MicroRNAs act as decoy molecules to inhibit the function of RNA binding proteins

Professional Biological Sciences: 3rd Place (The Ohio State University Edward F. Hayes Graduate Research Forum)Altered microRNA (miRNA) expression contributes to aberrant post-transcriptional gene regulation in several types of cancers; however, their role in the progression of chronic myeloid leukemia (CML) from chronic phase (CML-CP) to blast crisis (CML-BC) is still largely unknown. To gain further insight into the role of miRNAs in CML disease progression, we used microarray-based techniques to analyze miRNA expression in CML-BCCD34+ compared to CML-CPCD34+ progenitors and in BCR/ABL-expressing myeloid cell lines compared to untransformed controls. Using this method, we identified a discrete number of miRNAs either upregulated (34 miRNAs) or downregulated (14 miRNAs) in both BCR/ABL+ cell lines and primary patient samples. Among the downregulated miRNAs, we focused our attention on miR-223 because of its reported role in myelopoiesis, miR-15a/16-1 because of their reported role as tumor suppressors, and miR-328, a miRNA with no currently known function. Northern blot and qRT-PCR analyses validated the results of our microarray analysis, revealing a marked reduction in miR-223, miR-328, miR-15a, and miR-16-1 expression in 32D-BCR/ABL and K562 cells (50-75% inhibition), and expression of these miRNAs was rescued upon treatment of cells with the tyrosine kinase inhibitor imatinib (2 mM; 24h). Interestingly, sequence analysis of both miR-223 and miR-328 revealed homology with the hnRNP E2-mRNA binding site contained in the uORF spacer region of the CEBPA 5’ UTR (CEBPA 5’ uORF). hnRNP E2 is the RNA binding protein responsible for block of myeloid differentiation in CML-BCCD34+ progenitors, and does so by binding to the CEBPA 5’ uORF to block its translation. Indeed, REMSA experiments revealed that synthetic miR-223 and, to a greater extent, miR-328, could bind efficiently to recombinant hnRNP E2 protein in vitro and compete for its binding to an oligoribonucleotide containing the CEBPA 5’ uORF. Similarly, REMSA and UV-crosslinking showed that both miR-223 and miR-328 could bind endogenous hnRNP E2 from lysates of BCR/ABL-expressing but not parental cells, as well as from lysates of parental 32Dcl3 cells ectopically expressing a Flag-tagged hnRNP E2 protein. These results suggested that miR-223 and miR-328 may act as decoy molecules to interfere with the translation-regulatory activity of hnRNP E2. Consistent with this hypothesis, ectopic expression of miR-223 and, to a greater extent, miR-328, was capable of restoring G-CSF-driven granulocytic maturation of differentiation-arrested 32D-BCR/ABL cells and CML-BCCD34+ cells. Furthermore, C/EBP-alphaprotein levels were markedly increased in both miR-223- and miR-328-expressing 32D-BCR/ABL cells as well as in primary CML-BCCD34+ patient samples. Finally, while ectopic expression of miR-223, miR-328, miR-15a, or miR-16-1 had no significant effect on the viability of normal or BCR/ABL-expressing cells, ectopic expression of miR-15a/16-1 or miR-328 significantly impaired BCR/ABL-dependent colony forming ability (miR-15a/16-1: 65-75% inhibition, P<0.001; miR-328: 40-50% inhibition, P<0.01). Altogether, these data reinforce the importance of BCR/ABL-dependent post-transcriptional gene regulation during CML disease progression. Furthermore, they suggest a novel function for miRNAs as regulators of RNA binding protein activity and provide a paradigm-shift for miRNA function as currently understood.A three-year embargo was granted for this item

Combined STAT3 and BCR-ABL1 inhibition induces synthetic lethality in therapy-resistant chronic myeloid leukemia

manuscriptSupporting information to Combined STAT3 and BCR-ABL1 Inhibition Induces Synthetic Lethality in Therapy-Resistant Chronic Myeloid Leukemi

Kartlegging av aktiviteter ved sjøbaserte oppdrettsanlegg

Bacheloroppgaven er laget og utgitt av Kontali som et pilotprosjekt. Kontali foreslo denne oppgaven som et konseptbevis, og planlegger utvidelser av systemet for globalt bruk. Oppgaven har til hensikt å besvare problemstillingen: Kan man gjøre antagelser på hva slags aktivitet som foregår ved en lokasjon,ved hjelp av geografiske- og AIS-data? Analysen gjøres på offentlig tilgjengelig informasjon om posisjoner for forskjellige fartøy og maritime lokasjoner. Oppdragsgiver stilte ingen krav om bruk av spesifikke teknologiske verktøy. Oppgaven ble derfor løst med de verktøyene som tilfredstilte kravene fra problemstillingen på best måte etter gruppens mening. Rapporten begynner med en grunnleggende forklaring av prosjektets bakgrunn og grunnleggende informasjon om fiskeriindustrien. Deretter følger informasjon om valg av teknologier, språk og rammeverk valgt for å konstruere applikasjonen. Det vil også bli forklart hva slags alternativer gruppen vurderte, og hvorfor valgene ble tatt. Etter valg av utviklingsverktøy vil innsamling av data, konstruksjon av ap-plikasjonen og endelige algoritmer for aktivitets antakelser bli gjennomgått. Dette følges av en gjennomgang av hva slags revurderinger som har blitt tatt underveis, resultatene og refleksjon på prosjektets resultat mot oppgavens mål. Her vil det bli utdypet hvorfor noen av målene med oppgaven ikke ble nådd, og hva som kan gjøres for å oppnå de. Rapporten avsluttes med konklusjonen, hvor det blir reflektert over hvordan prosjektet gikk i sin helhet, og hvordan gjennomføringen kunne blitt forbedret.The bachelor thesis is made and published by Kontali as a pilot project. Kontali proposed this thesis as a concept proof, and is planning expansions of the system for global use. The thesis aims to answer the problem: Can one make assumptions about what kind of activity takes place at a location, using geographic and AIS data? The analysis is performed on publicly available information on positions for various vessels and maritime locations. The client did not require the use of specific technological tools. The task was therefore solved with the tools that satisfied the requirements given by the task in the best way possible. The report begins with a basic explanation of the project's background and basic information about the fishing industry. This is followed by information on the choice of technologies, languages ​​and frameworks selected to construct the application. It will also be explained what kind of alternatives the group considered and why the choices were made. After selecting development tools, data collection, construction of the application and final algorithms for activity assumptions are reviewed. This is followed by a review of the type of reassessments that have been taken along the way, the results and reflection on the project's result against the thesis goal. Here it will be elaborated why some of the goals of the thesis were not reached, and what can be done to achieve them. The report concludes with the conclusion, which reflects upon how the project went in its entirety, and how the implementation could have been improved

Kartlegging av aktiviteter ved sjøbaserte oppdrettsanlegg

Bacheloroppgaven er laget og utgitt av Kontali som et pilotprosjekt. Kontali foreslo denne oppgaven som et konseptbevis, og planlegger utvidelser av systemet for globalt bruk. Oppgaven har til hensikt å besvare problemstillingen: Kan man gjøre antagelser på hva slags aktivitet som foregår ved en lokasjon, ved hjelp av geografiske- og AIS-data? Analysen gjøres på offentlig tilgjengelig informasjon om posisjoner for for- skjellige fartøy og maritime lokasjoner. Oppdragsgiver stilte ingen krav om bruk av spesifikke teknologiske verktøy. Oppgaven ble derfor løst med de verktøyene som tilfredstilte kravene fra problemstillingen på best måte etter gruppens mening. Rapporten begynner med en grunnleggende forklaring av prosjektets bak- grunn og grunnleggende informasjon om fiskeriindustrien. Deretter følger informasjon om valg av teknologier, språk og rammeverk valgt for å konstruere applikasjonen. Det vil også bli forklart hva slags al- ternativer gruppen vurderte, og hvorfor valgene ble tatt. Etter valg av utviklingsverktøy vil innsamling av data, konstruksjon av ap- plikasjonen og endelige algoritmer for aktivitets antakelser bli gjennomgått. Dette følges av en gjennomgang av hva slags revurderinger som har blitt tatt underveis, resultatene og refleksjon på prosjektets resultat mot oppgavens mål. Her vil det bli utdypet hvorfor noen av målene med oppgaven ikke ble nådd, og hva som kan gjøres for å oppnå de. Rapporten avsluttes med konklusjonen, hvor det blir reflektert over hvordan prosjektet gikk i sin helhet, og hvordan gjennomføringen kunne blitt forbedret.The bachelor thesis is made and published by Kontali as a pilot project. Kontali proposed this thesis as a concept proof, and is planning expansions of the system for global use. The thesis aims to answer the problem: Can one make assumptions about what kind of activity takes place at a location, using geographic and AIS data? The analysis is performed on publicly available information on positions for various vessels and maritime locations. The client did not require the use of specific technological tools. The task was therefore solved with the tools that satisfied the requirements from the problem in the best way in the group's opinion. The report begins with a basic explanation of the project's background and basic information about the fishing industry. This is followed by information on the choice of technologies, languages ​​and frameworks selected to construct the application. It will also be explained what kind of alternatives the group considered and why the choices were made. After selecting development tools, data collection, construction of the application and final algorithms for activity assumptions are reviewed. This is followed by a review of the type of reassessments that have been taken along the way, the results and reflection on the project's result against the thesis goal. Here it will be elaborated why some of the goals of the thesis were not reached, and what can be done to achieve them. The report concludes with the conclusion, which reflects on how the project went in its entirety, and how the implementation could have been improved

Archaeological Monitoring and Test Excavations at the 1722 Presidio San Antonio de Bexar (Plaza de Armas Buildings), San Antonio, Bexar County, Texas

From April 2013 to November 2014, the Center for Archaeological Research (CAR) at The University of Texas at San Antonio (UTSA) conducted archaeological monitoring and test excavations at the site of the 1722 Presidio San Antonio de Bexar, also known in the nineteenth and twentieth centuries as the Plaza de Armas Buildings (Vogel Belt Complex) within Military Plaza in San Antonio, Bexar County, Texas. The project was performed for Ford, Powell and Carson, Architects and Planners, Inc. under contract with the City of San Antonio in anticipation of renovations and improvements to the Plaza de Armas Buildings (Vogel Belt Complex) to serve as offices and studios for the City of San Antonio. The complex is listed as contributing to the Main and Military Plaza National Register of Historic Places District, with the buildings listed individually on the National Register of Historic Places (NRHP). In addition to the above, the property is owned by the City of San Antonio. Compliance with the Antiquities Code of Texas was required. As such, the State Antiquities Code and Chapter 35 of the San Antonio Local Government Code that require coordination with the City Office of Historic Preservation and the Texas Historical Commission Divisions of Archaeology and Architecture govern the undertakings. CAR, therefore, conducted the work under Texas Antiquities Committee Permit No. 6526. Dr. Steve A. Tomka served as the Principal Investigator for the majority of the fieldwork, the initial analysis, and the description of materials collected. Kristi Nichols served as the Project Archaeologist during this initial monitoring and testing, assisted by Lindy Martinez. Both Dr. Tomka and Ms. Nichols left UTSA in 2014, and Dr. Raymond Mauldin assumed the Principal Investigator role for the project. Clinton McKenzie and Leonard Kemp were the Project Archaeologists for the final phases of monitoring, as well as for assembling the final report. Leonard Kemp oversaw additional test excavation. Trinomial 41BX2088 was assigned to the location. Principal activities during the project included monitoring trenches on the complex’s exterior, monitoring soil removal in sections of the interior, and hand excavations of a series of units in the basement. These basement excavations produced a variety of materials. CAR staff documented eight features, including several trash pits, recovered a variety of Spanish Colonial, Native American, and European/English ceramics, along with faunal material, chipped stone tools and debitage, and construction related items. It was concluded that much of this material was intact, and that additional features and midden deposits are present. The project provides direct evidence of materials associated with the Presidio de Bexar, built by the Spanish at this general location in 1722, as well as occupation in this area through the early twentieth century. CAR recommends that prior to any impacts in the basements, or any external impacts greater than 2.0 m in depth at the rear of the Plaza de Armas Buildings (Vogel Belt Complex), a comprehensive, systematic effort to recover significant data be initiated

Archaeological Investigations within San Pedro Springs Park (41BX19), San Antonio, Bexar County, Texas

The University of Texas at San Antonio Center for Archaeological Research (UTSA-CAR) contracted with Adams Environmental, Inc. to provide archaeological services to Capital Improvement Management (CIMS) of the City of San Antonio (COSA) related to the archaeological investigation of selected areas of San Pedro Springs Park in San Antonio, Bexar County, Texas. The CAR conducted archaeological testing at this National Register Site, 41BX19, from early December 2013 to mid-January of 2014. The goals of archaeological investigations were to identify and investigate any proto-historic and historic archaeological deposits associated with Colonial Period occupants of the area, including evidence of the first acequia and associated dam, and the location of the first presidio and villa. In addition, CAR was tasked with the investigation of any prehistoric cultural deposits encountered. This project was performed by staff archaeologists from the CAR. It was conducted under Texas Antiquities Permit No. 6727, with Dr. Steve Tomka serving as Principal Investigator (PI), and Kristi Nichols and Stephen Smith serving as Project Archaeologists. Dr. Tomka departed from UTSA shortly after the completion of fieldwork. At that time, Dr. Raymond Mauldin of CAR assumed PI responsibilities for the project. One hundred and eleven shovel tests, eleven 1-x-1 m test units, two 50-x-50 cm units, two backhoe trenches, and several auger holes were excavated during this effort. Minimal artifactual evidence of colonial occupants was noted during the archaeological investigations. Several Native American bone tempered sherds that could reflect either Late Prehistoric Leon Plain or Goliad ware were recovered. However, no Spanish Majolicas or lead glazed wares were uncovered, and no gunflints were identified in the lithic assemblage. Due to various utility lines and other obstructions, backhoe trenches to search for the acequia and associated dam could not be excavated. It is likely that areas proposed for investigation of the acequia and associated dam have been disturbed by aforementioned utility lines as well as earlier construction within the park. No evidence of the specific location of the first presidio or villa was located. Shovel testing and test units revealed the presence of historic and prehistoric use of the park, though mixing of historic and prehistoric material, as well as other disturbances (e.g., rodents), was common in the deposits. However, there was an increase in prehistoric material with depth as revealed in shovel testing results. Shovel testing located Feature 1, a burned rock feature that possibly was associated with a sheet midden, as well as several areas with high densities of prehistoric materials. Test excavations, based on these shovel tests, suggest that Feature 1 is a discrete feature that lies below a widespread, low-density distribution of burned rock. Shovel testing also identified a high-density cluster of lithic, bone, and burned rock. The excavation of a 1-x-1 m test unit (TU 4) in this area produced over 4,000 pieces of debitage, with over 50% of this total coming from three levels. Burned rock, a variety of tools, faunal material, and charcoal were present throughout these levels. Temporal placement of deposits relied on artifact typologies (e.g., ceramic types, lithic projectile points, lithic tool types) as well as two charcoal and four bone collagen radiocarbon dates. Artifact typologies suggest occupation as early as the Early Archaic as reflected by a possible Guadalupe tool. A series of Late Archaic Points (Castroville, Frio, Marcos, and Montell) and Late Prehistoric point forms (Edwards, Perdiz, and Scallorn) are present from several areas. In addition, a possible Middle Archaic La Jita point was recovered. The bone tempered Native American wares could date as early as AD 1250, though they could also reflect proto-historic or colonial age materials. Other ceramics primarily suggest a mid-nineteenth- to midtwentieth-century occupation. Using the midpoints of the 1-sigma distribution, calibrated radiocarbon dates show use of San Pedro Park from as early as 100 AD (CAR 345; 1905 +/- 22 Radiocarbon Years Before Present [RCYBP]) to as recently as the early twentieth century. The more recent end of that range is a function of two late dates from two different areas of the park. The first of these is on a bison bone (CAR 344) that returned a date of 158 +/- 23 RCYBP. The second is on a bone consistent with a bison-sized animal (CAR 346) that produced a date of 155 +/- 23 RCYBP. The corrected, calibrated dates for these two samples range from AD 1670 to the early 1940s using the 1-sigma spread. The wide range of these dates is related to the flat calibration curve late in time. However, the most probable date range (ca. 36% probability) for these two dates is between AD 1729 and 1779, with a roughly 48% probability that they date prior to AD 1779. Limited testing suggests that, with a few specific exceptions, the upper 30-40 cm of San Pedro Park is extensively disturbed. However, though some disturbances are present, at least three areas have materials in what appears to be good context. These include material dating to the Late Archaic, Late Prehistoric, and possibly the Proto-historic or Colonial Period. Based on historic maps, previous work, and the current investigation, CAR proposes a series of management areas for San Pedro Park. If work in these management areas follows these suggestions for various limits on subsurface impacts, CAR recommends that. renovation activities within the park be allowed to proceed. The Texas Historical Commission (THC), in a letter dated February 4, 2015, agreed with these recommendations. Finally, CAR provides several recommendations for public education facilities within the park. In accordance with the THC Permit specifications and the Scope of Work for this project, all field notes, analytical notes, photographs, and other project related documents, along with a copy of the final report, will be curated at the CAR. After quantification and completion of analysis, and in consultation with THC and the COSA Office of Historic Preservation, artifacts possessing little scientific value were discarded pursuant to Chapter 26.27(g)(2) of the Antiquities Code of Texas. Artifact classes discarded specific to this project included samples of burned rock and snail shell, all unidentifiable metal, soil samples, and recent (post-1950) material

MicroRNAs: the primary cause or a determinant of progression in leukemia?

available in PMC 2011 October 10.Leukemia is a complex disease with many different types and subtypes caused by a huge diversity of genetic and epigenetic aberrations. Until recently, alterations of protein-coding genes were thought to be the sole cause of tumorigenesis. With the recent discovery of multiple types of non-coding RNAs, it has become evident that mutations in these also contribute to the development of cancer. Among the non-coding RNAs, microRNAs play a crucial role in cancer owing to their involvement in fundamental processes such as apoptosis, differentiation and proliferation. MicroRNAs are small noncoding RNAs (approximately 19–25 nucleotides in length) that bind to and downregulate multiple mRNA targets; in mammals, the production of over a third of all proteins is regulated by microRNAs [3]. Several studies demonstrated that microRNAs are involved in leukemia progression but their role as the primary cause or a determinant of progression in leukemia has been unclear. Some have been identified as oncogenes or tumor suppressor genes, which suggests that they are playing a central role in tumorigenesis, while others appear to be associated with a specific stage in disease progression. Deciphering the exact role of microRNAs in oncogenesis is important in order to improve the diagnosis and treatment of leukemia patients.National Institutes of Health (U.S.) (NIH grant DK068348)National Institutes of Health (U.S.) (NIH Grant 5P01 HL066105)Leukemia & Lymphoma Society of America (Recherche sur le Cancer (ARC) fellowship

Platelet lamellipodium formation is not required for thrombus formation and stability

During platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. Controversial data have been published on the role of lamellipodia in thrombus formation and stability. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex, which has been shown in other cells to drive lamellipodium formation by enhancing actin nucleation via the actin-related protein 2/3 (Arp2/3) complex, is activated by Ras-related C3 botulinum toxin substrate 1 (Rac1) interaction with the WAVE complex subunit cytoplasmic fragile X mental retardation 1–interacting protein 1 (Cyfip1). We analyzed Cyfip1flox/floxPf4-Cre mice to investigate the role of Cyfip1 in platelet function. These mice displayed normal platelet counts and a slight reduction in platelet volume. Activation of mutant platelets was only moderately reduced to all tested agonists as measured by αIIbβ3 integrin activation and P-selectin surface exposure. However, lamellipodium formation of mutant platelets was completely abolished on different matrices. Nevertheless, Cyfip1-deficient platelets formed stable thrombi on collagen fibers ex vivo and in 2 models of occlusive arterial thrombosis in vivo. Similarly, the hemostatic function and maintenance of vascular integrity during inflammation of the skin and lung were unaltered in the mutant mice. Investigation of platelet morphology in an induced thrombus under flow revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodium-like structures when in direct contact to collagen fibers at the bottom of the thrombus. We provide for the first time direct evidence that platelet lamellipodium formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow

La enhances IRES-mediated translation of laminin B1 during malignant epithelial to mesenchymal transition

The majority of transcripts that harbor an internal ribosome entry site (IRES) are involved in cancer development via corresponding proteins. A crucial event in tumor progression referred to as epithelial to mesenchymal transition (EMT) allows carcinoma cells to acquire invasive properties. The translational activation of the extracellular matrix component laminin B1 (LamB1) during EMT has been recently reported suggesting an IRES-mediated mechanism. In this study, the IRES activity of LamB1 was determined by independent bicistronic reporter assays. Strong evidences exclude an impact of cryptic promoter or splice sites on IRES-driven translation of LamB1. Furthermore, no other LamB1 mRNA species arising from alternative transcription start sites or polyadenylation signals were detected that account for its translational control. Mapping of the LamB1 5′-untranslated region (UTR) revealed the minimal LamB1 IRES motif between −293 and −1 upstream of the start codon. Notably, RNA affinity purification showed that the La protein interacts with the LamB1 IRES. This interaction and its regulation during EMT were confirmed by ribonucleoprotein immunoprecipitation. In addition, La was able to positively modulate LamB1 IRES translation. In summary, these data indicate that the LamB1 IRES is activated by binding to La which leads to translational upregulation during hepatocellular EMT

Advances in the treatment of chronic myeloid leukemia

Although imatinib is firmly established as an effective therapy for newly diagnosed patients with chronic myeloid leukemia (CML), the field continues to advance on several fronts. In this minireview we cover recent results of second generation tyrosine kinase inhibitors in newly diagnosed patients, investigate the state of strategies to discontinue therapy and report on new small molecule inhibitors to tackle resistant disease, focusing on agents that target the T315I mutant of BCR-ABL. As a result of these advances, standard of care in frontline therapy has started to gravitate toward dasatinib and nilotinib, although more observation is needed to fully support this. Stopping therapy altogether remains a matter of clinical trials, and more must be learned about the mechanisms underlying the persistence of leukemic cells with treatment. However, there is good news for patients with the T315I mutation, as effective drugs such as ponatinib are on their way to regulatory approval. Despite these promising data, accelerated or blastic phase disease remains a challenge, possibly due to BCR-ABL-independent resistance