In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter

<p>Abstract</p> <p>Background</p> <p>Endothelial cells are a target for gene therapy because they are implicated in a number of vascular diseases. Recombinant baculovirus have emerged as novel gene delivery vectors. However, there is no information available concerning the use of endothelial-specific promoters in the context of the baculovirus genome. In the present study, we have generated a recombinant baculovirus containing the human flt-1 promoter (BacFLT-GFP) driving the expression of the green fluorescent protein. Transcriptional gene targeting was analyzed <it>in vitro </it>in different mammalian cell lines and <it>in vivo </it>in adult rat retinal vasculature.</p> <p>Results</p> <p>BacFLT-GFP evoked the highest levels of expression in the endothelial cell line BUVEC-E6E7-1, similar to those reached by recombinant baculovirus carrying the CMV promoter (112% relative to BacCMV-GFP, <it>n </it>= 4). Interestingly, BacFLT-GFP directed high levels of expression in rat glioma C6 and in human glioblastoma CH235 cells (34.78% and 47.86% relative to BacCMV-GFP, respectively). Histone deacetylase inhibitors such as butyrate or trichostatin A enhanced the transcriptional activity of both BacCMV-GFP and BacFLT-GFP. Thus, in this study histone deacetylation appears to be a central mechanism for the silencing of baculovirus, independently of the promoter utilized. <it>In vivo </it>transcriptional targeting was demonstrated in adult rat retinal vasculature by intravitreal delivery of BacFLT-GFP and immunohistochemical staining with von Willebrand factor (vWF). Analysis by fluorescence microscopy and deconvolved three-dimensional confocal microscopy of retinal whole mounts obtained after 3 days of baculovirus injection showed that most GFP-expressing cells localized to the inner limiting membrane (ILM) and ganglion cell layer (GCL) and colocalize with vWF (70%, <it>n </it>= 10) in blood vessels, confirming the endothelial phenotype of the transduced cells.</p> <p>Conclusion</p> <p>Taken together, our results indicate that the restricted expression in endothelial cells mediated by the flt-1 promoter is not affected by the context of the baculovirus genome and demonstrate the potential of using recombinant baculovirus for transcriptional targeted gene expression into the eye vasculature.</p

Anti‑inflammatory drugs and uterine cervical cancer cells: Antineoplastic effect of meclofenamic acid

Uterine cervical cancer (UCC) is one of the main causes of cancer-associated mortality in women. Inflammation has been identified as an important component of this neoplasia; in this context, anti-inflammatory drugs represent possible prophylactic and/or therapeutic alternatives that require further investigation. Anti-inflammatory drugs are common and each one may exhibit a different antineoplastic effect. As a result, the present study investigated different anti-inflammatory models of UCC in vitro and in vivo. Celecoxib, sulindac, nimesulide, dexamethasone, meclofenamic acid, flufenamic acid and mefenamic acid were tested in UCC HeLa, VIPA, INBL and SiHa cell lines. The cytotoxicity of the drugs was evaluated in vitro. Celecoxib, sulindac, nimesulide, mefenamic acid and flufenamic acid presented with slight to moderate toxicity (10–40% of cell death corresponding to 100 µM) in certain cell lines, while meclofenamic acid exhibited significant cytotoxicity in all essayed cell lines (50–90% of cell death corresponding to 100 µM). The meclofenamic acid was tested in murine models (immunodeficient and immunocompetent) of UCC, which manifested a significant reduction in tumor growth and increased mouse survival. It was demonstrated that of the evaluated anti-inflammatory drugs, meclofenamic acid was the most cytotoxic, with a significant antitumor effect in murine models. Subsequent studies are necessary to evaluate the clinical utility of this drug

Country-level gender inequality is associated with structural differences in the brains of women and men

男女間の不平等と脳の性差 --男女間の不平等は脳構造の性差と関連する--. 京都大学プレスリリース. 2023-05-10.Gender inequality across the world has been associated with a higher risk to mental health problems and lower academic achievement in women compared to men. We also know that the brain is shaped by nurturing and adverse socio-environmental experiences. Therefore, unequal exposure to harsher conditions for women compared to men in gender-unequal countries might be reflected in differences in their brain structure, and this could be the neural mechanism partly explaining women’s worse outcomes in gender-unequal countries. We examined this through a random-effects meta-analysis on cortical thickness and surface area differences between adult healthy men and women, including a meta-regression in which country-level gender inequality acted as an explanatory variable for the observed differences. A total of 139 samples from 29 different countries, totaling 7, 876 MRI scans, were included. Thickness of the right hemisphere, and particularly the right caudal anterior cingulate, right medial orbitofrontal, and left lateral occipital cortex, presented no differences or even thicker regional cortices in women compared to men in gender-equal countries, reversing to thinner cortices in countries with greater gender inequality. These results point to the potentially hazardous effect of gender inequality on women’s brains and provide initial evidence for neuroscience-informed policies for gender equality

Country-level gender inequality is associated with structural differences in the brains of women and men

Gender inequality across the world has been associated with a higher risk to mental health problems and lower academic achievement in women compared to men. We also know that the brain is shaped by nurturing and adverse socio-environmental experiences. Therefore, unequal exposure to harsher conditions for women compared to men in gender-unequal countries might be reflected in differences in their brain structure, and this could be the neural mechanism partly explaining women's worse outcomes in gender-unequal countries. We examined this through a random-effects meta-analysis on cortical thickness and surface area differences between adult healthy men and women, including a meta-regression in which country-level gender inequality acted as an explanatory variable for the observed differences. A total of 139 samples from 29 different countries, totaling 7,876 MRI scans, were included. Thickness of the right hemisphere, and particularly the right caudal anterior cingulate, right medial orbitofrontal, and left lateral occipital cortex, presented no differences or even thicker regional cortices in women compared to men in gender-equal countries, reversing to thinner cortices in countries with greater gender inequality. These results point to the potentially hazardous effect of gender inequality on women's brains and provide initial evidence for neuroscience-informed policies for gender equality

In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter-1

<p><b>Copyright information:</b></p><p>Taken from "In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter"</p><p>http://www.virologyj.com/content/4/1/88</p><p>Virology Journal 2007;4():88-88.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2034561.</p><p></p>es -748 to +284 bp); EGFP, enhanced green-fluorescent protein; BGHpA, bovine growth hormone poly adenylation sequence. (b) (Left panel), Representative histogram obtained by flow cytometry 48 h after transduction of BUVEC-E6E7-1 cells with 100 MOI of BacFLT-GFP and 5 mM of butyrate. The percentage of GFP+ cells is reported in the inset, and was calculated by subtracting the background from mock transduced cells (see Methods). (Right panel) Levels of expression obtained with BacCMV-GFP and BacFLT-GFP in BUVEC-E6E7-1 cells, representative of four independent experiments, mean ± SD. (c) Mean fluorescence intensity (MFI) measured 48 h after transduction with BacFLT-GFP relative to BacCMV-GFP. The percentage of GFP+ cells are indicate above of each bar. Cells were transduced with 100 MOI of BacFLT-GFP or BacCMV-GFP. Data are from four independent experiments ± SD

In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter-3

<p><b>Copyright information:</b></p><p>Taken from "In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter"</p><p>http://www.virologyj.com/content/4/1/88</p><p>Virology Journal 2007;4():88-88.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2034561.</p><p></p>ye vitreous chamber. Three days after the virus injection, the rats were sacrificed and the retinas processed for immunohistochemistry using an antibody against von Willebrand factor (red) and DAPI (blue) to highlight nuclei (see Methods). Representative retinas from eyes injected with 1 × 10pfu of BacFLT-GFP (a, b, c, d) or vehicle (PBS) (e, f, g, h). The same sections were evaluated by phase-contrast microscopy (a, e) and by anti-von Willebrand factor antibodies coupled to TRITC (red) and GFP green fluorescence. Merge images illustrate the colocalization of red and green fluorescence (in yellow) in capillary-like structures (big white rectangles in c, d, g) obtained from the areas in small white rectangle insets from b, f. Confocal three-dimensional reconstruction images of retinas injected with vehicle (h) or with BacFLT-GFP (i). In red is shown the localization of von Willebrand factor (red) within characteristic blood vessel structures. The colocalization of red (vWF) and green (GFP) fluorescence is shown in yellow (white arrowheads in i). (j) Percentage of vWF and GFP pixel co-localization in retinas injected with BacFLT-GFP, (= 10 animals). Abbreviations: RPE, retinal pigment epithelium GCL, ganglion cell layer; INL, inner nuclear layer; and ONL, outer nuclear layer; ILM inner limiting membrane

In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter-2

<p><b>Copyright information:</b></p><p>Taken from "In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter"</p><p>http://www.virologyj.com/content/4/1/88</p><p>Virology Journal 2007;4():88-88.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2034561.</p><p></p>FP and treated with increasing concentrations (shown in the figure) of butyrate or trichostatin A (TSA). The percentage of GFP+ cells (shown inside green rectangles) is reported in the insets, and was calculated by subtracting the background from mock transduced cells. (b) Fold induction in GFP expression mediated by BacFLT-GFP with increasing concentrations of butyrate or TSA. (c) Fold induction of the expression of GFP in transfected cells with the transfer plasmid pBlueFLT-GFP (see Methods) and treated with increasing concentrations of butyrate or TSA. Results from four independent experiments ± SD. NS = non significant

In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter-4

<p><b>Copyright information:</b></p><p>Taken from "In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter"</p><p>http://www.virologyj.com/content/4/1/88</p><p>Virology Journal 2007;4():88-88.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2034561.</p><p></p> of BacCMV-GFP. Abbreviations: CMV, cytomegalovirus immediately-early promoter/enhancer (nucleotides -655 to +106); EGFP, enhanced green-fluorescent protein; BGHpA, bovine growth hormone poly adenylation sequence. (b) Representative histograms obtained by flow cytometry after 48 h post-transduction. The percentage of GFP+ cells is reported in the insets, and was calculated by subtracting the background obtained with mock transduced cells (see Methods). Numbers in the inset refer to the percentage of GFP+ cells ± SD without (above) or with (below) treatment with butyrate. (c) Percentage of GFP+ cells determined by FACS analysis in either the presence or absence of butyrate. *< 0.01, **< 0.05, ***= 0.7244 versus cells non-treated with butyrate. NS = non significant. d) Levels of expression of GFP and induction ration (indicated on the right) as determined by the mean fluorescence intensity in the presence or absence of butyrate. Values are means ± SD of four independent experiments