The Cellular Distribution of Serotonin Transporter Is Impeded on Serotonin-Altered Vimentin Network

BACKGROUND:The C-terminus of the serotonin transporter (SERT) contains binding domains for different proteins and is critical for its functional expression. In endogenous and heterologous expression systems, our proteomic and biochemical analysis demonstrated that an intermediate filament, vimentin, binds to the C-terminus of SERT. It has been reported that 5HT-stimulation of cells leads to disassembly and spatial reorientation of vimentin filaments. METHODOLOGY/PRINCIPAL FINDINGS:We tested the impact of 5HT-stimulation on vimentin-SERT association and found that 5HT-stimulation accelerates the translocation of SERT from the plasma membrane via enhancing the level of association between phosphovimentin and SERT. Furthermore a progressive truncation of the C-terminus of SERT was performed to map the vimentin-SERT association domain. Deletion of up to 20, but not 14 amino acids arrested the transporters at intracellular locations. Although, truncation of the last 14 amino acids, did not alter 5HT uptake rates of transporter but abolished its association with vimentin. To understand the involvement of 5HT in phosphovimentin-SERT association from the plasma membrane, we further investigated the six amino acids between Delta14 and Delta20, i.e., the SITPET sequence of SERT. While the triple mutation on the possible kinase action sites, S(611), T(613), and T(616) arrested the transporter at intracellular locations, replacing the residues with aspartic acid one at a time altered neither the 5HT uptake rates nor the vimentin association of these mutants. However, replacing the three target sites with alanine, either simultaneously or one at a time, had no significant effect on 5HT uptake rates or the vimentin association with transporter. CONCLUSIONS/SIGNIFICANCE:Based on our findings, we propose that phosphate modification of the SITPET sequence differentially, one at a time exposes the vimentin binding domain on the C-terminus of SERT. Conversely, following 5HT stimulation, the association between vimentin-SERT is enhanced which changes the cellular distribution of SERT on an altered vimentin network

Bifrontal Osteoplastic Flap: An Option to Decrease Infection in Bifrontal Craniotomies with Skull Base Osteotomies

Infection can be a common complication following bifrontal craniotomy with skull base osteotomies given the potential violation of sinuses and entry into the nasal structures. Our objective was to examine our series of patients who underwent a bifrontal craniotomy with skull base osteotomies and describe the infection rate. We propose the bifrontal osteoplastic flap as an adjunct to infection prevention. A retrospective single-center study of a patient database was performed. Twenty patients were identified. Fifty-five percent were male. The mean age was 55.7 ± 13.9 years. The most common indications for surgery were esthesioneuroblastomas (35%) and anterior skull base meningiomas (30%). Six patients (30%) developed an infection, 1 patient (5%) developed a CSF leak, and no patients developed a mucocele. All 6 infected cases had nasal pathology with intracranial extension, they all received chemoradiation post-operatively and were all combined cases with otorhinolaryngology. Eighty-three percent of these patients required a craniectomy and all of them required long-term IV antibiotics. Infection is not uncommon after a bifrontal craniotomy with skull base osteotomies and the use of the bifrontal osteoplastic flap in cases where the risk of infection is high, i.e., esthesioneuroblastomas surgery, may help reduce said risk and lead to better patient outcomes

Gene Ontology Mining Tool functional analysis.

<p>Gene list created using genes FC>1.5 and p-values≤0.05 relative to saline-infused MK pop.</p

Expression level of genes involved in GPCR-mediated platelet activation.

<p>Expression level of genes involved in GPCR-mediated platelet activation.</p

Cell-surface expression of surrogate markers of platelet activation.

<p>(<b>A</b>) FITC-labeled P-selectin (CD62P) and (<b>B</b>) Alexa Fluor 448-labeled Gp1b show increased and decreased mean fluorescence intensity (unshaded area), respectively, following <i>in vivo</i> 5-HT treatment in contrast to saline-infused littermates (gray shaded area); n = 5–6. Increased staining indicates active exocytosis of alpha-granules in platelet membranes whereas decreased expression of Gp1b is consistent with enhanced platelet-platelet binding in the presence of high 5-HT. The assay was performed in triplicate.</p

Hierarchical clustering analysis of genes differentially expressed under elevated 5-HT conditions in MK cells.

<p>The expression pattern of 233 genes whose expression was changed significantly (FC>2, p<0.001) in saline (n = 3, denoted as C1–C3) and 5-HT-treated (n = 4, denoted as T1–T4) samples are shown. The data were clustered using the standard hierarchical method with average linkage and using the Pearson correlation to determine the distance function. The normalized expression index for each gene (rows) in each sample (columns) is indicated by a color code (see Expression index bar at top left of figure). The genes shown represent the genes that were up-regulated (red) and down-regulated (green) in the sample sets. Samples with similar patterns of expression of the genes studied will cluster together, as indicated by the dendogram.</p

Functional classification of differentially expressed genes.

<p>Genes (FC>1.5, p<0.05) are broadly grouped into selected functional categories: (<b>A</b>) cytoskeletal remodeling, (<b>B</b>) G-protein signaling, (<b>C</b>) vesicular transport, and (<b>D</b>) apoptosis and survival. The abscissa refers to the signal log ratio (SLR), representing the log₂ of the average change between transcript expression in saline- and 5-HT-infused megakaryocyte populations (SLR = 1 or log₂ of 1  =  FC of 2).</p

Dense-granule secretion assay.

<p>(<b>A</b>) Platelets isolated from mice after 24 hours of 5-HT infusion exhibit increased dense-granule exocytosis, as indicated by increased levels of secreted 5-HT. Dense granule secretion was monitored by measuring the level of fluorescent material which was created by OPT as described in the Materials and Method section. The measurements were done at excitation wavelength 355 nm and emission wavelength 475 nm. Data represent mean ± SD; Asterisk (*) indicates statistical significance compared to Saline+SLO by Student’s <i>t</i>-test, <i>p<0.05</i>. (<b>B</b>) Dense-granule secretion was monitored following plasma membrane staining for granulophysin (CD63), a surrogate marker of exocytosis. FACS analysis demonstrates increased mean fluorescence intensity following 5-HT infusion compared to saline-infused littermates.</p