Coronavirus mRNA synthesis: identification of novel transcription initiation signals which are differentially regulated by different leader sequences.

The mRNA synthesis of mouse hepatitis virus (MHV) has been proposed to be the result of interaction between the leader RNA and the intergenic sites. Previously, we have identified a transcription initiation site (for mRNA 2-1), which is more efficiently transcribed by viruses containing two copies of UCUAA sequence in the leader RNA than by those with three copies. In this study, we have identified several sites which are regulated in the opposite way, namely, they are efficiently transcribed by the leader RNA with three UCUAA copies but not by those with two copies. These sites were characterized by primer extension and amplification by polymerase chain reaction. One of these sites is in the gene 3 region of a recombinant virus between A59 and JHM strains of MHV. Another is in the gene 2 region of MHV-1 strain. Both of these sites have a sequence similar to but different from the consensus transcription initiation signal (UCUAAUCUAUC and UUUAAUCUU, as opposed to UCUAAAC). These two novel intergenic sequences are not present in the genome of the JHM strain, consistent with the absence of these mRNAs in the JHM-infected cells. The discovery of this type of transcription initiation site provides additional evidence for the importance of the leader RNA in the transcription initiation of MHV mRNAs

Biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus.

We have previously shown that gp65 (E3) is a virion structural protein which varies widely in quantity among different strains of mouse hepatitis virus (MHV). In this study, the biosynthetic pathway and possible biological activities of this protein were examined. The glycosylation of gp65 in virus-infected cells was inhibited by tunicamycin but not by monensin, suggesting that it contains an N-glycosidic linkage. Glycosylation is cotranslational and appears to be complete before the glycoprotein reaches the Golgi complex. Pulse-chase experiments showed that this protein decreased in size after 30 min of chase, suggesting that the carbohydrate chains of gp65 undergo trimming during its transport across the Golgi. This interpretation is supported by the endoglycosidase treatment of gp65, which showed that the peptide backbone of gp65 did not decrease in size after pulse-chase periods. This maturation pathway is distinct from that of the E1 or E2 glycoproteins. Partial endoglycosidase treatment indicated that gp65 contains 9 to 10 carbohydrate side chains; thus, almost all of the potential glycosylation sites of gp65 were glycosylated. In vitro translation studies coupled with protease digestion suggest that gp65 is an integral membrane protein. The presence of gp65 in the virion is correlated with the presence of an acetylesterase activity. No hemagglutinin activity was detected

Complexity of qualitative timeline-based planning

The timeline-based approach to automated planning was originally developed in the context of space missions. In this approach, problem domains are expressed as systems consisting of independent but interacting components whose behaviors over time, the timelines, are governed by a set of temporal constraints, called synchronization rules. Although timeline-based system descriptions have been successfully used in practice for decades, the research on the theoretical aspects only started recently. In the last few years, some interesting results have been shown concerning both its expressive power and the computational complexity of the related planning problem. In particular, the general problem has been proved to be EXPSPACE-complete. Given the applicability of the approach in many practical scenarios, it is thus natural to ask whether computationally simpler but still expressive fragments can be identified. In this paper, we study the timeline-based planning problem with the restriction that only qualitative synchronization rules, i.e., rules without explicit time bounds in the constraints, are allowed. We show that the problem becomes PSPACE-complete

Attractive Interactions Between Rod-like Polyelectrolytes: Polarization, Crystallization, and Packing

We study the attractive interactions between rod-like charged polymers in solution that appear in the presence of multi-valence counterions. The counterions condensed to the rods exhibit both a strong transversal polarization and a longitudinal crystalline arrangement. At short distances between the rods, the fraction of condensed counterions increases, and the majority of these occupy the region between the rods, where they minimize their repulsive interactions by arranging themselves into packing structures. The attractive interaction is strongest for multivalent counterions. Our model takes into account the hard-core volume of the condensed counterions and their angular distribution around the rods. The hard core constraint strongly suppresses longitudinal charge fluctuations.Comment: 4 figures, uses revtex, psfig and epsf. The new version contains a different introduction, and the bibliography has been expande

Retinal Ganglion Cells with Distinct Directional Preferences Differ in Molecular Identity, Structure, and Central Projections

The retina contains ganglion cells (RGCs) that respond selectively to objects moving in particular directions. Individual members of a group of ON-OFF direction-selective RGCs (ooDSGCs) detect stimuli moving in one of four directions: ventral, dorsal, nasal, or temporal. Despite this physiological diversity, little is known about subtype-specific differences in structure, molecular identity, and projections. To seek such differences, we characterized mouse transgenic lines that selectively mark ooDSGCs preferring ventral or nasal motion as well as a line that marks both ventral- and dorsal-preferring subsets. We then used the lines to identify cell surface molecules, including Cadherin 6, CollagenXXVα1, and Matrix metalloprotease 17, that are selectively expressed by distinct subsets of ooDSGCs. We also identify a neuropeptide, CART (cocaine- and amphetamine-regulated transcript), that distinguishes all ooDSGCs from other RGCs. Together, this panel of endogenous and transgenic markers distinguishes the four ooDSGC subsets. Patterns of molecular diversification occur before eye opening and are therefore experience independent. They may help to explain how the four subsets obtain distinct inputs. We also demonstrate differences among subsets in their dendritic patterns within the retina and their axonal projections to the brain. Differences in projections indicate that information about motion in different directions is sent to different destinations

Small Cell Lung Cancer Transformation as a Resistance Mechanism to Osimertinib in Epidermal Growth Factor Receptor-Mutated Lung Adenocarcinoma: Case Report and Literature Review

Introduction: Small cell lung cancer (SCLC) transformation represents a mechanism of resistance to osimertinib in EGFR-mutated lung adenocarcinoma, which dramatically impacts patients' prognosis due to high refractoriness to conventional treatments. Case Description: We present the case of a patient who developed a SCLC phenotypic transformation as resistance mechanism to second-line osimertinib for T790M-positive EGFR-mutated NSCLC. Our patient received platinum–etoposide doublet following SCLC switch and achieved a modest clinical benefit which lasted 4 months. NGS and IHC analyses for p53 and Rb were performed on subsequent liver biopsies, revealing baseline TP53 mutation and complete absence of p53 and Rb expression. Primary cell cultures were established following a liver biopsy at the time of SCLC transformation, and drug sensitivity assays showed meaningful cell growth inhibition when osimertinib was added to platinum–etoposide compared with control (p < 0.05). A review of the current literature regarding SCLC transformation after failure of osimertinib was performed. Conclusions: Based on retrospective data available to date, platinum–etoposide chemotherapy is the preferred treatment choice in the occurrence of SCLC transformation after osimertinib failure. The extension of osimertinib in combination with chemotherapy in the occurrence of SCLC transformation as resistance mechanism to osimertinib is a matter of debate. The combination of osimertinib and platinum–etoposide was effective in inhibiting cell growth in our primary cell cultures. Clinical studies are needed to further explore this combination in the occurrence of SCLC transformation as a resistance mechanism to osimertinib

Analysis of some physical-chemical and biological water parameters in two thermal seasons at Ensenada stream, Diamante, Entre Ríos

Las aguas superficiales continentales son fácilmente contaminadas por lo que es conveniente conocer su estado ambiental para detectar las agresiones al sistema y brindar conocimiento para su preservación. El río Paraná, uno de los principales componentes del la Cuenca del Plata, recibe los aportes de numerosos ríos y arroyos a lo largo de su curso. En su tramo medio, y sobre la margen izquierda, desemboca el arroyo de la Ensenada ubicado en el Departamento Diamante de la provincia de Entre Ríos. Este trabajo tiene como objetivo ampliar la información acerca del estado de las aguas de la cuenca del citado arroyo para lo que se realizaron muestreos en dos estaciones del año 2010 con marcadas diferencias térmicas. Se efectuaron muestreos en nueve sitios del curso y tributarios, y se analizaron las muestras, por métodos estandarizados, en sus parámetros físicos-químicos y microbiológicos. Los resultados mostraron que los parámetros físico-químicos como pH, CE y clorofila “a” se encuentran en rangos comparable con otros cursos de la región, no así el fosforo que muestra altos valores en su curso principal, lo que señala los efectos de las actividades antrópicas en la cuenca. Los altos valores de recuento en coliformes totales y termotolerantes, que mayormente fueron encontrados en los tributarios, superan los límites establecidos para aguas recreativas. El análisis de conglomerados aplicado al conjunto de parámetros físico-químicos mostró la formación de cinco clúster en las dos estaciones climáticas, destacándose el conformado por dos tributarios que se caracterizaron por presentar altos contenidos de fósforo. El análisis de la comunidad de cianobacterias mostró la presencia de Microcystis capaz de afectar la calidad recreacional y de consumo del agua, con valores no significativos de cianotoxinas.Inland surface waters are easily contaminated so it is convenient to know the environmental status to detect aggression and provide knowledge for preservation. The Parana River, one of the main components of the River Plate Basin, receives input from numerous rivers and streams along its course. In the Medium Paraná River on its left margin, the Ensenada stream ends, located in the Diamante Department, province of Entre Ríos. This work objective is to increase the information about the status of the waters of the stream basin considering samples obtained during two well differentiated thermal seasons in 2010. Samplings were made in nine sites of the course and tributary streams and were analyzed by standard methods, in their physical-chemical and microbiological parameters. The results show that the physical-chemical parameters such as pH, EC and chlorophyll "a" are in ranges comparable with other courses in the region, but not with phosphorus in the main course, with higher values showing the effects of human activities in the basin. The high values of total coliform count as well as thermotolerants, mostly found in the tributaries, exceed the limits for recreational waters. Cluster analysis applied to the set of physical-chemical parameters showed the formation of five clusters in two seasons, with the forming of two tributaries that were characterized by high contents of phosphorus. The analysis of cyanobacteria showed presence of Microcystis genus that can affect the quality of recreational and consumption water, with non significant values of cyanotoxins.Fil: Vivot, Eduardo Pedro. Universidad Nacional de Entre Rios; ArgentinaFil: Sanchez, Cecilia I.. Universidad Nacional de Entre Rios; ArgentinaFil: Kieffer, Luis Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Prosperi, Carlos Hugo. Universidad Nacional de Córdoba; ArgentinaFil: Gieco, Adriana M.. Universidad Nacional de Entre Rios; ArgentinaFil: Dragan, Analía N.. Universidad Nacional de Entre Rios; ArgentinaFil: Ormaechea, María V.. Universidad Nacional de Entre Rios; ArgentinaFil: de la Sierra, Patricia Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Guerra, Elio W.. Universidad Nacional de Entre Rios; Argentin

Expanding the arsenal of FGFR inhibitors: A novel chloroacetamide derivative as a new irreversible agent with anti-proliferative activity against FGFR1-amplified lung cancer cell lines

Fibroblast Growth Factor Receptors (FGFR1-4) have a critical role in the progression of several human cancers, including Squamous Non-Small-Cell Lung Cancer (SQCLC). Both non-selective and selective reversible FGFR inhibitors are under clinical investigation for the treatment of patients with tumors harboring FGFR alterations. Despite their potential efficacy, the clinical development of these drugs has encountered several challenges, including toxicity, and the appearance of drug resistance. Recent efforts have been directed at development of irreversible FGFR inhibitors, which have the potential to exert superior anti-proliferative activity in tumors carrying FGFR alterations. With this in mind, we synthetized, and investigated a set of novel inhibitors possessing a warhead potentially able to covalently bind a cysteine in the P-loop of FGFR. Among them, the chloroacetamide UPR1376 resulted able to irreversible inhibit FGFR1 phosphorylation in FGFR1 over-expressing cells generated from SQCLC SKMES-1 cells. In addition, this compound inhibited cell proliferation in FGFR1-amplified H1581 cells with a potency higher than the reversible inhibitor BGJ398 (infigratinib), while sparing FGFR1 low-expressing cells. The anti-proliferative effects of UPR1376 were demonstrated in both 2D and 3D systems and were associated with the inhibition of MAPK and AKT/mTOR signaling pathways. UPR1376 inhibited cell proliferation also in two BGJ398-resistant cell clones generated from H1581 by chronic exposure to BGJ398, although at concentrations higher than those effective in the parental cells, likely due to the persistent activation of the MAPK pathway associated to NRAS amplification. Combined blockade of FGFR1 and MAPK signaling, by UPR1376 and trametinib respectively, significantly enhanced the efficacy of UPR1376, providing a means of circumventing resistance to FGFR1 inhibition. Our findings suggest that the insertion of a chloroacetamide warhead on a suitable scaffold, as exemplified by UPR1376, is a valuable strategy to develop a novel generation of FGFR inhibitors for the treatment of SQCLC patients with FGFR alterations

Intakes of culinary herbs and spices from a food frequency questionnaire evaluated against 28-days estimated records

<p>Abstract</p> <p>Background</p> <p>Worldwide, herbs and spices are much used food flavourings. However, little data exist regarding actual dietary intake of culinary herbs and spices. We developed a food frequency questionnaire (FFQ) for the assessment of habitual diet the preceding year, with focus on phytochemical rich food, including herbs and spices. The aim of the present study was to evaluate the intakes of herbs and spices from the FFQ with estimates of intake from another dietary assessment method. Thus we compared the intake estimates from the FFQ with 28 days of estimated records of herb and spice consumption as a reference method.</p> <p>Methods</p> <p>The evaluation study was conducted among 146 free living adults, who filled in the FFQ and 2-4 weeks later carried out 28 days recording of herb and spice consumption. The FFQ included a section with questions about 27 individual culinary herbs and spices, while the records were open ended records for recording of herbs and spice consumption exclusively.</p> <p>Results</p> <p>Our study showed that the FFQ obtained slightly higher estimates of total intake of herbs and spices than the total intake assessed by the Herbs and Spice Records (HSR). The correlation between the two assessment methods with regard to total intake was good (r = 0.5), and the cross-classification suggests that the FFQ may be used to classify subjects according to total herb and spice intake. For the 8 most frequently consumed individual herbs and spices, the FFQ obtained good estimates of median frequency of intake for 2 herbs/spices, while good estimates of portion sizes were obtained for 4 out of 8 herbs/spices.</p> <p>Conclusions</p> <p>Our results suggested that the FFQ was able to give good estimates of frequency of intake and portion sizes on group level for several of the most frequently used herbs and spices. The FFQ was only able to fairly rank subjects according to frequency of intake of the 8 most frequently consumed herbs and spices. Other studies are warranted to further explore the intakes of culinary spices and herbs.</p