Expression pattern of <Emphasis Type="Italic">cdkl5</Emphasis> during zebrafish early development: implications for use as model for atypical Rett syndrome

Atypical Rett syndrome is a child neurodevelopmental disorder induced by mutations in CDKL5 gene and characterized by a progressive regression in development with loss of purposeful use of the hands, slowed brain and head growth, problems with walking, seizures, and intellectual disability. At the moment, there is no cure for this pathology and little information is available concerning animal models capable of mimicking its phenotypes, thus the development of additional animal models should be of interest to gain more knowledge about the disease. Zebrafish has been used successfully as model organism for many human genetic diseases; however, no information is available concerning the spatial and temporal expression of cdkl5 orthologous in this organism. In the present study, we identified the developmental expression patterns of cdkl5 in zebrafish by quantitative PCR and whole-mount in situ hybridization. cdkl5 is expressed maternally at low levels during the first 24 h of development. After that the expression of the gene increases significantly and it starts to be expressed mainly in the nervous system and in several brain structures, such as telencephalon, mesencephalon and diencephalon. The expression patterns of cdkl5 in zebrafish is in accordance with the tissues known to be affected in humans and associated to symptoms and deficits observed in Rett syndrome patients thus providing the first evidence that zebrafish could be an alternative model to study the molecular pathways of this disease as well as to test possible therapeutic approaches capable of rescuing the phenotype.     

Rapid selection and characterization of Cry1F resistance in a Brazilian strain of fall armyworm

Transgenic maize (Zea mays L., Poaceae) event TC1507, producing the Cry1F protein of Bacillus thuringiensis Berliner, has been used for management of the fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), in Brazil since 2009. A strain of S. frugiperda, obtained from field collections of larvae in TC1507 maize in Minas Gerais state in 2010, was selected in the laboratory for resistance to Cry1F using leaves of TC1507 maize in two selection regimes. Continuous exposure of larvae to Cry1F was more effective than exposure for 6, 8, and 10 days in the selection of resistant S. frugiperda individuals. With only four generations of laboratory selection, a strain with high levels of resistance to Cry1F was obtained, as indicated by the survival of insects reared on leaves of TC1507 maize plants and by the more than 300‐fold resistance level measured in bioassays with the purified Cry1F protein. Importantly, reciprocal crosses between control and the Cry1F‐selected strains revealed that the resistance is autosomal and incompletely recessive, and the response obtained in the backcross of the F₁ generation with the resistant strain was consistent with simple monogenic inheritance. Additionally, there were no apparent fitness costs associated with resistance either for survival or larval growth on non‐Bt maize leaves. Our findings provide experimental evidence for rapid evolution of Cry1F resistance in S. frugiperda in the laboratory and further reinforce the potential of this species to evolve field resistance to the TC1507 maize as previously reported. The resistant strain isolated in this study provides an opportunity to estimate the resistance allele frequency in the field and to determine the biochemical and molecular basis of the resistance, which should provide further information to assist in the resistance management of S. frugiperda on transgenic maize producing B. thuringiensis proteins.     

Selection of Escherichia coli K-12 chromosomal mutants that prevent expression of F-plasmid functions.

Chromosomal mutants of Escherichia coli deficient in the expression of F-plasmid functions were selected by mutagenizing F- cells, introducing an F' plasmid into the mutagenized cells by conjugation, and identifying transconjugants resistant to the donor-specific bacteriophage Q beta by a simple spray test. All but 1 of 25 mutants were defective in an extracellular stage of Q beta infection, suggesting that they fail to elaborate F-pili. At least six of these were also deficient as deoxyribonucleic acid donors. More than half of the mutants appear to be altered in peviously undetected chromosomal genes required for the expression of F-related cellular functions.     

Neuroinvasion of the Highly Pathogenic Influenza Virus H7N1 Is Caused by Disruption of the Blood Brain Barrier in an Avian Model

Influenza A virus (IAV) causes central nervous system (CNS) lesions in avian and mammalian species, including humans. However, the mechanism used by IAV to invade the brain has not been determined. In the current work, we used chickens infected with a highly pathogenic avian influenza (HPAI) virus as a model to elucidate the mechanism of entry of IAV into the brain. The permeability of the BBB was evaluated in fifteen-day-old H7N1-infected and non-infected chickens using three different methods: (i) detecting Evans blue (EB) extravasation into the brain, (ii) determining the leakage of the serum protein immunoglobulin Y (IgY) into the brain and (iii) assessing the stability of the tight-junction (TJ) proteins zonula occludens-1 and claudin-1 in the chicken brain at 6, 12, 18, 24, 36 and 48 hours post-inoculation (hpi). The onset of the induced viremia was evaluated by quantitative real time RT-PCR (RT-qPCR) at the same time points. Viral RNA was detected from 18 hpi onward in blood samples, whereas IAV antigen was detected at 24 hpi in brain tissue samples. EB and IgY extravasation and loss of integrity of the TJs associated with the presence of viral antigen was first observed at 36 and 48 hpi in the telencephalic pallium and cerebellum. Our data suggest that the mechanism of entry of the H7N1 HPAI into the brain includes infection of the endothelial cells at early stages (24 hpi) with subsequent disruption of the TJs of the BBB and leakage of virus and serum proteins into the adjacent neuroparenchyma.     

SopD acts cooperatively with SopB during Salmonella enterica serovar Typhimurium invasion

The intracellular bacterial pathogen, Salmonella enterica serovar Typhimurium (S. typhimurium), causes disease in a variety of hosts. To invade and replicate in host cells, these bacteria subvert host molecular machinery using bacterial proteins, called effectors, which they translocate into host cells using specialized protein delivery systems. One of these effectors, SopD, contributes to gastroenteritis, systemic virulence and persistence of S. typhimurium in animal models of infection. Recently, SopD has been implicated in invasion of polarized epithelial cells and here we investigate the features of SopD-mediated invasion. We show that SopD plays a role in membrane fission and macropinosome formation during S. typhimurium invasion, events previously shown to be mediated by the SopB effector. We further demonstrate that SopD acts cooperatively with SopB to promote these events during invasion. Using live cell imaging we show that a SopD-GFP fusion does not localize to HeLa cell cytosol as previously described, but instead is membrane associated. Upon S. typhimurium infection of these cells, SopD-GFP is recruited to the invasion site, and this recruitment required the phosphatase activity of SopB. Our findings demonstrate a role for SopD in manipulation of host-cell membrane during S. typhimurium invasion and reveal the nature of its cooperative action with SopB.