Antiviral activity of an N-allyl acridone against dengue virus

Background

Dengue virus (DENV), a member of the family Flaviviridae, is at present the most widespread causative agent of a human viral disease transmitted by mosquitoes. Despite the increasing incidence of this pathogen, there are no antiviral drugs or vaccines currently available for treatment or prevention. In a previous screening assay, we identified a group of N-allyl acridones as effective virus inhibitors. Here, the antiviral activity and mode of action targeted to viral RNA replication of one of the most active DENV-2 inhibitors was further characterized.

Results

The compound 10-allyl-7-chloro-9(10H)-acridone, designated 3b, was active to inhibit the in vitro infection of Vero cells with the four DENV serotypes, with effective concentration 50% (EC₅₀) values in the range 12.5-27.1 μM, as determined by virus yield inhibition assays. The compound was also effective in human HeLa cells. No cytotoxicity was detected at 3b concentrations up to 1000 μM. Mechanistic studies demonstrated that virus entry into the host cell was not affected, whereas viral RNA synthesis was strongly inhibited, as quantified by real time RT-PCR. The addition of exogenous guanosine together with 3b rescued only partially the infectivity of DENV-2.

Conclusions

The acridone derivative 3b selectively inhibits the infection of Vero cells with the four DENV serotypes without a direct interaction with the host cell or the virion but interfering specifically with the intracellular virus multiplication. The mode of antiviral action for this acridone apparently involves the cellular enzyme inosine-monophospahe dehydrogenase together with another still unidentified target related to DENV RNA synthesis.     

Satellite‐derived NDVI,LST, and climatic factors driving the distribution and abundance of Anopheles mosquitoes in a former malarious area in northwest Argentina

Distribution and abundance of disease vectors are directly related to climatic conditions and environmental changes. Remote sensing data have been used for monitoring environmental conditions influencing spatial patterns of vector‐borne diseases. The aim of this study was to analyze the effect of the Normalized Difference Vegetation Index (NDVI) and Land Surface Temperature (LST) obtained from the Moderate Resolution Imaging Spectroradiometer (MODIS), and climatic factors (temperature, humidity, wind velocity, and accumulated rainfall) on the distribution and abundance of Anopheles species in northwestern Argentina using Poisson regression analyses. Samples were collected from December, 2001 to December, 2005 at three localities, Aguas Blancas, El Oculto and San Ramón de la Nueva Orán. We collected 11,206 adult Anopheles species, with the major abundance observed at El Oculto (59.11%), followed by Aguas Blancas (22.10%) and San Ramón de la Nueva Orán (18.79%). Anopheles pseudopunctipennis was the most abundant species at El Oculto, Anopheles argyritarsis predominated in Aguas Blancas, and Anopheles strodei in San Ramón de la Nueva Orán. Samples were collected throughout the sampling period, with the highest peaks during the spring seasons. LST and mean temperature appear to be the most important variables determining the distribution patterns and major abundance of An. pseudopunctipennis and An. argyritarsis within malarious areas.     

Junín virus. A XXI century update

Junín virus of the Arenaviridae family is the etiological agent of Argentine hemorrhagic fever, a febrile syndrome causing hematological and neurological symptoms. We review historical perspectives of current knowledge on the disease, and update information related to the virion and its potential pathogenic mechanisms.     

Influence of 400, 900, and 1900 MHz electromagnetic fields on Lemna minor growth and peroxidase activity

Increased use of radio and microwave frequencies requires investigations of their effects on living organisms. Duckweed (Lemna minor L.) has been commonly used as a model plant for environmental monitoring. In the present study, duckweed growth and peroxidase activity was evaluated after exposure in a Gigahertz Transversal Electromagnetic (GTEM) cell to electric fields of frequencies 400, 900, and 1900 MHz. The growth of plants exposed for 2 h to the 23 V/m electric field of 900 MHz significantly decreased in comparison with the control, while an electric field of the same strength but at 400 MHz did not have such effect. A modulated field at 900 MHz strongly inhibited the growth, while at 400 MHz modulation did not influence the growth significantly. At both frequencies a longer exposure mostly decreased the growth and the highest electric field (390 V/m) strongly inhibited the growth. Exposure of plants to lower field strength (10 V/m) for 14 h caused significant decrease at 400 and 1900 MHz while 900 MHz did not influence the growth. Peroxidase activity in exposed plants varied, depending on the exposure characteristics. Observed changes were mostly small, except in plants exposed for 2 h to 41 V/m at 900 MHz where a significant increase (41%) was found. Our results suggest that investigated electromagnetic fields (EMFs) might influence plant growth and, to some extent, peroxidase activity. However, the effects of EMFs strongly depended on the characteristics of the field exposure.     

Characteristics of in vivo murine erythropoietic response to sodium orthovanadate

Current knowledge about the effects of vanadium compounds on erythropoiesis is still reduced and even contradictory. The aim of this work was to evaluate the in vivo effects of a single dose of sodium orthovanadate (OV, 33 mg/kg i.p.) on CF-1 mice in a time course study (0-8 days). Murine erythropoiesis was assessed through a combinatory of experimental approaches. Classical peripheral and bone marrow (BM) hematological parameters were determined. Erythroid maturation in blood stream and hemopoietic tissues (59Fe uptake assays), BM erythroid progenitor frequency (clonogenic assays) and erythroid crucial protein expressions for commitment and survival: GATA-1, erythropoietin receptor (Epo-R) and Bcl-xL (immunoblottings) were evaluated. Neither BM cellularities nor BM viabilities changed noticeably during the study. Peripheral reticulocytes showed a biphasic increment on days 2 and 8 post-OV. hematocrits enhanced transiently between days 2 and 4. 59Fe uptake percentages enhanced in peripheral blood nearly two-fold over control values between 4 and 8 days (p<0.01) without changes in BM and spleen. Additionally, mature erythroid BM compartments: polychromatophilic erythroblasts and orthochromatic normoblasts increased by the eighth day. BFU-E colonies remained near basal values during the whole experience, whilst CFU-E colonies raised 60% over control at 8 days post-OV (p<0.05). GATA-1 and Epo-R were significantly over-expressed from the third until the end of the experimental protocol (p<0.01). Surprisingly, Bcl-xL showed a constitutive expression pattern without changes during the experience. Experimental data let us suggest that OV does not to cause bone marrow cytotoxicity and that it accelerates maturation of BM committed erythroid precursors. Moreover, there are significant correlations among erythroid-related protein expressions: GATA-1 and Epo-R and the frequency of CFU-E. In addition, Bcl-xL expression invariance during the time course study would indicate that the stimulatory effect of OV treatment on erythropoiesis was mainly exerted on the maturation of red cell precursors rather than on the antiapoptosis of erythroid terminal progenitors.     

Calsequestrin (CASQ1) rescues function and structure of calcium release units in skeletal muscles of CASQ1-null mice

Amplitude of Ca(2+) transients, ultrastructure of Ca(2+) release units, and molecular composition of sarcoplasmic reticulum (SR) are altered in fast-twitch skeletal muscles of calsequestrin-1 (CASQ1)-null mice. To determine whether such changes are directly caused by CASQ1 ablation or are instead the result of adaptive mechanisms, here we assessed ability of CASQ1 in rescuing the null phenotype. In vivo reintroduction of CASQ1 was carried out by cDNA electro transfer in flexor digitorum brevis muscle of the mouse. Exogenous CASQ1 was found to be correctly targeted to the junctional SR (jSR), as judged by immunofluorescence and confocal microscopy; terminal cisternae (TC) lumen was filled with electron dense material and its width was significantly increased, as judged by electron microscopy; peak amplitude of Ca(2+) transients was significantly increased compared with null muscle fibers transfected only with green fluorescent protein (control); and finally, transfected fibers were able to sustain cytosolic Ca(2+) concentration during prolonged tetanic stimulation. Only the expression of TC proteins, such as calsequestrin 2, sarcalumenin, and triadin, was not rescued as judged by Western blot. Thus our results support the view that CASQ1 plays a key role in both Ca(2+) homeostasis and TC structure.     

In vitro conditions affect photosynthetic performance and crassulacean acid metabolism in Mammillaria gracilis Pfeiff. tissues

Mammillaria gracilis Pfeiff. plants cultivated in the pot (pot plants, PP), as well as in vitro-grown normal shoots (NS), habituated callus (HC), hyperhydric shoots (HS), and tumour tissue (TT) were investigated in order to reveal the influence of in vitro culture on functionality of the photosynthetic apparatus and CAM photosynthesis in cactus M. gracilis Pfeiff. Photosynthetic pigments content as well as maximum (F _v/F _m) and effective (??_PSII) quantum yield of photosystem II (PSII) decreased in all in vitro-grown tissues in comparison to PP. The decrease observed in hyperhydric HC, HS and TT correlated with a low expression of Rubisco large subunit (RbcL) and ?? subunit of ATP synthase (?? ATP synt) and almost undetectable levels of protein D1, light-harvesting chlorophyll a/b-binding protein (LHCII) and cytochrome f protein of thylakoid Cyt b ₆ /f-complex (Cyt f) found in these tissues. As for crassulacean acid metabolism (CAM) pattern, PP and NS expressed diurnal acid fluctuation, while HC, HS and TT failed to show it. Nevertheless, all M. gracilis tissues exhibited diurnal changes of phosphoenolpyruvate carboxylase (PEPC) activity indicating the typical CAM physiology. In conclusion, the photosynthesis was down-regulated in all in vitro-grown tissues. NS maintained typical CAM photosynthesis, while HC, HS and TT withheld PEPC activity, but not acid accumulation specific for CAM. Minor changes observed in NS in comparison to PP could be attributed to the sugar supplementation while the more prominent deviations found in HC, HS and TT could be correlated with hyperhydricity and the loss of characteristic tissue organisation pattern.     

A practical validation approach for virus titer testing of avian infectious bursal disease live vaccine according to current regulatory guidelines

The method for virus titer determination of avian infectious bursal disease (IBD) live vaccine, developed long before regulatory validation guidelines is a cell culture based biological assay intended for use in vaccine release testing.The aim of our study was to perform a validation, based on fit-for-purpose principle, of an old 50% tissue culture infectious dose (TCID₅₀) method according to Guidelines of the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH).This paper addresses challenges and discusses some key aspects that should be considered when validating biological methods. A different statistical approach and non-parametric statistics was introduced in validation protocol in order to derive useful information from experimental data. This approach is applicable for a wide range of methods.In conclusion, the previous virus titration method had showed to be precise, accurate, linear, robust and in accordance with current regulatory standards, which indicates that there is no need for additional re-development or upgrades of the method for its suitability for intended use.     

Chitosan influence on glucose and calcium availability from yogurt: In vitro comparative study with plants fibre

Since chitosan complies with the definition of dietary fibre is necessary to study the interaction of this biopolymer with nutrients. Yogurt with fortified chitosan and different types of plants fibres like wheat, bamboo, apple, psyllium and inulin was used as a food model. The availabilities of glucose and calcium in this model were studied by an in vitro gastrointestinal tract simulation. Results showed that the different fibres decreased both glucose and calcium availabilities whereas the effect of chitosan was more pronounced. (17.7 ± 2.1% and 21.0 ± 2.5% depress respectively). This work demonstrated that the addition of chitosan to yogurts influences the availability of nutrients.