Tensin2 Is a Novel Diagnostic Marker in GIST, Associated with Gastric Location and Non-Metastatic Tumors

GIST is a rare soft tissue sarcoma, for which KIT and DOG1 are used as highly sensitive diagnostic markers. Other diagnostic markers include CD34, protein kinase C θ, deficiency of succinate dehydrogenase complex subunit B, carbonic anhydrase II, and type I insulin-like growth factor receptor. We investigated the role of TNS2 as a diagnostic biomarker by using immunohistochemistry in 176 GISTs and 521 other sarcomas. All GISTs expressed TNS2, with intermediate or high expression in 71.4% of samples. The majority (89.8%) of other sarcomas were negative for TNS2, and intermediate to strong staining was only seen in 2.9% of samples. Strong TNS2 staining was associated with gastric location (gastric 52.8% vs. non-gastric 7.2%; p p < 0.001), absence of metastases (non-metastatic tumors 44.3% vs. metastatic tumors 5.9%; p = 0.004), female sex (female 45.9% vs. male 33.8%; p = 0.029), and tumors of lower risk categories (very low or low 46.9% vs. intermediate 51.7% vs. high 29.0%; p = 0.020). TNS2 expression did not correlate with overall survival or metastasis-free survival. No associations between TNS2 expression and KIT/PDGFRA mutation status, tumor size, mitotic count, or age of the patient were detected. The results provide conclusive evidence for the value of TNS2 as a sensitive and specific diagnostic biomarker for GIST.Peer reviewe

Tensin2 Is a Novel Diagnostic Marker in GIST, Associated with Gastric Location and Non-Metastatic Tumors

GIST is a rare soft tissue sarcoma, for which KIT and DOG1 are used as highly sensitive diagnostic markers. Other diagnostic markers include CD34, protein kinase C θ, deficiency of succinate dehydrogenase complex subunit B, carbonic anhydrase II, and type I insulin-like growth factor receptor. We investigated the role of TNS2 as a diagnostic biomarker by using immunohistochemistry in 176 GISTs and 521 other sarcomas. All GISTs expressed TNS2, with intermediate or high expression in 71.4% of samples. The majority (89.8%) of other sarcomas were negative for TNS2, and intermediate to strong staining was only seen in 2.9% of samples. Strong TNS2 staining was associated with gastric location (gastric 52.8% vs. non-gastric 7.2%; p < 0.001), absence of metastases (non-metastatic tumors 44.3% vs. metastatic tumors 5.9%; p = 0.004), female sex (female 45.9% vs. male 33.8%; p = 0.029), and tumors of lower risk categories (very low or low 46.9% vs. intermediate 51.7% vs. high 29.0%; p = 0.020). TNS2 expression did not correlate with overall survival or metastasis-free survival. No associations between TNS2 expression and KIT/PDGFRA mutation status, tumor size, mitotic count, or age of the patient were detected. The results provide conclusive evidence for the value of TNS2 as a sensitive and specific diagnostic biomarker for GIST

Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues

The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging −RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2–3 h after infection and the translation shortly after at 3–4 h post-infection. The replication hotspots with newly emerging −RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of −RNA and +RNA strands was almost identical, and −RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively

Cellular distribution and amounts of positive and negative RNA strands of enterovirus during an infection in vitro

Enterovirukset ovat pieniä vaipattomia viruksia, joilla on positiivinen RNAgenomi. Ihmisille infektiivisiä enteroviruksia tunnetaan yli sata erilaista, ja ne voivat aiheuttaa monia tauteja normaalista flunssasta sydänlihastulehdukseen ja aivokalvontulehdukseen. Enterovirusten elämänkierto tunnetaan melko hyvin, mutta riittämättömien menetelmien takia niiden positiivisen RNA-genomin sekä negatiivisten RNA-juosteiden jakaumaa ja määrää solussa infektion aikana ei tunneta juurikaan. Viruslääkkeitä voitaisiin suunnitella estämään mitä tahansa viruksen elämänkierron vaihetta, mutta tätä varten tarvitaan uusia tekniikoita tutkia RNA-juosteita infektion aikana. Tässä tutkimuksessa kehitimme branched DNA in-situ fluoresenssi hybridisaatioon perustuvan uuden protokollan, ja käytimme tätä metodia käänteistranskriptio – kvantitatiivisen PCR:n ohella enterovirusten RNA-juosteiden jakauman ja määrän tutkimiseen infektion aikana. Tulostemme mukaan viruksen positiivisen-, negatiivisen- sekä kaksoisjuosteisen RNA:n määrä on lähes olematon ennen kolmea tuntia infektion jälkeen, ja niiden määrä kasvaa huomattavasti 4-5 tuntiin infektion jälkeen. Positiivinen RNA on jakautunut solun ulkoreunoille, kun taas negatiivinen ja kaksoisjuosteinen RNA on lähempänä solun tumaa. RNA juosteet eivät lokalisoidu viruksen kapsidin tai solun tuman kanssa. Testasimme myös kolmen eri viruslääkkeen vaikutusta RNAjuosteiden määrään ja jakaumaan solussa, ja tulostemme mukaan kaikki lääkkeet estivät virusinfektion ja viruksen RNA-synteesin lähes kokonaan. Nämä tulokset, kehittämämme protokollan ohella, auttavat tutkimaan enterovirusten elämänkiertoa tehokkaammin ja kehittämään uusia mahdollisia viruslääkkeitä.Enteroviruses are small, non-enveloped viruses with a positive-sense RNA genome. Over a hundred different serotypes of enteroviruses can infect humans, and they can cause a wide range of diseases from common colds to encephalitis and myocarditis. The life cycle of enteroviruses is relatively well known, but the distribution of the positive RNA genome and its complementary negative RNA strands during an infection are unknown, mostly due to lacking techniques. Certain antiviral drugs could target this step of the life cycle, inhibiting the synthesis of the negative strand and stopping the infection, but to study this in more detail, new methods are required to visualise the viral RNAs effectively. In this study, we developed a protocol to study the enterovirus RNAs using branched DNA fluorescence in-situ hybridization. With this method, along with reverse transcription quantitative PCR, we studied the distribution and the amounts of enterovirus RNAs during an infection in vitro. The results indicate that the amounts of positive-, negative- and double stranded RNAs are negligible before 3 hours post-infection, and gradually rise after 4-5 hours postinfection. The positive RNA is located peripherally in the cell with the negative RNA and double stranded RNA being located more centrally. Furthermore, the RNA molecules reside in different locations than the viral capsid proteins and the cell nuclei. We also tested the effect of three different antiviral drugs on the distribution and amounts of viral RNAs, and the results show that all tested molecules effectively inhibit the infection and the appearance of viral RNAs. These results, along with the branched DNA protocol we established, will be useful in more efficiently studying the life cycle of enteroviruses and in the development of antiviral drugs

Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues

The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging &minus;RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2&ndash;3 h after infection and the translation shortly after at 3&ndash;4 h post-infection. The replication hotspots with newly emerging &minus;RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of &minus;RNA and +RNA strands was almost identical, and &minus;RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively

Tensin2 Is a Novel Diagnostic Marker in GIST, Associated with Gastric Location and Non-Metastatic Tumors

GIST is a rare soft tissue sarcoma, for which KIT and DOG1 are used as highly sensitive diagnostic markers. Other diagnostic markers include CD34, protein kinase C &theta;, deficiency of succinate dehydrogenase complex subunit B, carbonic anhydrase II, and type I insulin-like growth factor receptor. We investigated the role of TNS2 as a diagnostic biomarker by using immunohistochemistry in 176 GISTs and 521 other sarcomas. All GISTs expressed TNS2, with intermediate or high expression in 71.4% of samples. The majority (89.8%) of other sarcomas were negative for TNS2, and intermediate to strong staining was only seen in 2.9% of samples. Strong TNS2 staining was associated with gastric location (gastric 52.8% vs. non-gastric 7.2%; p &lt; 0.001), absence of metastases (non-metastatic tumors 44.3% vs. metastatic tumors 5.9%; p = 0.004), female sex (female 45.9% vs. male 33.8%; p = 0.029), and tumors of lower risk categories (very low or low 46.9% vs. intermediate 51.7% vs. high 29.0%; p = 0.020). TNS2 expression did not correlate with overall survival or metastasis-free survival. No associations between TNS2 expression and KIT/PDGFRA mutation status, tumor size, mitotic count, or age of the patient were detected. The results provide conclusive evidence for the value of TNS2 as a sensitive and specific diagnostic biomarker for GIST

Uusien monoamiinioksidaasi B:n inhibiittorien hyödyntäminen lääkeainetutkimuksessa

Monoamiinioksidaasi (MAO) on entsyymi, jonka kahta eri alamuotoa (MAO-A ja MAO-B) esiintyy mitokondrioiden ulkokalvoilla. MAO-entsyymit katalysoivat monoamiinien, kuten dopamiinin, serotoniinin ja adrenaliinin, oksidatiivista deaminaatiota. MAO-entsyymien liiallinen aktiivisuus hermosoluissa aiheuttaa muun muassa Parkinsonin- ja Alzheimerin tautia, dementiaa ja masennusta. MAO-B:tä esiintyy lähinnä hermosoluissa, kun taas MAOA:ta esiintyy myös sisäelimissä, joten MAO-B:lle selektiiviset inhibiittorit ovat hyviä lääkeaineita monien neurologisten tautien hoidossa. Joitain MAO-B selektiivisiä inhibiittoreita, kuten pargyliiniä, käytetäänkin jo yhdessä lääkeaine Levodopan kanssa estämään Parkinsonin tautiin liittyvää liiallista dopamiinin hajotusta aivoissa. Näin ollen uusien, tehokkaiden ja turvallisten MAO-B selektiivisten inhibiittorien löytäminen on hyvin tärkeä aihe lääkeainetutkimuksessa. Tämän työn tarkoituksena oli tutkia 23 uutta MAO-B selektiivistä inhibiittoria ja niiden sitoutumis- ja inhibitiotehokkuutta MAO-B:n sitoutumistaskussa. Tutkimus toteutettiin sekä tietokoneella tehdyllä telakointisimulaatiolla että laboratoriossa suoritetulla fosfataasiaktiivisuusmittauksella. Tulokset osoittivat, että testimolekyylien inhibitiotehokkuus ei ollut samalla tasolla kaupallisen pargyliiniinhibiittorin kanssa. Telakointien ja fosfataasiaktiivisuusmittausten tulokset kuitenkin auttavat edistämään tulevaisuudessa tehtävien jatkotutkimusten menetelmien suunnittelua ja toteuttamista. Tulokset auttavat myös valitsemaan erilaisia inhibiittoreita tuleviin jatkotutkimuksiin.Monoamine oxidase (MAO) is an enzyme that is expressed in two different isoforms (MAOA and MAO-B) on the outer membranes of mitochondria. MAOs catalyse the oxidative deamination of monoamines such as dopamine, serotonin and adrenaline. The excessive activity of MAOs in nerve cells cause Parkinson’s- and Alzheimer’s diseases, dementia, depression and many other neurological disorders. MAO-B is predominantly expressed in nerve cells, but MAO-A is expressed also in the internal organs. This makes MAO-B selective inhibitors great drugs for the treatment of a variety of different neurological disorders and diseases. Some MAO-B selective inhibitors, such as pargyline, are already being used alongside the drug Levodopa to prevent the Parkinson’s disease associated excessive deamination of dopamine in the brain. This means that discovering new, effective and safe MAO-B selective inhibitors is an important subject in drug research. The aim of this study was to test the binding efficiency and inhibition efficiency of 23 new MAO-B selective inhibitors inside the binding site of MAO-B. The study was carried out by a docking simulation using computer software, and also in practice in the form of a phosphatase activity assay in the laboratory. The results showed that the inhibition efficiency of the test molecules were not on par with the commercial pargyline inhibitor. However, the results of the docking simulations and phosphatase activity assays do help in planning and executing future studies on the subject. The results also help in choosing different kinds of inhibitors for the future studies

<i>PDE3A</i> Is a Highly Expressed Therapy Target in Myxoid Liposarcoma

Liposarcomas (LPSs) are a heterogeneous group of malignancies that arise from adipose tissue. Although LPSs are among the most common soft-tissue sarcoma subtypes, precision medicine treatments are not currently available. To discover LPS-subtype-specific therapy targets, we investigated RNA sequenced transcriptomes of 131 clinical LPS tissue samples and compared the data with a transcriptome database that contained 20,218 samples from 95 healthy tissues and 106 cancerous tissue types. The identified genes were referred to the NCATS BioPlanet library with Enrichr to analyze upregulated signaling pathways. PDE3A protein expression was investigated with immunohistochemistry in 181 LPS samples, and PDE3A and SLFN12 mRNA expression with RT-qPCR were investigated in 63 LPS samples. Immunoblotting and cell viability assays were used to study LPS cell lines and their sensitivity to PDE3A modulators. We identified 97, 247, and 37 subtype-specific, highly expressed genes in dedifferentiated, myxoid, and pleomorphic LPS subtypes, respectively. Signaling pathway analysis revealed a highly activated hedgehog signaling pathway in dedifferentiated LPS, phospholipase c mediated cascade and insulin signaling in myxoid LPS, and pathways associated with cell proliferation in pleomorphic LPS. We discovered a strong association between high PDE3A expression and myxoid LPS, particularly in high-grade tumors. Moreover, myxoid LPS samples showed elevated expression levels of SLFN12 mRNA. In addition, PDE3A- and SLFN12-coexpressing LPS cell lines SA4 and GOT3 were sensitive to PDE3A modulators. Our results indicate that PDE3A modulators are promising drugs to treat myxoid LPS. Further studies are required to develop these drugs for clinical use

Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues

The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging −RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2–3 h after infection and the translation shortly after at 3–4 h post-infection. The replication hotspots with newly emerging −RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of −RNA and +RNA strands was almost identical, and −RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively.Science, Faculty ofOther UBCNon UBCMicrobiology and Immunology, Department ofReviewedFacult