Functional characterization of the sciarid BhC4-1 core promoter in transgenic Drosophila

<p>Abstract</p> <p>Background</p> <p>Core promoters are <it>cis</it>-regulatory modules to which bind the basal transcriptional machinery and which participate in the regulation of transcription initiation. Although core promoters have not been extensively investigated through functional assays in a chromosomal context, the available data suggested that the response of a given core promoter might vary depending on the promoter context. Previous studies suggest that a (-57/+40) fragment constitutes the core promoter of the <it>BhC4-1 </it>gene which is located in DNA puff C4 of the sciarid fly <it>Bradysia hygida</it>. Here we tested this (-57/+40) fragment in distinct regulatory contexts in order to verify if promoter context affects its core promoter activity.</p> <p>Results</p> <p>Consistent with the activity of a core promoter, we showed that in the absence of upstream regulatory sequences the (-57/+40) fragment drives low levels of reporter gene mRNA expression throughout development in transgenic <it>Drosophila</it>. By assaying the (-57/+40) fragment in two distinct regulatory contexts, either downstream of the previously characterized <it>Fbp1 </it>enhancer or downstream of the UAS element, we showed that the <it>BhC4-1 </it>core promoter drives regulated transcription in both the germline and in various tissues throughout development. Furthermore, the use of the <it>BhC4-1 </it>core promoter in a UAS construct significantly reduced salivary gland ectopic expression in third instar larvae, which was previously described to occur in the context of the GAL4/UAS system.</p> <p>Conclusions</p> <p>Our results from functional analysis in transgenic <it>Drosophila </it>show that the <it>BhC4-1 </it>core promoter drives gene expression regardless of the promoter context that was assayed. New insights into the functioning of the GAL4/UAS system in <it>Drosophila </it>were obtained, indicating that the presence of the SV40 sequence in the 3' UTR of a UAS construct does not preclude expression in the germline. Furthermore, our analysis indicated that ectopic salivary gland expression in the GAL4/UAS system does not depend only on sequences present in the GAL4 construct, but can also be affected by the core promoter sequences in the UAS construct. In this context, we propose that the sciarid <it>BhC4-1 </it>core promoter constitutes a valuable core promoter which can be employed in functional assays in insects.</p

Immunohistochemical approach to the pathogenesis of clinical cases of Bovine Herpesvirus type 5 infections

Meningoencephalitis by Herpesvirus type 5 (BoHV-5) in cattle has some features that are similar to those of herpetic encephalitis in humans and other animal species. Human Herpesvirus 3 (commonly known as Varicella-zoster virus 1), herpes simplex viruses (HSV), and equid Herpesvirus 1 (EHV-1) induce an intense inflammatory, vascular and cellular response. In spite of the many reports describing the histological lesions associated with natural and experimental infections, the immunopathological mechanisms for the development of neurological disorder have not been established. A total of twenty calf brains were selected from the Veterinary School, University of São Paulo State, Araçatuba, Brazil, after confirmation of BoHV-5 infection by virus isolation as well as by a molecular approach. The first part of the study characterized the microscopic lesions associated with the brain areas in the central nervous system (CNS) that tested positive in a viral US9 gene hybridization assay. The frontal cortex (Fc), parietal cortex (Pc), thalamus (T) and mesencephalon (M) were studied. Secondly, distinct pathogenesis mechanisms that take place in acute cases were investigated by an immunohistochemistry assay. This study found the frontal cortex to be the main region where intense oxidative stress phenomena (AOP-1) and synaptic protein expression (SNAP-25) were closely related to inflammatory cuffs, satellitosis and gliosis, which represent the most frequently observed neurological lesions. Moreover, MMP-9 expression was shown to be localized in the leptomeninges, in the parenchyma and around mononuclear infiltrates (p < 0.0001). These data open a new perspective in understanding the role of the AOP-1, MMP-9 and SNAP-25 proteins in mediating BoHV-5 pathogenesis and the strategies of host-virus interaction in order to invade de CNS

Photon-number-resolving segmented avalanche-photodiode detectors

We investigate the feasibility and performance of photon-number-resolved photodetection employing avalanche photodiodes (APDs) with low dark counts. The main idea is to split n photons over m modes such that every mode has no more than one photon, which is detected alongside propagation by an APD. We characterize performance by evaluating the purities of positive-operator-valued measurements (POVMs), in terms of APD number and photon loss.Comment: 5 pages, 7 figures, submitted for publicatio

Chronic Pancreatitis and Systemic Lupus Erythematosus: An Uncommon Association

The association between systemic lupus erythematosus (SLE) and chronic pancreatitis (CP) is extremely rare. Up to now, only six cases have been reported. We report the case of a young woman who presented her first episode of abdominal pain and hyperamylasemia at the onset of SLE and developed chronic calcifying pancreatitis after a two year period

Expanding our knowledge on African trypanosomes of the subgenus <i>Pycnomonas</i>:A novel Trypanosoma suis-like in tsetse flies, livestock and wild ruminants sympatric with Trypanosoma suis in Mozambique

Among the subgenera of African tsetse-transmitted trypanosomes pathogenic to livestock, the least known is the subgenus Pycnomonas, which contains a single species, Trypanosoma suis (TSU), a pathogen of domestic pigs first reported in 1905 and recently rediscovered in Tanzania and Mozambique. Analysis by Fluorescent Fragment Length Barcoding (FFLB) revealed an infection rate of 20.3% (108 out of 530 tsetse flies) in a recent study in the Gorongosa and Niassa wildlife reserves in Mozambique, and demonstrated two groups of Pycnomonas trypanosomes: one (14.1%, 75 flies) showing an FFLB profile identical to the reference TSU from Tanzania, and the other (6.2%, 33 flies) differing slightly from reference TSU and designated Trypanosoma suis-like (TSU-L). Phylogenetic analyses tightly clustered TSU and TSU-L from Mozambique with TSU from Tanzania forming the clade Pycnomonas positioned between the subgenera Trypanozoon and Nannomonas. Our preliminarily exploration of host ranges of Pycnomonas trypanosomes revealed TSU exclusively in warthogs while TSU-L was identified, for the first time for a member of the subgenus Pycnomonas, in ruminants (antelopes, Cape buffalo, and in domestic cattle and goats). The preferential blood meal sources of tsetse flies harbouring TSU and TSU-L were wild suids, and most of these flies concomitantly harboured the porcine trypanosomes T. simiae, T. simiae Tsavo, and T. godfreyi. Therefore, our findings support the link of TSU with suids while TSU-L remains to be comprehensively investigated in these hosts. Our results greatly expand our knowledge of the diversity, hosts, vectors, and epidemiology of Pycnomonas trypanosomes. Due to shortcomings of available molecular diagnostic methods, a relevant cohort of trypanosomes transmitted by tsetse flies to ungulates, especially suids, has been neglected or most likely misidentified. The method employed in the present study enables an accurate discrimination of trypanosome species and genotypes and, hence, a re-evaluation of the “lost” subgenus Pycnomonas and of porcine trypanosomes in general, the most neglected group of African trypanosomes pathogenic to ungulates.Brazilian grants from the PROAFRICA program (CNPq) and FAPESP (Process no 2016/07487-0). CMFR and HAG are postdoctoral fellows of CNPq (INCT-EpiAmo) and FAPESP (Process no 2016./03028-1)http://www.elsevier.com/locate/meegidhj2020Veterinary Tropical Disease

Cambios históricos en el aporte terrígeno de la cuenca del Río de la Plata sobre la plataforma interna Uruguaya

El Río de la Plata (RdlP) presenta significativas variaciones naturales (hidrodinámicas y oceanográficas) asociadas a diferentes condiciones climáticas. El propósito de este trabajo es inferir los cambios de aportes continentales de sedimentos y su relación con las variaciones hidrológicas del Río de la Plata, a través del análisis de proxies sedimentológicos y geoquímicos en testigos de sedimentos de la plataforma interna uruguaya que registran los últimos 100 años, aproximadamente. A partir de ladatación por 210Pb de dos testigos de sedimentos (GeoB 13813-4 y BAR1) se reconstruyó la geocronología del ambiente, y se relacionó con datos de las forzantes climáticas Pacific Decadal Oscillation, El Niño/La Niña Southern Oscillation, Atlantic MultidecadalOscillation, y las anomalías hidrológicas de los ríos Paraná y Uruguay. Los valores máspositivos y estables del Southern Oscillation Index, los cuales corresponden a fases La Niña,se observan en el periodo correspondiente entre 1910-1970, respecto al resto de la serie,donde se aprecia una mayor variabilidad y una tendencia hacia valores más negativos(eventos El Niño). Se hicieron dendrogramas (clustering) jerárquicos para ambos testigos. Para el testigo GeoB 13813-4, se utilizó la relación Ca/Ti y la granulometría, mientras que para BAR1 se recurrió a variables granulométricas y la tasa de sedimentación. El mayoraporte continental hacia la región de la plataforma adyacente al Río de la Plata registrado a partir del año 1970, podría ser el factor principal de los agrupamientos observados en los clusters para ambos testigos. Las agrupaciones mostraron una diferenciación en la década de 1970 lo que estaría asociado al aumento de los caudales de los ríos Paraná y Uruguay, durante las últimas tres décadas del siglo XX. Por otra parte se observa que la granulometría del testigo BAR1 presentó un mayor tamaño de grano y más variabilidad que en el caso del testigo GeoB 13813-4. También se determinó una mayor acumulación de sedimentos a través del tiempo en el cinturón de barro del Río de la Plata (plataforma continental adyacente), comparado con aquel registrado en la Barra del Indio (límite entre zona intermedia y externa del estuario). Estas diferencias podrían estar relacionadas con la influencia del Río de la Plata la cual genera un ambiente altamente dinámico sobre la Barra del Indio y un ambiente más estable sobre el cinturón de barro en la plataforma continental.Fil: Marrero, Analía. Universidad de la República. Facultad de Ciencias; UruguayFil: Tudurí, Adriana. Universidad de la República. Facultad de Ciencias; UruguayFil: Perez, Laura. Universidad de la República. Facultad de Ciencias; UruguayFil: Cuña Rodriguez, Carolina Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Ciencias de la Tierra. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Centro de Investigaciones en Ciencias de la Tierra; ArgentinaFil: Muniz, Pablo. Universidad de la República. Facultad de Ciencias; UruguayFil: Lopes Figueira, Rubens C.. Instituto Oceanografico; BrasilFil: Michcelovitch de Mahiques, Michel. Instituto Oceanografico; BrasilFil: Alves de Lima Ferreira, Paulo. Instituto Oceanografico; BrasilFil: Pittauerová, Daniela. Center For Marine Environmental Sciences, Marum; AlemaniaFil: Hanebuth, Till. Center For Marine Environmental Sciences, Marum; AlemaniaFil: Garcia Rodriguez, Felipe. Universidad de la República. Facultad de Ciencias; Urugua

Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate

Natural and synthetic phenazines are widely used in biomedical sciences. In dehydrogenase histochemistry, phenazine methosulfate (PMS) is applied as a redox reagent for coupling reduced coenzymes to the reduction of tetrazolium salts into colored formazans. PMS is also currently used for cytotoxicity and viability assays of cell cultures using sulfonated tetrazoliums. Under UV (340 nm) excitation, aqueous solutions of the cationic PMS show green fluorescence (λem: 526 nm), whereas the reduced hydrophobic derivative (methyl-phenazine, MPH) shows blue fluorescence (λem: 465 nm). Under UV (365 nm) excitation, cultured cells (LM2, IGROV-1, BGC-1, and 3T3-L1 adipocytes) treated with PMS (5 μg/mL, 30 min) showed cytoplasmic granules with bright blue fluorescence, which correspond to lipid droplets labeled by the lipophilic methyl-phenazine. After formaldehyde fixation blue-fluorescing droplets could be stained with oil red O. Interestingly, PMS-treated 3T3-L1 adipocytes observed under UV excitation 24 h after labeling showed large lipid droplets with a weak green emission within a diffuse pale blue-fluorescing cytoplasm, whereas a strong green emission was observed in small lipid droplets. This fluorescence change from blue to green indicates that reoxidation of methyl-phenazine to PMS can occur. Regarding cell uptake and labeling mechanisms, QSAR models predict that the hydrophilic PMS is not significantly membrane-permeant, so most PMS reduction is expected to be extracellular and associated with a plasma membrane NAD(P)H reductase. Once formed, the lipophilic and blue-fluorescing methyl-phenazine enters live cells and mainly accumulates in lipid droplets. Overall, the results reported here indicate that PMS is an excellent fluorescent probe to investigate labeling and redox dynamics of lipid droplets in cultured cells.Fil: Stockert, Juan C.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; ArgentinaFil: Carou, María Clara. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; ArgentinaFil: Casas, Adriana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; ArgentinaFil: Garcia Vior, María Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; ArgentinaFil: Ezquerra Riega, Sergio Dario. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Blanco, María M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; ArgentinaFil: Espada, Jesús. Universidad Bernardo O'Higgins; ChileFil: Blázquez Castro, Alfonso. Universidad Autónoma de Madrid. Facultad de Ciencias. Departamento de Biología; EspañaFil: Horobin, Richard W.. University of Glasgow; Reino UnidoFil: Lombardo, Daniel Marcelo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentin

Discovery and targeting of a noncanonical mechanism of sarcoma resistance to ADI-PEG20 mediated by the microenvironment

PURPOSE: Many cancers lack argininosuccinate synthetase 1 (ASS1), the rate-limiting enzyme of arginine biosynthesis. This deficiency causes arginine auxotrophy, targetable by extracellular arginine-degrading enzymes such as ADI-PEG20. Long-term tumor resistance has thus far been attributed solely to ASS1 reexpression. This study examines the role of ASS1 silencing on tumor growth and initiation and identifies a noncanonical mechanism of resistance, aiming to improve clinical responses to ADI-PEG20. EXPERIMENTAL DESIGN: Tumor initiation and growth rates were measured for a spontaneous Ass1 knockout (KO) murine sarcoma model. Tumor cell lines were generated, and resistance to arginine deprivation therapy was studied in vitro and in vivo. RESULTS: Conditional Ass1 KO affected neither tumor initiation nor growth rates in a sarcoma model, contradicting the prevalent idea that ASS1 silencing confers a proliferative advantage. Ass1 KO cells grew robustly through arginine starvation in vivo, while ADI-PEG20 remained completely lethal in vitro, evidence that pointed toward a novel mechanism of resistance mediated by the microenvironment. Coculture with Ass1-competent fibroblasts rescued growth through macropinocytosis of vesicles and/or cell fragments, followed by recycling of protein-bound arginine through autophagy/lysosomal degradation. Inhibition of either macropinocytosis or autophagy/lysosomal degradation abrogated this growth support effect in vitro and in vivo. CONCLUSIONS: Noncanonical, ASS1-independent tumor resistance to ADI-PEG20 is driven by the microenvironment. This mechanism can be targeted by either the macropinocytosis inhibitor imipramine or the autophagy inhibitor chloroquine. These safe, widely available drugs should be added to current clinical trials to overcome microenvironmental arginine support of tumors and improve patient outcomes

KIAA1549: BRAF gene fusion and FGFR1 hotspot mutations are prognostic factors in pilocytic astrocytomas

Up to 20% of patients with pilocytic astrocytoma (PA) experience a poor outcome. BRAF alterations and Fibroblast growth factor receptor 1 (FGFR1) point mutations are key molecular alterations in Pas, but their clinical implications are not established. We aimed to determine the frequency and prognostic role of these alterations in a cohort of 69 patients with PAs. We assessed KIAA1549:BRAF fusion by fluorescence in situ hybridization and BRAF (exon 15) mutations by capillary sequencing. In addition, FGFR1 expression was analyzed using immunohistochemistry, and this was compared with gene amplification and hotspot mutations (exons 12 and 14) assessed by fluorescence in situ hybridization and capillary sequencing. KIAA1549:BRAF fusion was identified in almost 60% of cases. Two tumors harbored mutated BRAF. Despite high FGFR1 expression overall, no cases had FGFR1 amplifications. Three cases harbored a FGFR1 p.K656E point mutation. No correlation was observed between BRAF and FGFR1 alterations. The cases were predominantly pediatric (87%), and no statistical differences were observed in molecular alterations-related patient ages. In summary, we confirmed the high frequency of KIAA1549:BRAF fusion in PAs and its association with a better outcome. Oncogenic mutations of FGFR1, although rare, occurred in a subset of patients with worse outcome. These molecular alterations may constitute alternative targets for novel clinical approaches, when radical surgical resection is unachievable.This study was partially supported by CNPq/Universal (475358/2011-2), and FAPESP (2012/19590-0) grants to RMR and to the NIH- P30CA046934 (CCSG Molecular Pathology/Cytogenetics) to MVG and DL