A faster immunofluorescence assay for tracking infection progress of human cytomegalovirus

Immunofluorescence assay (IFA) is one of the most frequently used methods in the biological sciences and clinic diagnosis, but it is expensive and time-consuming. To overcome these limitations, we developed a faster and more cost-effective IFA (f-IFA) by modifying the standard IFA, and applied this method to track the progression of human cytomegalovirus (HCMV) infection in different cells. The f-IFA that we developed not only saves time, but also dramatically reduces the quantity of antibody (Ab), which will facilitate the application of IFA in clinic diagnosis. f-IFA requires only 15 min for blocking, 10 min incubation for each primary and secondary Abs, followed by 1 min extensive wash after each incubation. Only 25 μl of diluted Ab solution was needed for each coverslip at the primary and secondary Ab incubation steps. In addition, all steps were performed at room temperature. This f-IFA has been applied successfully to follow virion entry (pp65) and expression of viral genes (IE1, UL44, and pp65) in order to track the details of HCMV infection process. We found that ～0.5% HCMV-infected T98G cells formed multiple-micronuclei (IE1 and nucleus staining) and had virus shedding (pp65 staining) by f-IFA, which could not be detected by the traditional IFA. Our results indicated that f-IFA is a sensitive, convenient, fast, and cost-effective method for investigating the details of virus infection progress, especially HCMV infection. The faster and cost-effective feature with higher sensitivity and specificity implies that f-IFA has potential applications in clinical diagnosis.     

Computational model of EGFR and IGF1R pathways in lung cancer: a Systems Biology approach for Translational Oncology

In this paper we propose a Systems Biology approach to understand the molecular biology of the Epidermal Growth Factor Receptor (EGFR, also known as ErbB1/HER1) and type 1 Insulin-like Growth Factor (IGF1R) pathways in non-small cell lung cancer (NSCLC). This approach, combined with Translational Oncology methodologies, is used to address the experimental evidence of a close relationship among EGFR and IGF1R protein expression, by immunohistochemistry (IHC) and gene amplification, by in situ hybridization (FISH) and the corresponding ability to develop a more aggressive behavior. We develop a detailed in silico model, based on ordinary differential equations, of the pathways and study the dynamic implications of receptor alterations on the time behavior of the MAPK cascade down to ERK, which in turn governs proliferation and cell migration. In addition, an extensive sensitivity analysis of the proposed model is carried out and a simplified model is proposed which allows us to infer a similar relationship among EGFR and IGF1R activities and disease outcome.     

miR-24 triggers epidermal differentiation by controlling actin adhesion and cell migration

During keratinocyte differentiation and stratification, cells undergo extensive remodeling of their actin cytoskeleton, which is important to control cell mobility and to coordinate and stabilize adhesive structures necessary for functional epithelia. Limited knowledge exists on how the actin cytoskeleton is remodeled in epithelial stratification and whether cell shape is a key determinant to trigger terminal differentiation. In this paper, using human keratinocytes and mouse epidermis as models, we implicate miR-24 in actin adhesion dynamics and demonstrate that miR-24 directly controls actin cable formation and cell mobility. miR-24 overexpression in proliferating cells was sufficient to trigger keratinocyte differentiation both in vitro and in vivo and directly repressed cytoskeletal modulators (PAK4, Tks5, and ArhGAP19). Silencing of these targets recapitulated the effects of miR-24 overexpression. Our results uncover a new regulatory pathway involving a differentiation-promoting microribonucleic acid that regulates actin adhesion dynamics in human and mouse epidermis.     

Silicon Suppresses Fusarium Wilt Development in Banana Plants

This study aimed to determine the effect of silicon (Si) in reducing the symptoms of Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), on banana plants. Banana seedlings of Grand Nain (resistant) and Maçã (susceptible) were grown in plastic trays amended with 0 (?Si) or 0.39 g Si (+Si) per kg of soil and inoculated with Foc at 60 days after transplanting. The Si concentration in the roots and rhizome‐pseudostem significantly increased by 30.26 and 58.82%, respectively, for the +Si treatment compared with ?Si treatment. The Si concentration in the roots and rhizome‐pseudostem of Grand Nain plants was, respectively, 11.57 and 37.04% greater than that found in Maçã. The +Si plants showed a reduction of 12.37, 49.81, 51.87 and 20.39%, respectively, for the area under reflex leaf symptoms progress curve, the area under root symptoms progress curve, the area under disease progress curve and the area under asymptomatic fungal colonization of tissue progress curve compared with ‐Si plants. The area under darkening of rhizome‐pseudostem progress curve (AUDRPPC) of Maçã significantly increased by 15.98% for the ?Si treatment in comparison with the +Si treatment. For the +Si treatment, the AUDRPPC of the plants from the Maçã cultivar significantly decreased by 20.59% in comparison with the plants from the Grand Nain cultivar. The area under relative lesion length progress curve (AURLLPC) of the plants from the Maçã cultivar significantly decreased by 41.54% for the +Si treatment in comparison with the ?Si treatment. There was no significant difference between the ‐Si and +Si treatments in the AUDRPPC and AURLLPC of Grand Nain. For the +Si treatment, the AURLLPC of Grand Nain significantly decreased by 9.23% in comparison with Maçã. There was no significant difference between the Grand Nain and Maçã for the AUDRPPC and AURLLPC in the ?Si treatment. The findings of this study show that supplying Si to banana plants, especially to a susceptible cultivar to Foc, had a great potential in reducing the intensity of Fusarium wilt and may play a key role in disease management when banana plants are cultivated in Si‐deficient soils infested by this pathogen.     

Genome size rather than content might affect call properties in toads of three ploidy levels (Anura: Bufonidae: Bufo viridis subgroup)

In vertebrates, genome size has been shown to correlate with nuclear and cell sizes, and influences phenotypic features, such as brain complexity. In three different anuran families, advertisement calls of polyploids exhibit longer notes and intervals than diploids, and difference in cellular dimensions have been hypothesized to cause these modifications. We investigated this phenomenon in green toads (Bufo viridis subgroup) of three ploidy levels, in a different call type (release calls) that may evolve independently from advertisement calls, examining 1205 calls, from ten species, subspecies, and hybrid forms. Significant differences between pulse rates of six diploid and four polyploid (3n, 4n) green toad forms across a range of temperatures from 7 to 27 °C were found. Laboratory data supported differences in pulse rates of triploids vs. tetraploids, but failed to reach significance when including field recordings. This study supports the idea that genome size, irrespective of call type, phylogenetic context, and geographical background, might affect call properties in anurans and suggests a common principle governing this relationship. The nuclear‐cell size ratio, affected by genome size, seems the most plausible explanation. However, we cannot rule out hypotheses under which call‐influencing genes from an unexamined diploid ancestral species might also affect call properties in the hybrid‐origin polyploids. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 584–590.     

Dufour’s gland possible role in the evolution of sting morphology and function in hover wasps (Hymenoptera Stenogastrinae)

The sting is the most effective defense of social Hymenoptera against vertebrate predators but in the hover wasps (subfamily Stenogastrinae) it is scarcely used. In these wasps a quite enlarged Dufour’s gland and the extensive use of its secretion in the peculiar rearing of the larvae and defense determined important morphological modifications of the sting structure. Connecting anatomical and morphological data with behavioral observations we determined that in these wasps the Dufour’s gland secretion is attached to the egg during oviposition but can be also channeled to the outside via the sting when it is collected by adult females for larval rearing or construction of the nest ant guards. The anatomical modifications of the sting reduced the function of the sting as a defensive weapon in hover wasps.     

Human cytomegalovirus (HCMV) and hearing impairment: infection of fibroblast cells with HCMV induces chromosome breaks at 1q23.3, between loci DFNA7 and DFNA49 -- both involved in dominantly inherited, sensorineural, hearing impairment

Human cytomegalovirus (HCMV) infection is the most common congenital infection in developed countries and is responsible for a substantial fraction of sensorineural hearing impairment (SNHI) in children. The risk of hearing impairment is associated with viral load in urine and blood collected during the first postnatal month. However, although inner ear abnormalities are observed in some children with HCMV-induced SNHI, the exact mechanism whereby congenital HCMV infection causes hearing impairment is unknown. Earlier studies using standard cytogenetic mapping techniques showed that infection of S-phase human fibroblast cells with HCMV resulted in two specific, site-directed, chromosome breaks at band positions 1q21 and 1q42 which include loci involved in dominantly and recessively inherited hearing impairment, respectively. These findings suggested that cells infected with HCMV might provide a reservoir for genetic damage and, in a clinical perspective, a scenario could be envisioned whereby hearing impairment could result from early DNA damage of dividing fetal cells rather than viral replication and cell lysis. In this work we demonstrate, using fine mapping techniques, that HCMV infection in S-phase fibroblast cells induces genetic damage at 1q23.3, within a maximal region of 37 kb, containing five low copy repeat (LCR) elements. The breakpoint is situated between two hearing impairment (HI) loci, DFNA49 and DFNA7, and in close proximity to the MPZ gene previously shown to be involved in autosomal dominant Charcot-Marie-Tooth syndrome (CMT1B) with auditory neuropathy.     

Phylogenetic relevance of the genital sclerites of Neuropterida (Insecta: Holometabola)

Abstract Segment 9 of male Raphidioptera, comprising tergite, sternite, gonocoxites, gonostyli and gonapophyses, is a benchmark for homologies in the male and female terminalia of the three Neuropterida orders Raphidioptera, Megaloptera and Neuroptera. The segments relating to genitalia are 9, 10 and 11 in males and 7, 8 and 9 in females. Results from holomorphological and recent molecular cladistic analyses of Neuropterida agree in supporting the sister‐group relationships between: (1) the Raphidioptera and the clade Megaloptera + Neuroptera, and (2) the suborder Nevrorthiformia and all other Neuroptera. The main discrepancy between the results of these studies is the nonmonophyly of the suborder Hemerobiiformia in the molecular analysis. The monophyly of the Megaloptera (which has been repeatedly questioned) is further corroborated by a hitherto overlooked ground pattern autapomorphy: the presence of eversible sacs within the complex of the fused gonocoxites 11 in Corydalidae and Sialidae. The recently discovered paired complex of gonocoxites 10 (parameres) in Nipponeurorthus (Nevrorthidae) indicates that the curious apex of sternite 9 of Nevrorthus and Austroneurorthus is the amalgamation of the sclerites of gonocoxites 10 with sternite 9, interpreted as synapomorphic. In the molecular study, the Nevrorthidae, Sisyridae and Osmylidae branch off in consecutive splitting events, a result that is supported by the analysis of male genital sclerites reported here. Extraordinary parallel apomorphies (e.g. excessive enlargement and modification of gonocoxites 10 ending in a thread‐like ‘penisfilum’) in derived representatives of Coniopterygidae, Berothidae, Rhachiberothidae and Mantispidae corroborate the dilarid clade of the morphological analysis and leads us to hypothesize a sister‐group relationship of the Coniopterygidae with the dilarid clade. A re‐interpretation of the tignum of Chrysopidae as gonocoxites 11 means that the structure previously called the gonarcus represents the fused gonocoxites 9. In Hemerobiidae, the corresponding sclerite is consequently also homologized as fused gonocoxites 9. The enlargement of the lateral wings of the gonocoxites in both families is interpreted as a synapomorphy. Excessive enlargement of gonostyli 11 in the Polystoechotid clade and Myrmeleontiformia supports a sister‐group relationship of these two clades. The occurrence of certain serial homologues of female genitalia structures (gonocoxites and gonapophyses), such as the digitiform processus together with the flat appendices in segment 8 of certain Myrmeleontidae, or the wart‐like processus together with the flat circular sclerites in segment 7 of certain Berothidae, as well as the presence of gonocoxites 8 as pseudosternites in certain Nemopteridae and Coniopterygidae, are probably character reversals. The digitiform processus of tergite 9 (pseudogonocoxites) in Rhachiberothidae and Austroberothella (Berothidae) are either independently developed acquisitions with a function in oviposition, or are homologous sclerites, possibly of epipleurite origin.     

Genetic regulation of amniotic fluid TNF-alpha and soluble TNF receptor concentrations affected by race and preterm birth

Racial disparity in spontaneous preterm birth (PTB) between African Americans and Caucasians in the US is unexplained, but is probably related to differences in amniotic fluid (AF) inflammatory cytokine profiles. Therefore, this study analyzed the association of 34 single nucleotide polymorphisms (SNPs) in TNF-α and its receptor genes (TNFR1 and TNFR2) with AF TNF-α and soluble TNF receptor (R1 and R2) concentrations in PTB. Samples consisted of African American and Caucasian cases (PTB), and controls (term birth) for which both cytokine, and maternal and fetal genotype data were available. Analyses were performed with genotype, case, and maker-status interaction in the model for log transformed cytokine concentrations. In Caucasians, two interactions between genotype and pregnancy outcome associated with cytokine concentrations, whereas 14 gene variants in African Americans showed interactions with pregnancy outcome, and 13 showed association with genetic markers. In conclusion, cytokine concentrations in African American preterm births can be partially explained by interactions between pregnancy outcome, SNPs and infection. This does not appear to be the case in Caucasians. These findings may be important in understanding disparity in rates of PTB between the two populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Abnormal Skeletal Muscle Regeneration plus Mild Alterations in Mature Fiber Type Specification in Fktn-Deficient Dystroglycanopathy Muscular Dystrophy Mice

Glycosylated α-dystroglycan provides an essential link between extracellular matrix proteins, like laminin, and the cellular cytoskeleton via the dystrophin-glycoprotein complex. In secondary dystroglycanopathy muscular dystrophy, glycosylation abnormalities disrupt a complex O-mannose glycan necessary for muscle structural integrity and signaling. Fktn-deficient dystroglycanopathy mice develop moderate to severe muscular dystrophy with skeletal muscle developmental and/or regeneration defects. To gain insight into the role of glycosylated α-dystroglycan in these processes, we performed muscle fiber typing in young (2, 4 and 8 week old) and regenerated muscle. In mice with Fktn disruption during skeletal muscle specification (Myf5/Fktn KO), newly regenerated fibers (embryonic myosin heavy chain positive) peaked at 4 weeks old, while total regenerated fibers (centrally nucleated) were highest at 8 weeks old in tibialis anterior (TA) and iliopsoas, indicating peak degeneration/regeneration activity around 4 weeks of age. In contrast, mature fiber type specification at 2, 4 and 8 weeks old was relatively unchanged. Fourteen days after necrotic toxin-induced injury, there was a divergence in muscle fiber types between Myf5/Fktn KO (skeletal-muscle specific) and whole animal knockout induced with tamoxifen post-development (Tam/Fktn KO) despite equivalent time after gene deletion. Notably, Tam/Fktn KO retained higher levels of embryonic myosin heavy chain expression after injury, suggesting a delay or abnormality in differentiation programs. In mature fiber type specification post-injury, there were significant interactions between genotype and toxin parameters for type 1, 2a, and 2x fibers, and a difference between Myf5/Fktn and Tam/Fktn study groups in type 2b fibers. These data suggest that functionally glycosylated α-dystroglycan has a unique role in muscle regeneration and may influence fiber type specification post-injury.