Effect of VSV M2 and M3 expression on nucleus-cytoplasm transport of mRNAs.

<p>BHK-T7 cells were transfected for 2 h with pTM1 empty (mock) or pTM1 encoding M1, M2 or M3 proteins. Cells were fixed 6 hpt and <i>in situ</i> hybridization with fluorescein-labeled oligo (dT) probe was carried out to detect cellular mRNAs. VSV M proteins were visualized by immunofluorescence using specific monoclonal antibodies against M1 (αM) and the corresponding mouse secondary antibody conjugated to Alexa 555. To-Pro3 was used as a nuclear marker. Images were acquired with a confocal microscope. Merged images are shown on the right.</p

Subcellular localization of VSV M proteins.

<p>BHK-T7 cells were transfected for 2 h with pTM1 encoding VSV M1 (A-D), M2 (E-F) or M3 (G-H), and fixed 6 h later. Expression and localization of viral proteins were analyzed by immunofluorescence using specific monoclonal antibodies against M1 that also recognize M2 and M3, and the corresponding mouse secondary antibody conjugated to Alexa 488. Localization of M1 in intracellular membranes and dot-like structures at the nuclear envelope or at the cell surface is shown in panels A, B and C, respectively. Localization of M2 and M3 in intracellular compartments (E and G) or surrounding the nucleus (F and H) is shown. Images were acquired with an Axiovert microscope connected to a digital camera.</p

Expression of 2A^pro provokes nuclear-cytoplasmic redistribution of splicing factors.

<p>(A) Huh7-T7 cells were transfected with pTM1-2A, pTM1-2AM, PV-Rep or empty plasmid. Subcellular distributions of the indicated proteins were analyzed by immunoblot using the respective antibodies. <i>T</i> refers to total, <i>N</i> refers to nuclear, and <i>C</i> refers to cytoplasmic fractions. GAPDH and Ref1 were used as controls for cytoplasmic and nuclear location respectively. (B) The densitometry of the bands was used to calculate the cytoplasm/nucleus ratio for each protein shown. Data are mean ± s.d. (n = 3; *<i>P</i><0.05; **<i>P</i><0.01 by Student’s <i>t</i>-test). (C-E) Immunofluorescence staining of TIA1, TIAR and HuR proteins in Huh-7-T7 cells. Cells transfected as in (A) were immunolabelled with the indicated antibodies. Merge/Topro-3 refers to simultaneous visualization of images. Scale bars: 10 µm.</p

Induction of vesicle budding from the plasma membrane by VSV M proteins.

<p>BHK-T7 cells were transfected for 2 h with pTM1 encoding M1 (A-F), pTM1 empty (G), M2 (H) or M3 (I). Cells were fixed 6 h after transfection and immunodetection of VSV M proteins was performed using specific monoclonal antibodies and the corresponding mouse-secondary antibodies coupled to gold particles. Cells were visualized with a transmission electron microscope. Arrows indicate sites of vesicle budding at the plasma membrane (A-E) where M1 protein is concentrated, as well as vesicles already released from the cells (F). Statistical analyses of the gold granules distribution was carried out by unpaired (two-tailed) Student <i>t</i>-test. p < 0.01 using the Stata Program Version 11.0.</p

TIA1 and TIAR overexpression promotes Fas exon 6 inclusion in 2A^pro-expressing cells.

<p>(A) Workflow: Huh7-T7 cells were transfected with plasmids expressing MS2BP, MS2-TIA1 or MS2-TIAR. At 12 hpt the cells were co-transfected with Fas minigene and pTM1-2A, 2AM or empty pTM1 plasmid. Cells were analyzed at 3 hpt. (B) Cells from (A) were fractionated as described above and immunoblot analysis using the antibodies indicated was performed (from top to bottom: eIF4GI, MS2BP, TIA1, TIAR, HuR and GAPDH). (C) RT-PCR analysis of alternatively spliced products from cells processed in (B). (D) Intensity of the bands was calculated by densitometry and the values of ratios between 5–7 and 5–6–7 amplification products were expressed as mean ± s.d. (n = 3; *<i>P</i><0.05; **<i>P</i><0.01 by Student’s <i>t</i>-test). The dotted line indicates the ratio of the control sample.</p

VSV M proteins do not alter cell membrane permeability.

<p>(A) BHK-T7 cells were transfected with pTM1 empty plasmid or pTM1 constructs encoding M1, M2 or M3 proteins. As a positive control, cells were transfected with pTM1-2B (encoding poliovirus 2B viroporin). The medium was removed 2 h later and fresh DMEM containing 5% FCS was added. Cells were pre-treated 15 h after transfection with 0.5 mM of the translation inhibitor hygromycin B (HB) for 15 min. Then, cells were metabolically labelled with [³⁵S]Met/Cys for 45 minutes in the presence or absence of HB. Samples were processed by SDS-PAGE (17.5%), fluorography and autoradiography. (B) BHK-21 cells were mock transfected or electroporated with SV-derived mRNA replicons: Rep C, Rep C+M1 or Rep C+6K, obtained by <i>in vitro</i> transcription from their corresponding DNA templates. Cells were pre-treated with HB and metabolically labeled as indicated in (A). Numbers below each lane indicate the percentages of protein synthesis calculated by dividing the densitometric values for HB-treated cells by the values for untreated cells. A cellular protein band in mock transfected cells, or the band corresponding to the SV C protein in replicon transfected cells, was quantified by densitometric scanning, respectively. Bands corresponding to SV C and VSV M1 protein are indicated with arrows. Detection of α-tubulin served as loading control.</p

Cytotoxic effect mediated by expression of VSV M proteins.

<p>BHK-T7 cells were transfected with pTM1 empty (mock), pTM1-M1, pTM1-M2 or pTM1-M3 for 2 h. Cells were then washed and incubated in DMEM containing 5% FCS until they were fixed at 6, 18 and 24 h post transfection (hpt). Cell morphology was examined with a phase-contrast microscope.</p

The URE6 sequence mediates skipping of Fas exon 6 in 2A^pro-expressing cells.

<p>(A) Schematic diagram of the human Fas minigene (Fas genomic sequence from exon 5–7) and mutations used (Izquierdo, 2010). (B and C) Huh7-T7 cells were transfected with, pTM1-2A or pTM1-2AM or empty (mock) plasmid and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073723#pone-0073723-g001" target="_blank">Figure 1B</a>. (D) Intensity of the bands from panels B and C was calculated by densitometry and the values of ratios between 5–7 and 5–6–7 amplification products were expressed as mean ± s.d. (n = 2). The dotted line indicates the ratio of the control sample.</p

Model for human Fas exon 6 splicing.

<p>(A) In the mock, TIA1 (1) and TIAR (R) are located mainly in the nucleus. In these conditions, the amount of TIA1/R is slightly higher that amount of HuR. The binding of TIA1 and TIAR to their corresponding binding site localized in Fas intron 6 promotes exon 6 inclusion. (B) In 2A^pro-expressing Huh7-T7 cells, TIA1 and TIAR are translocated to the cytoplasm. The binding of HuR (H) to its site promotes Fas exon 6 skipping.</p

Table_6_Infection of Fungi and Bacteria in Brain Tissue From Elderly Persons and Patients With Alzheimer’s Disease.pdf

<p>Alzheimer’s disease (AD) is the leading cause of dementia in elderly people. The etiology of this disease remains a matter of intensive research in many laboratories. We have advanced the idea that disseminated fungal infection contributes to the etiology of AD. Thus, we have demonstrated that fungal proteins and DNA are present in nervous tissue from AD patients. More recently, we have reported that bacterial infections can accompany these mycoses, suggesting that polymicrobial infections exist in AD brains. In the present study, we have examined fungal and bacterial infection in brain tissue from AD patients and control subjects by immunohistochemistry. In addition, we have documented the fungal and bacterial species in brain regions from AD patients and control subjects by next-generation sequencing (NGS). Our results from the analysis of ten AD patients reveal a variety of fungal and bacterial species, although some were more prominent than others. The fungal genera more prevalent in AD patients were Alternaria, Botrytis, Candida, and Malassezia. We also compared these genera with those found in elderly and younger subjects. One of the most prominent genera in control subjects was Fusarium. Principal component analysis clearly indicated that fungi from frontal cortex samples of AD brains clustered together and differed from those of equivalent control subjects. Regarding bacterial infection, the phylum Proteobacteria was the most prominent in both AD patients and controls, followed by Firmicutes, Actinobacteria, and Bacteroides. At the family level, Burkholderiaceae and Staphylococcaceae exhibited higher percentages in AD brains than in control brains. These findings could be of interest to guide targeted antimicrobial therapy for AD patients. Moreover, the variety of microbial species in each patient may constitute a basis for a better understanding of the evolution and severity of clinical symptoms in each patient.</p