A Bemisia tabaci midgut protein interacts with begomoviruses and plays a role in virus transmission

Begomoviruses are a major group of plant viruses, transmitted exclusively by Bemisia tabaci (Gennadius) in a persistent circulative non‐propagative manner. The information regarding molecular and cellular basis underlying Begomovirus – whitefly interaction is very scarce. Evidences have suggested that the insect gut possesses some crucial protein receptors that allow specific entry of virus into the insect haemolymph. We have performed yeast two hybrid gut cDNA expression library screening against coat protein of Tomato leaf curl New Delhi virus (ToLCV) and Cotton leaf curl Rajasthan virus (CLCuV) as bait. Midgut protein (MGP) was the common protein found interacting with both ToLCV and CLCuV. MGP was localized in whole mount B. tabaci as well as in dissected guts through confocal microscopy. Pull down and dot blot assays confirmed in vitro interaction between ToLCV/CLCuV coat protein and MGP. Immunolocalization analysis also showed colocalization of ToLCV/CLCuV particles and MGP within insect's gut. Finally, anti‐MGP antibody fed B. tabaci, exhibited 70% reduction in ToLCV transmission, suggesting a supportive role for MGP in virus transmission.     

Revelation of Influencing Factors in Overall Codon Usage Bias of Equine Influenza Viruses

Equine influenza viruses (EIVs) of H3N8 subtype are culprits of severe acute respiratory infections in horses, and are still responsible for significant outbreaks worldwide. Adaptability of influenza viruses to a particular host is significantly influenced by their codon usage preference, due to an absolute dependence on the host cellular machinery for their replication. In the present study, we analyzed genome-wide codon usage patterns in 92 EIV strains, including both H3N8 and H7N7 subtypes by computing several codon usage indices and applying multivariate statistical methods. Relative synonymous codon usage (RSCU) analysis disclosed bias of preferred synonymous codons towards A/U-ended codons. The overall codon usage bias in EIVs was slightly lower, and mainly affected by the nucleotide compositional constraints as inferred from the RSCU and effective number of codon (ENc) analysis. Our data suggested that codon usage pattern in EIVs is governed by the interplay of mutation pressure, natural selection from its hosts and undefined factors. The H7N7 subtype was found less fit to its host (horse) in comparison to H3N8, by possessing higher codon bias, lower mutation pressure and much less adaptation to tRNA pool of equine cells. To the best of our knowledge, this is the first report describing the codon usage analysis of the complete genomes of EIVs. The outcome of our study is likely to enhance our understanding of factors involved in viral adaptation, evolution, and fitness towards their hosts.     

Effect of Melatonin in Epithelial Mesenchymal Transition Markers and Invasive Properties of Breast Cancer Stem Cells of Canine and Human Cell Lines

Cancer stem cells (CSCs) have been associated with metastasis and therapeutic resistance and can be generated via epithelial mesenchymal transition (EMT). Some studies suggest that the hormone melatonin acts in CSCs and may participate in the inhibition of the EMT. The objectives of this study were to evaluate the formation of mammospheres from the canine and human breast cancer cell lines, CMT-U229 and MCF-7, and the effects of melatonin treatment on the modulation of stem cell and EMT molecular markers: OCT4, E-cadherin, N-cadherin and vimentin, as well as on cell viability and invasiveness of the cells from mammospheres. The CMT-U229 and MCF-7 cell lines were subjected to three-dimensional culture in special medium for stem cells. The phenotype of mammospheres was first evaluated by flow cytometry (CD44⁺/CD24^low/- marking). Cell viability was measured by MTT colorimetric assay and the expression of the proteins OCT4, E-cadherin, N-cadherin and vimentin was evaluated by immunofluorescence and quantified by optical densitometry. The analysis of cell migration and invasion was performed in Boyden Chamber. Flow cytometry proved the stem cell phenotype with CD44⁺/CD24^low/- positive marking for both cell lines. Cell viability of CMT-U229 and MCF-7 cells was reduced after treatment with 1mM melatonin for 24 h (P<0.05). Immunofluorescence staining showed increased E-cadherin expression (P<0.05) and decreased expression of OCT4, N-cadherin and vimentin (P<0.05) in both cell lines after treatment with 1 mM melatonin for 24 hours. Moreover, treatment with melatonin was able to reduce cell migration and invasion in both cell lines when compared to control group (P<0.05). Our results demonstrate that melatonin shows an inhibitory role in the viability and invasiveness of breast cancer mammospheres as well as in modulating the expression of proteins related to EMT in breast CSCs, suggesting its potential anti-metastatic role in canine and human breast cancer cell lines.     

Characterization of a spontaneous mutant of Azotobacter vinelandii in which vanadium-dependent nitrogen fixation is not inhibited by molybdenum

A spontaneous mutant derivative of Azotobacter vinelandii CA12 (ΔnifHDK), in which vanadium-dependent nitrogen fixation is not inhibited by molybdenum (A. vinelandii CARR), grows profusely on BNF-agar containing 1 μM Na₂MoO₄, alone or supplemented with 1 μM V₂O₅. The expression of A. vinelandii vnfH::lacZ and vnfA::lacZ fusions in A. vinelandii CARR was not inhibited by 1 mM Na₂MoO₄, whereas molybdenum at much lower concentration inhibited the expression of vnfH::lacZ and vnfA::lacZ fusions in A. vinelandii CA12. The mutant also exhibited normal acetylene reduction activity in the presence of 1 μM Na₂MoO₄. The expression of A. vinelandii nifH::lacZ fusion in A. vinelandii CARR was low even though the cells were cultured under non-repressing conditions with urea as nitrogen source in the presence of Na₂MoO₄. The molybdenum content of A. vinelandii CARR cells was found to be about one-fourth that of A. vinelandii CA12. No nitrate reductase activity could be detected in A. vinelandii CARR when the cells were cultured in the presence of 10 μM Na₂MoO₄, whereas A. vinelandii CA12 exhibited some activity even with 100 pM Na₂MoO₄.     

Relationship between timing of development,morula morphology,and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos

Noninvasive measurements of bovine embryo quality, such as timing of cleavage, morula morphology, blastocyst formation, and hatching ability, were linked with the number of inner cell mass (ICM) cells and trophectoderm (TE) cells of the resulting embryos. First, it was confirmed that fast-cleaving embryos proved to have significantly higher chances to reach advanced developmental stages vs. intermediate and slow cleavers (P = 0.01). They also showed significantly less fragmentation at the morula stage, implying the presence of more excellent morulae among fast-cleaving embryos (P < 0.05). Second, the quality of hatched blastocysts, resulting from morulae of different morphological grades, was examined by differential staining. The total cell and ICM cell numbers were significantly lower for hatched blastocysts developed from poor morulae compared to hatched blastocysts developed from excellent, good, or fair morulae. However, hatched blastocysts with <10 ICM cells were seen in embryos belonging to all four morphological scores. Finally, it was found that timing of first cleavage was not significantly correlated with timing of blastocyst formation or with cell number of blastocysts. Timing of blastocyst formation, however, was significantly correlated with cell number: day 8 blastocysts had significantly lower total cell and ICM cell numbers than day 6 and day 7 blastocysts (P < 0.001). These results suggest that the quality of in vitro-produced bovine embryos is very variable and cannot be linked with a single criterion such as embryo morphology and/or hatching ability. Timing of blastocyst formation was the most valuable criterion with regard to embryonic differentiation. Mol. Reprod. Dev. 47:47–56, 1997. © 1997 Wiley-Liss, Inc.     

Method for collecting and immobilizing individual cumulus cells enabling quantitative immunofluorescence analysis of proteins

Most immunofluorescence methods rely on techniques dealing with a very large number of cells. However, when the number of cells in a sample is low (e.g., when cumulus cells must be analyzed from individual cumulus–oocyte complexes), specific techniques are required to conserve, fix, and analyze cells individually. We established and validated a simple and effective method for collecting and immobilizing low numbers of cumulus cells that enables easy and quick quantitative immunofluorescence analysis of proteins from individual cells. To illustrate this technique, we stained proprotein of a disintegrin and metalloproteinase with thrombospondin-like repeats-1 (proADAMTS-1) and analyzed its levels in individual porcine cumulus cells.