior parietal lobule. Since no visual and auditory feedback were provided during orofacial actions, these results suggest somatosensory-motor adaptive control of intransitive and silent orofacial actions in these premotor and parietal regions. Single-unit neurophysiological recordings in nonhuman primates have provided evidence for movement selectivity and action goal coding of visually guided transitive manual motor acts  in posterior parietal and ventral premotor areas Although largely discussed in terms of action goal coding in the above-mentioned studies, RS has been proposed to reflect possibly enhanced adaptive updates/learning and increased processing efficiency in specific neural populations Although action goal coding and forward motor-to-sensory control processes have been extensively studied in the context of limb/hand movements, feed-forward control is also a central idea in speech production research , native French speakers, participated in the study after giving their informed consent. All were right-handed according to standard handedness inventory Three orofacial motor tasks were performed independently and without phonation: a lip protrusion movement, a tongue retraction movement (the tongue turned in the back of the mouth) and a jaw lowering movement. A resting condition, without any movement, served as baseline. For all motor conditions, participants were instructed to initiate and end each movement from a resting state position, with the mouth closed and the tongue and jaw relaxed. In each trial, a 1000 ms visual instruction informed the participants about the motor condition  or the resting baseline (“pause”) and indicated the onset and offset of the movement. Participants were instructed to initiate each motor task as soon as they perceived the visual instruction and to maintain the movement until the visual cue disappeared. Apart from articulatory movements, participants were instructed not to move during the whole experimental session to avoid head-movement artifacts. They were trained a few days prior to the scanning session and all the motor tasks were practiced again just before entering into the scanner. No participant reported any difficulty performing the tasks. This procedure was similar to the one used in Grabski et al.'s study 2012. , except Magnetic resonance images were acquired with a 3T whole-body MRI scanner (Bruker Medspec S300) with a transmit/receive quadrature head coil. Participants laid supine in the scanner with head movements minimized with a standard birdcage and foam cushions. Visual instructions were presented using the Presentation software  and displayed on a screen situated behind the scanner and viewed on a mirror fixed above the subject's eyes.The fMRI experiment consisted of one functional run and one anatomical run. Functional images were obtained using a T2*-weighted, echoplanar imaging (EPI) sequence with whole-brain coverage . Each functional scan comprised forty axial slices parallel to the anteroposterior commisural plane acquired in interleaved order . A high-resolution T1-weighted whole-brain structural image was acquired for each participant after the third functional run .In order to avoid movement artefacts, a “sparse sampling” acquisition paradigm was used e.g., . This acThe motor or resting conditions were performed in three sets of six consecutive trials in a pseudorandom sequence. This RS structure allows measuring changes in BOLD signal for repeated compared to novel performed actions. Altogether, 72 functional scans were therefore acquired . In addition, three “dummy” scans at the beginning of each run were added to allow for equilibration of the MRI signal and were removed from the analyses.http://www.sph.sc.edu/comd/rorden/mricron/). In both group analyses, all activation peaks were first determined in each cluster. The location of maximum activation peaks were then labeled according to probabilistic cytoarchitectonic maps Data were analyzed using the SPM5 software package  running on Matlab 7.1 . Brain activated regions were labeled using the SPM Anatomy toolbox The first three volumes (‘dummy’ scans) were discarded. For each participant, the functional series were first realigned by estimating the 6 movement parameters of a rigid-body transformation in order to control for head movements between scans. After segmentation of the T1 structural image  and 6 realignment parameters, with the silent trials forming an implicit baseline. Individual statistical maps were calculated for each motor repetition with the related baseline and subsequently used for group statistics. A one-way repeated measures analysis of variance (ANOVA) was performed with the “repetition” condition (6 levels: RS1 to RS6) as within-subject factor and the subjects treated as a random factor. Six t-contrasts were first calculated to determine brain regions specifically activated for each of the six consecutive actions compared to the resting condition .Three different time-courses of adaptation across the six repetitions were tested, corresponding to a linear decrease, an exponential decrease and a categorical decrease  of the BOLD response see . The timSurface rendering of brain activity and maximum activation peaks observed for the three motor tasks, the conjunction analysis and differences between tasks are provided in In addition, significant changes in activity between the motor tasks were observed see . Higher Surface rendering of brain activity and maximum activation peaks observed in the RS analysis are provided in The use of the repetition suppression paradigm allowed us to determine brain regions sensitive to sensory-motor adaptation, among all brain regions classically involved in orofacial motor control. Linear RS across the 6 consecutive productions over all types of orofacial actions was observed in the left intraparietal sulcus (IPS) and adjacent anterior dorsal IPL, the left superior parietal lobule  extending to the posterior dorsal IPL, and in the precentral and postcentral gyri (with the maximum activation peak for this cluster located in the most dorsal part of the left vPM; see Using sparse temporal acquisition, the goal of this fMRI-adaptation was to investigate RS during repeated intransitive silent lip, tongue and jaw movements. Irrespective of RS, orofacial movements activated a set of largely overlapping, common brain areas forming a core neural network classically involved in orofacial motor control. Crucially, suppressed neural responses during repeated orofacial actions were observed in the left ventral premotor cortex, the intraparietal sulcus, the inferior parietal lobule and the superior parietal lobule. Because lip, tongue and jaw movements were devoid of visual and auditory feedback, these results strongly suggest motor-to-somatosensory adaptive changes during repeated orofacial actions in posterior parietal and ventral premotor cortices.As provided by the conjunction analysis and apart from RS see , the lipCrucially, linear and exponential RS across the six consecutive productions over all types of orofacial actions were observed in the left vPM, the IPS and adjacent antero-dorsal IPL, as well as the SPL extending to the postero-dorsal IPL see , 4 and 5Altogether, these results appear exquisitely in line with previous studies on manual action goal coding in both nonhuman and human primates. As previously mentioned, neurophysiological recordings of single neurons in nonhuman primate have shown neuronal selectivity for action goal coding of transitive manual motor acts in posterior parietal and ventral premotor areas As in the present study, although parietal and premotor areas were the most consistent regions showing RS in previous fMRI-adaptation studies on repeated motor acts, suppressed response in the SPL has also been observed during repeated manual actions Given that the left ventral premotor cortex, the intraparietal sulcus, the inferior parietal lobule and the superior parietal lobule have been previously shown to be sensitive to RS during repeated produced and/or observed manual actions in both nonhuman and human primates, these findings further extend the role of these regions in action goal coding and forward sensory-motor control to silent intransitive orofacial actions. Since no visual or auditory feedback occurred during orofacial actions, this likely suggests a supramodal function of these regions in forward predictive sensory-motor control, being visuo-motor and/or somatosensory-motor. As previously mentioned, while action goal coding and forward sensory-motor control processes have been extensively studied in the context of upper limb movements, generative forward models of speech production also argue for a role of the auditory and somatosensory cortices in online predictive/corrective control mechanisms in which sensory consequence of the speech motor act are evaluated with actual sensory input in order to further control production e.g.,  and percDespite strong evidence for RS in left frontal and parietal areas, some methodological issues which could impact on the interpretation of the data have to be finally addressed. First, force parameters were not monitored during the scanning session, we therefore cannot exclude that the observed RS might partly derive from force decrease or muscle fatigue during repeated movements. From this possibility, a previous fMRI study on hand muscle fatigue provided evidence for BOLD decrease in the precuneus This fMRI-adaptation study was designed to determine the neural correlates of action goal coding and forward predictive processes during repeated silent and intransitive lip, tongue and jaw movements. In line with previous studies on manual actions, suppressed neural responses during repeated orofacial actions were specifically observed in the left ventral premotor cortex, the intraparietal sulcus and adjacent antero-dorsal inferior parietal lobule as well as the superior parietal lobule extending to the postero-dorsal inferior parietal lobule. These results provide evidence for motor-to-somatosensory adaptive changes in this fronto-parietal circuit.


Colorectal cancer (CRC) is a heterogeneous disease that, on the molecular level, can be characterized by inherent genomic instabilities; chromosome instability and microsatellite instability. In the present study we analyze genome-wide disruption of pre-mRNA splicing, and propose transcriptome instability as a characteristic that is analogous to genomic instability on the transcriptome level.Exon microarray profiles from two independent series including a total of 160 CRCs were investigated for their relative amounts of exon usage differences. Each exon in each sample was assigned an alternative splicing score calculated by the FIRMA algorithm. Amounts of deviating exon usage per sample were derived from exons with extreme splicing scores.P = 0.001), while a combination of the two characteristics showed the strongest association with poor survival in the validation series (P = 0.03).There was great heterogeneity within both series in terms of sample-wise amounts of deviating exon usage. This was strongly associated with the expression levels of approximately half of 280 splicing factors (54% and 48% of splicing factors were significantly correlated to deviating exon usage amounts in the two series). Samples with high or low amounts of deviating exon usage, associated with overall transcriptome instability, were almost completely separated into their respective groups by hierarchical clustering analysis of splicing factor expression levels in both sample series. Samples showing a preferential tendency towards deviating exon skipping or inclusion were associated with skewed transcriptome instability. There were significant associations between transcriptome instability and reduced patient survival in both sample series. In the test series, patients with skewed transcriptome instability showed the strongest prognostic association (We have described transcriptome instability as a characteristic of CRC. This transcriptome instability has associations with splicing factor expression levels and poor patient survival. BRAF mutations, CIMP is considered an epigenetically rather than genetically acting phenotype, and is characterized by extensive DNA methylation at promoter regions and associated gene silencing [Colorectal cancer (CRC) is a prevalent disease with a world-wide incidence of more than one million new cases each year, making it the third most commonly diagnosed cancer among men and women . Colorecilencing ,7. Despiilencing .Tumor staging remains the most important factor for determining the prognosis of CRC patients . Coloreccis-acting elements (exonic and intronic splicing silencers and enhancers) as well as the numerous trans-acting factors constituting the splicing machinery [Alternative pre-mRNA splicing is an important source of functional diversity in the majority of mammalian genes . Nearly achinery ,15. The BCL-XL) of the apoptosis regulator BCL-X [Aberrant splicing patterns have been associated with cancer . One exaor BCL-X . Cancer-or BCL-X ,19. Receor BCL-X , and preor BCL-X ,22. Moreor BCL-X . For somor BCL-X . DespiteIn this study, we describe the disruption of alternative splicing as a global event in CRC tissue using exon microarray analysis. We show a great extent of variation in the amount of deviating exon usage among the samples. This transcriptome instability is found to be associated with prognosis in CRC patients, and also has clear associations with the expression levels of approximately half of all splicing factors.Two independent series comprising a total of 160 stage II and III CRC tissue samples were included in this study. The two series are referred to as test and validation series (Table ® Whole Transcript (WT) Sense Target Labeling Assay [RNA (1 μg) from each sample was individually amplified, reverse transcribed, fragmented, and labeled using the Affymetrix GeneChipng Assay . Labeledng Assay . Each arng Assay . A probeScanning of the microarrays and preprocessing of raw image intensity data were controlled by the Affymetrix GeneChip Command Console software version 1.0). For each microarray, the software generated cell intensity (CEL) files storing probe-level intensity data calculated from scanned image files containing pixel intensity values. CEL data files were used as input for preprocessing and alternative splicing detection with the Finding Isoforms using Robust Multichip Analysis (FIRMA) method  .For further statistical analyses, the software SPSS 15.0  was used. This includes t-statistics, multinomial logistic regression, generation of Kaplan-Meier plots, Cox regression analyses for calculation of hazard ratios (HR) and corresponding 95% confidence intervals (CI), Fisher's exact test, and Mantel-Cox test for equality of survival distributions. A list of 280 human splicing factors  - are considered to have a skewed TIN (sTIN) subtype. Tumors where the overall amounts of deviating exon usage differ from the average  are considered to have an overall TIN (oTIN) subtype. The patients showed no significant associations between sTIN or oTIN and MSI status, tumor stage, patient age and gender, or tumor location .We consider tumors with large amounts of deviating exon usage as tumors with transcriptome instability (TIN). Tumors showing preferential exon skipping or inclusion - that is, having a skewed distribution in the amounts of deviating skipping and inclusion events  in each permutation ranged from -0.17 to 0.18 . Also, the increase in amounts of negatively compared to positively correlated splicing factor genes was higher for the observed deviating exon usage amounts than for 99% of the permutations  showed a significant correlation to the total relative amounts of deviating exon usage . Groups of samples with high and low total relative amounts of deviating exon usage were mainly separated into different clusters Figure . RestricP = 0.001 by log rank test for equality of survival distributions; Figure P = 0.002).Patients with sTIN tumors Figure  had signP = 0.004). Among the other variables, only tumor stage showed significant associations with patient prognosis in this model.In a multivariate model including tumor stage, MSI status, patient age and gender, as well as tumor location, sTIN was the strongest independent prognostic predictor  had significantly poorer survival than patients with TIN-negative cancers . This difference was significant also in a multivariate model including tumor stage, MSI status, patient age and gender, as well as tumor location . Two patients had tumors with overlapping subtypes of TIN, that is, characterized by both sTIN and oTIN. These patients died from metastatic disease 0.8 and 4.3 years after surgical removal of their primary tumor.Patients whose tumors were characterized by oTIN had a slightly poorer survival rate than patients with average amounts of deviating exon usage, although this finding was not significant .P < 0.05; Figure S4A in Additional file Also in the validation series, there was a strong association between oTIN and expression levels of splicing factors. Here, the sample-wise expression levels of 48% of splicing factors 133 of 280) were significantly correlated to the total relative amounts of deviating exon usage , the oTIN samples were almost completely separated into the two respective groups  had a significantly poorer 5-year survival rate (64%) than patients whose tumors were characterized with neither or only one of the TIN subtypes .The strong associations between TIN and disease-specific survival among the patients in the test series were not as clearly indicated in the validation series . These patients received adjuvant chemotherapy according to the current standard, whereas patients in the historical test series received no other treatment than surgery. The mean age at diagnosis was also different between the two patient series (66 versus 73 years). These factors may have contributed to the observed different associations between TIN and patient survival. Although we found no associations between MSI and TIN, the different amounts of patients with MSI tumors in the two series (16% versus 31%) may also have influence in this regard. However, both series showed significantly reduced survival for patients stratified according to TIN, suggesting that high amounts of inter-tumor deviations in exon usage patterns may indeed be associated with poor patient survival. We would welcome additional effort to verify the prognostic value of this molecular characteristic.The amounts of samples assigned to either or both of the TIN subtypes varied between the two sample series. In the test series, 29% (24 of 83) of the tumors were characterized by TIN, compared to 56% in the validation series (43 of 77). Although resulting in an increased amount of TIN-positive samples, we chose to keep a fixed scoring threshold for TIN to avoid introducing subjective bias into the validation. Despite the different frequencies, the reproducibility of the strong associations between oTIN and expression levels of splicing factor genes provides strong evidence for a biological explanation of the observed variability in deviating exon usage amounts. However, it is not certain to what extent this has influenced the associations between TIN and patient survival in the two series.SLC39A14, however, is reported to have high cancer sensitivity [The amounts of deviating exon usage were significantly higher in the cancer samples compared to paired normal colonic mucosa. A possible bias may have been introduced to these comparisons by the use of different RNA extraction protocols for the two sample groups. However, when analyzing known splicing events, 11 of 13 exons were found to have a mean difference in alternative splicing scores between the paired samples according to expectation. Also, 36% of the probe sets across the 13 CRC samples included in this analysis of known splicing events were designated as differentially spliced . Accordingly, it seems evident that alternative exon usage is indeed reflected in the analytical approach, where individual exons in individual samples are scored according to the likelihood of differential splicing. For the majority of the known splicing events tested, the recurrence rate in CRC tissue is uncertain, due to limited sample numbers used for discovery . The cansitivity . In accoIt is striking that the expression levels of the majority of splicing factors negatively correlated to the numbers of deviating exon usage. Irrespective of the role of the splicing factor, whether it is predominantly a splicing enhancer or silencer, and irrespective of the type of exon usage event (exon skipping or inclusion), this was true for more than 95% of the significantly correlated splicing factor genes in two independent sample series. In fact, the correlations among splicing factor genes were significantly stronger in the direction of negative correlation than among random gene sets of equal size. This strong correlation is further supported by unsupervised hierarchical clustering analyses, which were based on the expression levels of the total set of splicing factors and separated the samples according to the oTIN phenotype in both series. Therefore, the association between low expression levels of splicing factors and increased variability in exon usage seems to be indicative of a critical role of splicing factor activity for the maintenance of a stable transcriptome. This suggests a biological rationale for the differences in exon usage observed in this study.Consistent with this great variation in exon usage among CRC samples, other recent studies have also shown that the extent of alternative splicing far exceeds previous estimates. On average, individual multi-exon genes are suggested to undergo at least seven alternative splicing events across various human tissues and cell lines ,39. ThisAlthough the approach taken here to analyze sample-wise amounts of deviating exon usage was shown to correctly classify known splicing events, the main purpose of the study was to describe splicing on a genome-wide scale and does not allow for detailed analysis of individual transcript structures and individual splicing events. Furthermore, the current analyses did not intend to provide insights into the functional consequences of individual splicing events, that is, whether the predicted splicing events yield functionally different protein isoforms. There is increasing evidence that a great amount of expressed transcripts result from splicing noise ,40,41. TThis study provides evidence for a high degree of variation among CRC samples with regards to amounts of differential exon usage. Based on this, we suggest TIN as a characteristic of CRC, which can be further dissected into the oTIN and sTIN subclasses. The oTIN subtype, reflecting sample-wise total relative amounts of deviating exon usage, is negatively correlated to the expression level of the majority of splicing factor encoding genes. Furthermore, analyses of corresponding clinical data demonstrate that TIN is associated with poor patient survival.CEL: cell intensity; CI: confidence interval; CIMP: CpG island methylator phenotype; CIN: chromosomal instability; CRC: colorectal cancer; FIRMA: Finding Isoforms using Robust Multichip Analysis; HR: hazard ratio; MSI: microsatellite instability; oTIN: overall transcriptome instability; RMA: robust multi-array average; sTIN: skewed transcriptome instability; TIN: transcriptome instability.AS and THÅ conducted the exon microarray experiments and data analyses, and AS drafted the manuscript. AN and TOR collected and contributed the clinical specimens and data. RAL and RIS conceived and directed the project. All authors participated in drafting the manuscript and have read and approved the final manuscript.The authors declare that they have no competing interests.Supplementary methods and results.Click here for fileSupplementary Figures 1 to 4, and Supplementary Tables 1 to 5.Click here for file


Yeast-like fungi inhabit soils throughout all climatic zones in a great abundance. While recent estimations predicted a plethora of prokaryotic taxa in one gram of soil, similar data are lacking for fungi, especially yeasts.We assessed the diversity of soil yeasts in different forests of central Germany using cultivation-based techniques with subsequent identification based on rDNA sequence data. Based on experiments using various pre-cultivation sample treatment and different cultivation media we obtained the highest number of yeasts by analysing mixed soil samples with a single nutrient-rich medium. Additionally, several species richness estimators were applied to incidence-based data of 165 samples. All of them predicted a similar range of yeast diversity, namely 14 to 16 species. Randomized species richness curves reached saturation in all applied estimators, thus indicating that the majority of species is detected after approximately 30 to 50 samples analysed.In this study we demonstrate that robust species identification as well as mathematical approaches are essential to reliably estimate the sampling effort needed to describe soil yeast communities. This approach has great potential for optimisation of cultivation techniques and allows high throughput analysis in the future. Soils display a remarkable heterogeneity throughout all climate zones, thus providing a multitude of diverse habitats of different scales and properties. Many studies on the biological diversity associated with particular soil types suggest that soil, in general, may well be a megadiverse habitat dominated by invertebrates, prokaryotes and fungi Cryptococcus albidus, Cr. humicola, Cr. laurentii, are taxonomically complex and may in fact be difficult to distinguish Soil yeasts are known from the Polar Regions to the tropics and in total up to 130 species were reported worldwide. However, evidence for a strong association with soil-related substrates is lacking for many of these species Yeast species richness studies in forest soils of the temperate zone that were based on conventional phenotypic methods reported 18 to 26 yeast species Because microbial diversity differs largely across habitats, no meaningful comparison of biodiversity assessments can be performed without understanding the ranges of alpha-diversity, i.e. species richness, in a substrate or biotope. We used soil yeast community data to address three basic questions, which have previously not been considered within a single study: (1) Does soil heterogeneity have an effect on the observed species richness? (2) Is cultivation success of different yeast species influenced by different cultivation media and thereby by differences in nutrient availability? (3) What is the expected diversity estimated in relation to sampling effort? Special attention was given to robust species identification by sequencing rDNA and subsequent phylogenetic analysis in order to address species recognition and its impact on estimations of the yeast species richness in soils.http://www.biodiversity-exploratories.de). An overview of the German Biodiversity Exploratories is given by Fischer at al. Fagus sylvatica) forests of two different management types. The near-natural unmanaged forest consisted of 100 years old beech stands sometimes mixed with Fraxinus excelsior and Acer pseudoplatanus. The managed forest type consisted of 40-year-old age-class planted forests, with the tree species being Fagus sylvatica, and beech selection cutting forest. Details on the study sites, their properties and the field permits are provided in Fischer et al. The study has been performed in the Biodiversity Exploratory Hainich-Dün (h horizon) were collected using a steel ring (volume: 100 cm3), placed in sterile paper bags, transferred to the laboratory and kept at −20°C before analysis. Soil from one plot (five core samples) was mixed in equal proportion and sieved consequently through 10 mm, 2 mm, 1 mm and 0.065 mm meshes  1∶5, 1∶10, and 1∶20 in sterile water and shaken on an orbital shaker at 200 rpm for 1 hour. An aliquot of 0.15 ml was distributed on the surface of solid media. Glucose-yeast extract-peptone agar (GPYA) acidified with lactate , MYP agar 5, (ATG)5 and (GAC)5 as single PCR primer DNA was isolated from 3–4 days old cultures using a technique described by Hoffman and Winston www.ncbi.nih.gov) and CBS (www.cbs.knaw.nl) databases, respectively.PCR products were purified with the my-Budget Double Pure kit  and sequenced on an ABI3130xl sequencer using the same primers as for PCR amplification. Chromatograms were checked and corrected with Sequencher 4.8–4.10 . For species identification the obtained nucleotide sequences were compared with sequences deposited in the NCBI  per gram of soil at natural humidity. The community structure was characterised by frequency of occurrence (incidence) of every observed species in the sample, which was calculated as the relative occurrence of the species to the total number of species in the sample. Statistical evaluations were performed with STATISTICA 8.0 . The reliability of the different sampling assays was statistically tested by the comparison of results obtained from mixed and individual samples both in near-natural and managed forests using one-way analysis of variance. Only data that passed normality test were used for further analyses. Effects were considered to be statistically significant at the level p<0.05. Significant effects were additionally confirmed with Chi-square test. Species accumulation curves were calculated with EstimateS 8.0 using 50 randomizations, sampling without replacement and default settings for upper incidence limit for infrequent species 2 to 104 CFU/g. However, there was no significant difference related to the distinct sample treatments. 1.2×103 CFU/g of yeasts were observed in mixed and 1.0×103 CFU/g in individual samples . The frequency of appearance of Trichosporon porosum was slightly higher in the individual samples. Nevertheless, in both cases it was the second dominating species . Modified Brown's media was the most selective one and yielded isolates of only one species, G. pullulans  to represent the soil cover of a biotope. Total yeast counts in our study are comparable with the average numbers previously found for soil-related substrates in temperate climate zone Lipomyces because these soil yeasts have the rare ability among yeasts to utilize nitrogen from heterocyclic compounds, such as imidazole, pyrimidine, and pyrazine Lipomyces yeasts from soils Lipomyces and their anamorphs Myxozyma  display a typical oligotrophic behaviour, slow growth on standard nutrient-rich media coupled with the ability to assimilate complex substrates. The ability of other yeasts to grow on medium containing heterocyclic compounds as the only source on nitrogen has been recorded only once Four different media, representing a gradient of nutrient availability were used in the cultivation experiment. Although various authors have suggested using selective media in order to obtain higher diversity In addition to lower observed diversity, modified Browns' agar and TMV media seems not suitable for soil diversity studies in contrast to nutrient-rich media, because yeast colonies of different species on nitrogen-depleted media looked very similar, either extremely mucous or dimorphic (with developed substrate mycelium). After transfer from these media to GPYA they often split in several morphologically and taxonomically distinct cultures. Therefore, it is very difficult to make assumptions about the observed species abundance using these media. Additionally, we often observed strong development of moulds on our samples although TMV agar has been reported to prevent overgrowth of yeast colonies by filamentous fungi Trichosporon dulcitum, T. porosum, Cryptococcus terricola) accounting for the majority of isolates, and a large number of minor or rare species. Interestingly, the same pattern, a species-poor yeast community with uneven distribution of species, is known for yeasts inhabiting floral nectar Rarefaction curves were close to saturation and we applied species richness estimators to predict the number of species to be expected from the soils using the traditional plating technique . All of Although all randomised species accumulation curves reached saturation with our sampling effort, ICE and Chao 2 richness estimators approach the plateau earlier than first-order Jackknife or Bootstrap . Our stuMolecular studies on yeast diversity have mainly replaced physiological assimilation tests to identify species as commonly used assimilation tests are not necessarily able to distinguish between closely related species. Additionally, the application of molecular markers has also vastly increased the species numbers in this informal group of fungi Clavispora reshetovae and Barnettozyma vustinii, were found during this survey Cryptococcus. However, only Cryptococcus ramirezgomezianus and Cr. musci  can be interpreted as real cryptic species, as they are difficult to distinguish morphologically Cryptococcus terricola - Cr. gastricus can pose only a minor problem in identification. Although both species belong to the Filobasidiales they are not closely related to each other and exhibit distinct physiological profiles, e.g. assimilation of nitrogen sources Trichosporon dulcitum and T. porosum differ phenotypically as they belong to two different clades within the order Trichosporonales, which were suggested to be reclassified into distinct anamorphic genera In this study, the used plating strategy resulted in an average of 4.7 colonies per plate, including plates with no yeast colonies. This enabled a reliable differentiation of colonies and adequate isolation of representative strains. Consequently, two new species, described as Proper identification of soil fungi using molecular markers is important for diversity estimations and provides a solid background for reliable comparison of results achieved in different surveys. Our study demonstrated that conventional culture-based experiments can be successfully optimised by using mixed soil samples and a single nutrient-rich medium in order to reduce sampling effort in a given substrate or biotope. Additionally, our results demonstrate the usefulness of quantitative estimations to calculate optimal sampling effort in biodiversity- and monitoring-orientated studies. All species richness estimations performed in this study were based on solid species identification, which should be considered in future high throughput analyses as well. The dissimilarities in the yeast community composition observed at the two different sampling dates might hint to seasonal variation in species occurrence and this observation should be addressed in additional studies with more time points.Figure S1Yeast quantity (CFU/g) observed after different pre-cultivation soil treatments. Whiskers correspond to confidential interval and asterisks to outliers.(EPS)Click here for additional data file.Table S1Average frequency of occurrence of yeasts in samples after different pre-cultivation treatments  and in relation to the two different management types .(DOC)Click here for additional data file.Table S2Species-plot relationship: the number of samples allocated for the analysed combination of plots and the species richness values (Mean and standard deviation).(DOC)Click here for additional data file.


Mycobacterium bovis) system to investigate the landscape-scale potential for bovine tuberculosis transmission within a nonmigratory white-tailed deer (Odocoileus virginianus) and elk (Cervus canadensis) community. Using landscape population genetics, we tested for genetic and spatial structure in white-tailed deer. We then compared these findings with the sympatric elk population that is structured and which has structure that correlates spatially and genetically to physiognomic landscape features. Despite genetic structure that indicates the white-tailed deer population forms three sympatric clusters, the absence of spatial structure suggested that intraspecific pathogen transmission is not likely to be limited by physiognomic landscape features. The potential for intraspecific transmission among subpopulations of elk is low due to spatial population structure. Given that white-tailed deer are abundant, widely distributed, and exhibit a distinct lack of spatial population structure, white-tailed deer likely pose a greater threat as bovine tuberculosis vectors among elk subpopulations than elk.Sympatric populations of phylogenetically related species are often vulnerable to similar communicable diseases. Although some host populations may exhibit spatial structure, other hosts within the community may have unstructured populations. Thus, individuals from unstructured host populations may act as interspecific vectors among discrete subpopulations of sympatric alternate hosts. We used a cervid-bovine tuberculosis ( One of the key goals of disease ecology is to understand pathogen transmission and disease spread over space and time  and elk (Cervus canadensis).Studying multi-host endemic disease systems is often complicated because they commonly involve highly mobile and widely distributed wildlife species with low prevalence of infection . Limited connectivity among elk subpopulations in the region indicates a low potential for long-distance disease spread through the movement and dispersal of infected elk  and the Duck Mountain Provincial Park and Forest (DMPP&F 3800 km2)  . The reg800 km2)  and in l800 km2)  and elk 800 km2) ; however800 km2) . Over th800 km2) .Bos taurus) herds around RMNP have tested positive for bTB between 1991 and 2010 , the effects of hunting in the estimates of population subdivision are likely to be negligible. Also critical is obtaining sufficiently large and widespread distribution of the samples used. In our study, both strategies yielded widespread and large samples sizes.For this study, we used two different sampling techniques to collect white-tailed deer and elk genetic samples. Elk samples were collected through the winter using a net gun fired from a helicopter  came from hunt-harvested deer, a few of them  were obtained by Parks Canada using a helicopter-deployed net gun  following the animal care protocol 472602 of the University of Alberta. Although most of the samples  and observed (HO) heterozygosity estimates, and tests of Hardy–Weinberg equilibrium (HWE) were obtained using the ADEGENET package  is inferred using sequential K-means and model selection  spatial population structure, white-tailed deer are typically panmictic . Then we used DAPC to assign individuals into populations, retaining the number of principal components using 85% of the cumulative deviance.DAPC transforms data using a principal component (PC) analysis before summarizing genetic variance between and within groups  all corroborated the lack of any spatial structure in the white-tailed deer dataset.Spatial Principal Component Analysis (sPCA) scores can detect clines and spatial groups  as well as strong genetic differences between neighbors . A global permutation test on the eigenvalues derived from a sPCA with a minimum neighbor connection network did not find any significant global or local structures . However, the bootstrap results were not significant at P = 0.05. Rather the average mean nearest-neighbor distance for white-tailed deer occurred at the 90th percentile of the distribution of resampled elk mean nearest-neighbor distances . Infected white-tailed deer appeared to be more dispersed than infected elk. For the Mann–Whitney istances B.Differences in connectivity among subpopulations are known to affect the probability of landscape-scale disease transmission Hess . Here, wM. bovis into neighboring cattle farms  amount of genetic variation retained among clusters. We did not, however, sequence mtDNA haplotypes and cannot comment on whether these clusters represent vestiges of the colonization or expansion of distinct geographic lineages. Nor do we have information to suggest that assortative mating contributes to the maintenance of sympatric clusters. Notwithstanding this, our results underscore the high connectivity that characterizes white-tailed deer populations.In comparison with elk, white-tailed deer are well adapted to highly modified agricultural landscapes . Furthermore, appreciating that these samples were collected using mixed sampling methods . Thus, our results should be viewed as a trend, rather than a definitive result.Because of being highly connected, white-tailed deer may disperse bTB further from the bTB infection focus than elk. Our dispersion results are not fully conclusive as the mean distance to the nearest infected neighbor for white-tailed deer occurred at the 90th percentile of the bootstrapped mean infected-neighbor distance distribution for elk. Despite the intensive sampling , transmission needs to occur interspecifically. This can occur from animal to animal and by environmental contamination (Williams et al. We have highlighted that understanding the potential for pathogen transmission among wildlife at landscape scales requires knowledge of the population structures of all free-ranging hosts, particularly when connectivity among populations can be a function of intraspecific and/or heterospecific dispersals. Our study suggests that the role of white-tailed deer in bTB transmission is likely to be more critical than previously appreciated. This has important applications for ongoing intervention programs that so far have been largely elk-biased. Several behavioral and life history traits make managing disease in white-tailed deer especially problematical. For example, white-tailed deer are more likely to co-mingle with cattle on winter cattle feeding areas (Brook et al. Data for this study are available as supplementary material online.


Transcription factors play a central role in the gene regulatory networks that mediate various aspects of plant developmental processes and responses to environmental changes. Therefore, their activities are elaborately regulated at multiple steps. In particular, accumulating evidence illustrates that post-transcriptional control of mRNA metabolism is a key molecular scheme that modulates the transcription factor activities in plant responses to temperature fluctuations. Transcription factors have a modular structure consisting of distinct protein domains essential for DNA binding, dimerization, and transcriptional regulation. Alternative splicing produces multiple proteins having different structural domain compositions from a single transcription factor gene. Recent studies have shown that alternative splicing of some transcription factor genes generates small interfering peptides (siPEPs) that negatively regulate the target transcription factors via peptide interference (PEPi), constituting self-regulatory circuits in plant cold stress response. A number of splicing factors, which are involved in RNA binding, splice site selection, and spliceosome assembly, are also affected by temperature fluctuations, supporting the close association of alternative splicing of transcription factors with plant responses to low temperatures. In this review, we summarize recent progress on the temperature-responsive alternative splicing of transcription factors in plants with emphasis on the siPEP-mediated PEPi mechanism. Transcription factor is a critical component of the gene regulatory networks that mediate virtually all aspects of plant growth and developmental processes. It also plays a central role in plant responses to biotic and abiotic stresses. Therefore, the transcription factor activities are coordinately regulated at various steps to fine-tune signal transduction pathways in diverse cellular signaling networks for optimal growth and survival under given growth conditions  that is mediated by small interfering peptides (siPEPs)  mechanism  proteins have been identified as putative zinc finger (ZF) motif-containing transcription factors functioning in diverse growth hormone signaling and flower architecture  protein LONG HYPOCOTYL IN FAR-RED 1 (HFR1) retains the HLH domain, but has defects in the basic DNA-binding domain. It has been shown that the HFR1 protein plays a dominant-negative role in photomorphogenesis  proteins consisting of ~100 residues. The KDR proteins lack protein domains required for transcription factor activities. They interact with HFR1 through the HLH motif and prevent HFR1 from binding to the PIF transcription factors  and PISTILLATA (PI) in floral organogenesis /bHLH136, BNQ2/bHLH134, and BNQ3/bHLH161 proteins also interact with HFR1  gene homolog, produces three different transcripts through exon skipping at low temperatures  and LATE ELONGATED HYPOCOTYL (LHY) that encode MYB domain-containing members, produce differential isoforms at high ambient temperatures. At warm temperatures, whereas the alternative splicing of the CCA1 gene is enhanced, that of the LHY gene is suppressed  gene regulates starch metabolism under cold conditions  exhibit distinct growth alterations possibly by inducing the QUA-QUINE STARCH (QQS) gene, which is involved in starch degradation  genes and, thus, designated CBF-COR regulon, in higher plants  and its downstream targets CBF genes by binding directly to the gene promoters  gene links the circadian clock with ambient temperature responses in Neurospora crassa  gene is a vital player in the vernalization process in Arabidopsis  transcription factors regulate secondary cell wall biosynthesis by inducing their own genes and PtrSND1 gene is influenced by temperature changes. Secondary cell wall biosynthesis is influenced by cold temperatures  genes encode RNA-binding proteins that act as splicing regulators. It has been shown that the splice variants of the SR genes are targeted by NMD  genes undergo alternative splicing, which is regulated primarily by temperature signals  splicing factor genes, Le9G8-SR and LeSF2-SR1, is altered dramatically at low temperatures  in the N-terminal region and one arg/ser-rich domain in the C-terminal region, are highly conserved in plants  and are, thus, termed ‘‘NAGNAG acceptor’’ sites  and PRR9, in a temperature-dependent manner  domain-containing protein is a splicing factor. It regulates alternative splicing possibly by modulating the recognition or cleavage of the splice donor and acceptor sites  protein is a pre-mRNA splicing factor that is homologous to the human U5 small ribonucleoprotein-associated 102-kD protein (Lee et al. Alternative splicing is widely conserved in eukaryotes. Numerous examples illustrate that temperature-responsive alternative splicing is prevalent in plants and plays a role in a broad spectrum of plant responses to low temperatures by diversifying transcriptomes and proteome plasticity.CCA1 alternative splicing (Park et al. Arabidopsis and rice (Seo et al. Alternative splicing is also linked with diverse gene regulatory mechanisms, such as PEPi that regulates gene transcription by modulating transcription factor activities. Plant siPEPs produced by alternative splicing of transcription factor genes form a self-regulatory circuit, as exemplified by Alternative splicing is profoundly affected by low temperatures in plants. Therefore, it is perceived as a way of integrating temperature signals into plant development and endogenous cellular physiology. Despite its close association of alternative splicing with temperature signals, just a few responsible splicing factors have been characterized so far. It is also unknown how environmental stress signals affect the activities of splicing factors in most cases. Molecular and biochemical investigations on the splicing factors and phenotypic examinations of plants that are defective in the splicing factors and their target genes would help to understand the molecular mechanisms underlying temperature-responsive alternative splicing of cold-responsive transcription factor genes.Alternative splicing can be biotechnologically explored as a means of elaborate control of transcription factor activities in crop plants. Engineering of the alternative splicing patterns through mutations in splice sites can be applied for modifying plant development and responses to environmental stresses. Modulations of splicing factor activities would be an alternative approach to precisely control plant functions for improved stress tolerance.