mRNA Capture Sequencing and RT-qPCR for the Detection of Pathognomonic, Novel, and Secondary Fusion Transcripts in FFPE Tissue: A Sarcoma Showcase.

We assess the performance of mRNA capture sequencing to identify fusion transcripts in FFPE tissue of different sarcoma types, followed by RT-qPCR confirmation. To validate our workflow, six positive control tumors with a specific chromosomal rearrangement were analyzed using the TruSight RNA Pan-Cancer Panel. Fusion transcript calling by FusionCatcher confirmed these aberrations and enabled the identification of both fusion gene partners and breakpoints. Next, whole-transcriptome TruSeq RNA Exome sequencing was applied to 17 fusion gene-negative alveolar rhabdomyosarcoma (ARMS) or undifferentiated round cell sarcoma (URCS) tumors, for whom fluorescence in situ hybridization (FISH) did not identify the classical pathognomonic rearrangements. For six patients, a pathognomonic fusion transcript was readily detected, i.e., PAX3-FOXO1 in two ARMS patients, and EWSR1-FLI1, EWSR1-ERG, or EWSR1-NFATC2 in four URCS patients. For the 11 remaining patients, 11 newly identified fusion transcripts were confirmed by RT-qPCR, including COPS3-TOM1L2, NCOA1-DTNB, WWTR1-LINC01986, PLAA-MOB3B, AP1B1-CHEK2, and BRD4-LEUTX fusion transcripts in ARMS patients. Additionally, recurrently detected secondary fusion transcripts in patients diagnosed with EWSR1-NFATC2-positive sarcoma were confirmed (COPS4-TBC1D9, PICALM-SYTL2, SMG6-VPS53, and UBE2F-ALS2). In conclusion, this study shows that mRNA capture sequencing enhances the detection rate of pathognomonic fusions and enables the identification of novel and secondary fusion transcripts in sarcomas.

[Characterization of the problem of the therapeutic use of Cannabis Oil in Córdoba, Argentina]. / Caracterización de la problemática del uso terapéutico del Aceite de Cannabis en Córdoba, Argentina.

Introduction: The therapeutic use of the "cannabis" oil is a social problem that puts legal, health, scientific and cultural aspects under stress. Difficulty in access generates an emptiness exploited by the illegal market, to which patients and relatives resort to improve their health and quality of life. These oils, with unknown chemical composition, are used without therapeutic follow-up. An interdisciplinary team from the Universidad Nacional de Córdoba (UNC) started the study of this problem with the aim of characterizing the socio-therapeutic use of "cannabis" oil in Córdoba and establishing a relationship with the real content of cannabinoids. Methodology: Observational-descriptive and cross-sectional study approved by the Comité Institucional de Ética de las Investigaciones en Salud, Hospital Nacional de Clínicas from UNC (CIEIS-HNC-UNC): interviews with patients/caregivers of legal age who used the "cannabis" oil (year 2019). Experimental study: analysis of oil samples obtained from interviewees to determine their cannabinoid content, specifically delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), by High Performance Liquid Chromatography analysis (HPLC). Results: thirty-seven interviews were conducted, and 48 samples were analysed. The 73% were adults and older adults. The 92% started using the oil without prescription or medical suggestion, mainly due to the lack of effectiveness of other therapies (54%) and in the search for therapeutic alternatives (33%). The 84% perceived it to be effective (moderate to highly effective), and 78% reported no adverse events. Main uses: refractory epilepsy 27% and arthritis/arthrosis 24%. Fifteen percent of the samples showed no quantifiable content of CBD and THC, and 67% had only THC. The quantifiable content of cannabinoids was very low. Conclusions: This work allowed carrying out a preliminary information-gathering on several aspects (social and therapeutic) about the use of "cannabis" oil in Córdoba, and to analyze the chemical quality of the oils consumed. An important finding was the discrepancy between the effectiveness perceived by users and the low cannabinoid content detected.

Introducción: El uso terapéutico del aceite de "cannabis" es una problemática social que pone en tensión aspectos legales, sanitarios, científicos y culturales. La dificultad en el acceso genera un vacío aprovechado por el mercado ilegal, al que recurren pacientes y familiares para mejorar su salud y calidad de vida. Estos aceites, de composición química desconocida, se emplean sin un seguimiento terapéutico. Un equipo interdisciplinario de la UNC se involucró en esta problemática con el objetivo de aportar elementos para su caracterización en nuestro medio. Metodología: Estudio observacional-descriptivo y transversal (aprobado por el CIEIS-HNC-UNC): entrevistas a pacientes/cuidadores mayores de edad que usaban el aceite (2019). Estudio experimental: análisis de muestras de aceites de los entrevistados para determinar su contenido de cannabinoides (THC y CBD), mediante HPLC. Resultados: Se realizaron 37 entrevistas y analizaron 48 muestras. El 73% fueron adultos y adultos-mayores. El 79% empiezan a usar el aceite por recomendación de parientes/amigos o por iniciativa propia, principalmente por falta de efectividad de otras terapias (54%) y por búsqueda de alternativas (33%). El 84% lo percibe efectivo (moderado-muy efectivo) y el 78% no manifestó eventos adversos. Usos principales: epilepsia refractaria 27% y artritis/artrosis 24%. El 15% de las muestras no presentaron contenidos cuantificables de CBD y THC, y el 67% presentó THC sin CBD. El contenido de cannabinoides cuantificables fue muy bajo. Conclusiones: Se obtuvo una aproximación sobre el uso terapéutico del aceite de "cannabis" en Córdoba y su calidad. Se observó discrepancia entre la efectividad percibida y el bajo contenido de cannabinoides detectados.

1-((2,4-Dichlorophenethyl)Amino)-3-Phenoxypropan-2-ol Kills Pseudomonas aeruginosa through Extensive Membrane Damage.

The ever increasing multidrug-resistance of clinically important pathogens and the lack of novel antibiotics have resulted in a true antibiotic crisis where many antibiotics are no longer effective. Further complicating the treatment of bacterial infections are antibiotic-tolerant persister cells. Besides being responsible for the recalcitrant nature of chronic infections, persister cells greatly contribute to the observed antibiotic tolerance in biofilms and even facilitate the emergence of antibiotic resistance. Evidently, eradication of these persister cells could greatly improve patient outcomes and targeting persistence may provide an alternative approach in combatting chronic infections. We recently characterized 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol (SPI009), a novel anti-persister molecule capable of directly killing persisters from both Gram-negative and Gram-positive pathogens. SPI009 potentiates antibiotic activity in several in vitro and in vivo infection models and possesses promising anti-biofilm activity. Strikingly, SPI009 restores antibiotic sensitivity even in resistant strains. In this study, we investigated the mode of action of this novel compound using several parallel approaches. Genetic analyses and a macromolecular synthesis assays suggest that SPI009 acts by causing extensive membrane damage. This hypothesis was confirmed by liposome leakage assay and membrane permeability studies, demonstrating that SPI009 rapidly impairs the bacterial outer and inner membranes. Evaluation of SPI009-resistant mutants, which only could be generated under severe selection pressure, suggested a possible role for the MexCD-OprJ efflux pump. Overall, our results demonstrate the extensive membrane-damaging activity of SPI009 and confirm its clinical potential in the development of novel anti-persister therapies.

A putative de-N-acetylase of the PIG-L superfamily affects fluoroquinolone tolerance in Pseudomonas aeruginosa.

A major cause of treatment failure of infections caused by Pseudomonas aeruginosa is the presence of antibiotic-insensitive persister cells. The mechanism of persister formation in P. aeruginosa is largely unknown, and so far, only few genetic determinants have been linked to P. aeruginosa persistence. Based on a previous high-throughput screening, we here present dnpA (de-N-acetylase involved in persistence; gene locus PA14_66140/PA5002) as a new gene involved in noninherited fluoroquinolone tolerance in P. aeruginosa. Fluoroquinolone tolerance of a dnpA mutant is strongly reduced both in planktonic culture and in a biofilm model, whereas overexpression of dnpA in the wild-type strain increases the persister fraction. In addition, the susceptibility of the dnpA mutant to different classes of antibiotics is not affected. dnpA is part of the conserved LPS core oligosaccharide biosynthesis gene cluster. Based on primary sequence analysis, we predict that DnpA is a de-N-acetylase, acting on an unidentified substrate. Site-directed mutagenesis suggests that this enzymatic activity is essential for DnpA-mediated persistence. A transcriptome analysis indicates that DnpA primarily affects the expression of genes involved in surface-associated processes. We discuss the implications of these findings for future antipersister therapies targeted at chronic P. aeruginosa infections.

Integration of 'omics' data: does it lead to new insights into host-microbe interactions?

The interaction between both beneficial and pathogenic microbes and their host has been the subject of many studies. Although the field of systems biology is rapidly evolving, the use of a systems biology approach by means of high-throughput techniques to study host-microbe interactions is just beginning to be explored. In this review, we discuss some of the most recently developed high-throughput 'omics' techniques and their use in the context of host-microbe interaction. Moreover, we highlight studies combining several techniques that are pioneering the integration of 'omics' data related to host-microbe interactions. Finally, we list the major challenges ahead for successful systems biology research on host-microbe interactions.