Anaplasma marginale Infection of Dermacentor andersoni Primary Midgut Cell Culture Is Dependent on Fucosylated Glycans.

Tick midgut is the primary infection site required by tick-borne pathogens to initiate their development for transmission. Despite the biological significance of this organ, cell cultures derived exclusively from tick midgut tissues are unavailable and protocols for generating primary midgut cell cultures have not been described. To study the mechanism of Anaplasma marginale-tick cell interactions, we successfully developed an in vitro Dermacentor andersoni primary midgut cell culture system. Midgut cells were maintained for up to 120 days. We demonstrated the infection of in vitro midgut cells by using an A. marginale omp10::himar1 mutant with continued replication for up to 10 days post-infection. Anaplasma marginale infection of midgut cells regulated the differential expression of tick α-(1,3)-fucosyltransferases A1 and A2. Silencing of α-(1,3)-fucosyltransferase A2 in uninfected midgut cells reduced the display of fucosylated glycans and significantly lowered the susceptibility of midgut cells to A. marginale infection, suggesting that the pathogen utilized core α-(1,3)-fucose of N-glycans to infect tick midgut cells. This is the first report using in vitro primary D. andersoni midgut cells to study A. marginale-tick cell interactions at the molecular level. The primary midgut cell culture system will further facilitate the investigation of tick-pathogen interactions, leading to the development of novel intervention strategies for tick-borne diseases.

Midgut cells alteration in gamma-irradiated beetles (Blaps polycresta, Coleoptera: Tenebrionidae) / Alteração das células do intestino médio em besouros irradiados com radiação gama (Blaps polycresta, Coleoptera: Tenebrionidae)

Abstract This study was conducted to examine the effect of gamma radiation on biological specimens. Thus, our concept is to clarify that exposure to accumulated dose of 0.2 Gy gamma rays (0.66 rad/Sec. dose rate) from Cs137 source induces cellular perturbations in the midgut epithelium of the F1 progeny of Blaps polycresta, therefore affecting nutrition and growth. Beetles were reared in laboratory conditions and the newly emerged adults were irradiated with the aforementioned dose. Histological and ultrastructure anomalies of midgut cells (digestive and regenerative cells) were observed by 72 h after radiation exposure to ensure that the cells will not return to control state. Retardation in the development of the F1 progeny was also noticed and beetles died through two weeks. In the light of these observations, biological tissue act as an indicator to the continuous exposure to environmental radiation.

Resumo Este estudo foi conduzido para examinar o efeito da radiação gama em espécimes biológicos. Assim, nosso conceito é esclarecer que a exposição à dose acumulada de raios gama de 0,2 Gy (0,66 rad / seg. Dose) da fonte Cs137 induz perturbações celulares no epitélio do intestino médio da progênie F1 de Polycresta blaps, afetando a nutrição e crescimento. Besouros foram criados em condições de laboratório, e os adultos recém-emergidos foram irradiados com a dose acima mencionada. Anomalias histológicas e ultraestruturais das células do intestino médio (células digestivas e regenerativas) foram observadas 72 horas após a exposição à radiação, para garantir que as células não retornariam ao estado de controle. Retardo no desenvolvimento da progênie F1 também foi notado, e besouros morreram por duas semanas. À luz dessas observações, os tecidos biológicos atuam como um indicador para a exposição contínua à radiação ambiental.

How virus size and attachment parameters affect the temperature sensitivity of virus binding to host cells: Predictions of a thermodynamic model for arboviruses and HIV.

Virus binding to host cells involves specific interactions between viral (glyco)proteins (GP) and host cell surface receptors (Cr) (protein or sialic acid (SA)). The magnitude of the enthalpy of association changes with temperature according to the change in heat capacity (ΔCp) on GP/Cr binding, being little affected for avian influenza virus (AIV) haemagglutinin (HA) binding to SA (ΔCp = 0 kJ/mol/K) but greatly affected for HIV gp120 binding to CD4 receptor (ΔCp = -5.0 kJ/mol/K). A thermodynamic model developed here predicts that values of ΔCp from 0 to ~-2.0 kJ/mol/K have relatively little impact on the temperature sensitivity of the number of mosquito midgut cells with bound arbovirus, while intermediate values of ΔCp of ~-3.0 kJ/mol/K give a peak binding at a temperature of ~20 °C as observed experimentally for Western equine encephalitis virus. More negative values of ΔCp greatly decrease arbovirus binding at temperatures below ~20 °C. Thus to promote transmission at low temperatures, arboviruses may benefit from ΔCp ~ 0 kJ/mol/K as for HA/SA and it is interesting that bluetongue virus binds to SA in midge midguts. Large negative values of ΔCp as for HIV gp120:CD4 diminish binding at 37 °C. Of greater importance, however, is the decrease in entropy of the whole virus (ΔSa_immob) on its immobilisation on the host cell surface. ΔSa_immob presents a repulsive force which the enthalpy-driven GP/Cr interactions weakened at higher temperatures struggle to overcome. ΔSa_immob is more negative (less favourable) for larger diameter viruses which therefore show diminished binding at higher temperatures than smaller viruses. It is proposed that small size phenotype through a less negative ΔSa_immob is selected for viruses infecting warmer hosts thus explaining the observation that virion volume decreases with increasing host temperature from 0 °C to 40 °C in the case of dsDNA viruses. Compared to arboviruses which also infect warm-blooded vertebrates, HIV is large at 134 nm diameter and thus would have a large negative ΔSa_immob which would diminish its binding at human body temperature. It is proposed that prior non-specific binding of HIV through attachment factors takes much of the entropy loss for ΔSa_immob so enhancing subsequent specific gp120:CD4 binding at 37 °C. This is consistent with the observation that HIV attachment factors are not essential but augment infection. Antiviral therapies should focus on increasing virion size, for example through binding of zinc oxide nanoparticles to herpes simplex virus, hence making ΔSa_immob more negative, and thus reducing binding affinity at 37 °C.

Towards a thermodynamic mechanistic model for the effect of temperature on arthropod vector competence for transmission of arboviruses.

Arboviruses such as West Nile virus (WNV), bluetongue virus (BTV), dengue virus (DENV) and chikungunya virus (CHIKV) infect their arthropod vectors over a range of average temperatures depending on the ambient temperature. How the transmission efficiency of an arbovirus (i.e. vector competence) varies with temperature influences not only the short term risk of arbovirus outbreaks in humans and livestock but also the long term impact of climate change on the geographical range of the virus. The strength of the interaction between viral surface (glyco)protein (GP) and the host cell receptor (Cr) on binding of virus to host cell is defined by the thermodynamic dissociation constant Kd_receptor which is assumed to equal 10-3 M (at 37 °C) for binding of a sialic acid (SA) on the arthropod midgut epithelial cell surface to a SA-binding site on the surface of BTV, for example. Here virus binding affinity is modelled with increasing number of GP/Cr contacts at temperatures from 10 °C to 35 °C taking into account the change in entropy on immobilization of the whole virus on binding (ΔSa_immob). Based on published data, three thermodynamic GP/Cr binding scenarios, namely enthalpy-driven, entropy-assisted and entropy-driven, are shown to affect the temperature sensitivity of virus binding in different ways. Thus for enthalpy-driven GP/Cr binding, viruses bind host cells much more strongly at 10 °C than 35 °C. A mechanistic model is developed for the number of arthropod midgut cells with bound virus and by building in a kinetic component for the rate of arbovirus replication and subsequent spread to the arthropod salivary glands, a model for the effect of temperature on vector competence is developed. The model separates the opposing effects of temperature on midgut cell binding affinity from the kinetic component of virogenesis. It successfully accommodates both increases in vector competence with temperature as for DENV and WNV in mosquitoes and decreases as for the CHIKV 2010-1909 strain in various populations of Aedes albopictus mosquitoes. Enhanced cell binding at lower temperatures through enthalpy-driven GP/Cr binding compensates for the lower replication rate to some degree such that some transmission can still occur at lower temperatures. In contrast, the strength of entropy-driven GP/Cr binding diminishes at low temperatures although there is no minimum temperature threshold for transmission efficiency. The magnitude of ΔSa_immob is an important data gap. It is concluded that thermodynamic and kinetic data obtained at the molecular level will prove important in modelling vector competence with temperature.

Assessing the genotoxic effects of two lipid peroxidation products (4-oxo-2-nonenal and 4-hydroxy-hexenal) in haemocytes and midgut cells of Drosophila melanogaster larvae.

Lipid peroxidation products can induce tissue damage and are implicated in diverse pathological conditions, including aging, atherosclerosis, brain disorders, cancer, lung and various liver disorders. Since in vivo studies produce relevant information, we have selected Drosophila melanogaster as a suitable in vivo model to characterise the potential risks associated to two lipid peroxidation products namely 4-oxo-2-nonenal (4-ONE) and 4-hydroxy-hexenal (4-HHE). Toxicity, intracellular reactive oxygen species production, and genotoxicity were the end-points evaluated. Haemocytes and midgut cells were the evaluated targets. Results showed that both compounds penetrate the intestine of the larvae, affecting midgut cells, and reaching haemocytes. Significant genotoxic effects, as determined by the comet assay, were observed in both selected cell targets in a concentration/time dependent manner. This study highlights the importance of D. melanogaster as a model organism in the study of the different biological effects caused by lipid peroxidation products entering via ingestion. This is the first study reporting genotoxicity data in haemocytes and midgut cells of D. melanogaster larvae for the two selected compounds.

Midgut cells alteration in gamma-irradiated beetles (Blaps polycresta, Coleoptera: Tenebrionidae)

Abstract This study was conducted to examine the effect of gamma radiation on biological specimens. Thus, our concept is to clarify that exposure to accumulated dose of 0.2 Gy gamma rays (0.66 rad/Sec. dose rate) from Cs137 source induces cellular perturbations in the midgut epithelium of the F1 progeny of Blaps polycresta, therefore affecting nutrition and growth. Beetles were reared in laboratory conditions and the newly emerged adults were irradiated with the aforementioned dose. Histological and ultrastructure anomalies of midgut cells (digestive and regenerative cells) were observed by 72 h after radiation exposure to ensure that the cells will not return to control state. Retardation in the development of the F1 progeny was also noticed and beetles died through two weeks. In the light of these observations, biological tissue act as an indicator to the continuous exposure to environmental radiation.

Resumo Este estudo foi conduzido para examinar o efeito da radiação gama em espécimes biológicos. Assim, nosso conceito é esclarecer que a exposição à dose acumulada de raios gama de 0,2 Gy (0,66 rad / seg. Dose) da fonte Cs137 induz perturbações celulares no epitélio do intestino médio da progênie F1 de Polycresta blaps, afetando a nutrição e crescimento. Besouros foram criados em condições de laboratório, e os adultos recém-emergidos foram irradiados com a dose acima mencionada. Anomalias histológicas e ultraestruturais das células do intestino médio (células digestivas e regenerativas) foram observadas 72 horas após a exposição à radiação, para garantir que as células não retornariam ao estado de controle. Retardo no desenvolvimento da progênie F1 também foi notado, e besouros morreram por duas semanas. À luz dessas observações, os tecidos biológicos atuam como um indicador para a exposição contínua à radiação ambiental.

BmVDAC upregulation in the midgut of Rhipicephalus microplus, during infection with Babesia bigemina.

The molecular mechanisms involved during the infection of Rhipicephalus microplus midgut cells by Babesia bigemina are of great relevance and currently unknown. In a previous study, we found a voltage-dependent anion channel (VDAC)-like protein (BmVDAC) that may participate during parasite invasion of midgut cells. In this work, we investigated BmVDAC expression at both mRNA and protein levels and examined BmVDAC localization in midgut cells of ticks infected with B. bigemina at different times post-repletion. Based on the RT-PCR results, Bmvdac expression levels were significantly higher in infected ticks compared to uninfected ones, reaching their highest values at 24h post-repletion (p<0.0001). Similar results were obtained at the protein level (p<0.0001). Interestingly, BmVDAC immunolocalization showed that there was an important differential expression and redistribution of BmVDAC protein between the midgut cells of infected and uninfected ticks, which was more evident 24h post-repletion of infected ticks. This is the first report of BmVDAC upregulation and immunolocalization in R. microplus midgut cells during B. bigemina infection. Further studies regarding the function of BmVDAC during the infection may provide new insights into the molecular mechanisms between B. bigemina and its tick vector and could result in its use as an anti-tick and transmission-blocking vaccine candidate.

Drosophila comet assay: insights, uses, and future perspectives.

The comet assay, a very useful tool in genotoxicity and DNA repair testing, is being applied to Drosophila melanogaster since around 15 years ago, by several research groups. This organism is a valuable model for all kind of processes related to human health, including DNA damage response. The assay has been performed mainly in vivo using different larvae cell types (from brain, midgut, hemolymph, and imaginal disk), but also in vitro with the S2 cell line. Since its first application, it has been used to analyze the genotoxicity and action mechanisms of different chemicals, demonstrating good sensitivity and proving its usefulness. Moreover, it is the only assay that can be used to analyze DNA repair in somatic cells in vivo, comparing the effects of chemicals in different repair strains, and to quantitate repair activities in vitro. Additionally, the comet assay in Drosophila, in vivo and in vitro, has been applied to study the influence of protein overexpression on genome integrity and degradation. Although the assay is well established, it could benefit from some research to determine optimal experimental design to standardize it, and then to allow comparisons among laboratories independently of the chosen cell type.