Arch Sex Behav

Thirty years after the beginning of the HIV epidemic, gay, bisexual, and other men who have sex with men (collectively called MSM) bear a disproportionate burden of HIV in the United States and continue to acquire a distressingly high number and proportion of new infections. Historically, HIV prevention for MSM has been focused on individual-level behavior change, rarely intervening with MSM as part of a couple. Yet, an estimated 33\u201367% of HIV infections among MSM are acquired from primary sexual partners, suggesting that work with MSM as couples could be an important contributor to prevention. Given the emergence of high impact combination HIV prevention, it is timely to consider how work with the broad variety of male couples can improve both personal and community health. Couples HIV testing and counseling for MSM is an important advance for identifying men who are unaware that they are HIV-positive, identifying HIV-discordant couples, and supporting men who want to learn their HIV status with their partner. Once men know their HIV status, new advances in biomedical prevention, which can dramatically reduce risk of HIV transmission or acquisition, allow men to make prevention decisions that can protect themselves and their partners. This paper highlights the present-day challenges and benefits of using a couples-based approach with MSM in the era of combination prevention to increase knowledge of HIV status, increase identification of HIV discordant couples to improve targeting prevention services,and support mutual disclosure of HIV status.CC999999/Intramural CDC HHS/United States2017-01-09T00:00:00Z24233328PMC5221480vault:2090

Spatial-Temporal Analysis of Land Cover and Use Changes Using Gis Tools. Case Study Băneasa Neighborhood, Bucharest

The paper topic is related to the use of Geographic Information System and remote sensing in analysing the spatio-temporal dynamics of a post-socialist city. The purpose of this study is to highlight the changes in the land use of the Băneasa neighbourhood, located in the northern part of the capital city of Romania. The changes have been analysed from the nineteenth century to the present. The authors used historical mapping using old maps, as well as digitizing aerial photographs using the Geographic Information System. There has been an accelerated territorial evolution in the last 70 years, marked by the impact of the socialist policy of intensive urbanization, when the urban-type systematization was applied through the construction of collective dwellings, to which were added some important industrial units. After 1990, the dynamics of the tertiary sector generated significant changes in the physiognomy of the urban landscape, observing the increase of the built space (commercial spaces, new residential areas) at the expense of green spaces, tree nurseries, greenhouses and industrial sites. The most recent stage of evolution, however, has brought the most profound changes in the landscape of the Băneasa district, as a result of the new political and socio-economic context, marked by the collapse of communism. Thus, in the last 30 years, the capital city has registered an uncontrolled urban growth dynamic, this being characteristic of other large urban centres at the national level as well favoured by less strict, inconsistent and incomplete urban legislation

Polarized Trafficking of AQP2 Revealed in Three Dimensional Epithelial Culture

In renal collecting duct (CD) principal cells (PCs), vasopressin (VP) acts through its receptor, V2R, to increase intracellular cAMP leading to phosphorylation and apical membrane accumulation of the water channel aquaporin 2 (AQP2). The trafficking and function of basolaterally located AQP2 is, however, poorly understood. Here we report the successful application of a 3-dimensional Madin-Darby canine kidney (MDCK) epithelial model to study polarized AQP2 trafficking. This model recapitulates the luminal architecture of the CD and bi-polarized distribution of AQP2 as seen in kidney. Without stimulation, AQP2 is located in the subapical and basolateral regions. Treatment with VP, forskolin (FK), or 8-(4-Chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate monosodium hydrate (CPT-cAMP) leads to translocation of cytosolic AQP2 to the apical membrane, but not to the basolateral membrane. Treating cells with methyl-β-cyclodextrin (mβCD) to acutely block endocytosis causes accumulation of AQP2 on the basolateral membrane, but not on the apical membrane. Our data suggest that AQP2 may traffic differently at the apical and basolateral domains in this 3D epithelial model. In addition, application of a panel of phosphorylation specific AQP2 antibodies reveals the polarized, subcellular localization of differentially phosphorylated AQP2 at S256, S261, S264 and S269 in the 3D culture model, which is consistent with observations made in the CDs of VP treated animals, suggesting the preservation of phosphorylation dependent regulatory mechanism of AQP2 trafficking in this model. Therefore we have established a 3D culture model for the study of trafficking and regulation of both the apical and basolaterally targeted AQP2. The new model will enable further characterization of the complex mechanism regulating bi-polarized trafficking of AQP2 in vitro

Cyclic AMP stimulates apical V-ATPase accumulation, microvillar elongation and proton extrusion in kidney collecting duct A-intercalated cells

Kidney proton-secreting A-intercalated cells (A-IC) respond to systemic acidosis by accumulating the vacuolar ATPase (V-ATPase) in their apical membrane and by increasing the length and number of apical microvilli. We show here that the cell permeant cAMP analog CPT-cAMP, infused in vivo, results in an almost two-fold increase in apical V-ATPase accumulation in AE1-positive A-IC within 15 min, and that these cells develop an extensive array of apical microvilli compared to controls. In contrast, no significant change in V-ATPase distribution could be detected by immunocytochemistry in B-intercalated cells at the acute time point examined. To show a direct effect of cAMP on A-IC, we prepared cell suspensions from the medulla of transgenic mice expressing EGFP in IC (driven by the B1-subunit promoter of the V-ATPase) and exposed them to cAMP analogs in vitro. 3D-reconstructions of confocal images revealed that cAMP induced a time dependent growth of apical microvilli, starting within minutes after addition. This effect was blocked by the PKA inhibitor, myristoylated PKI. These morphological changes were paralleled by increased cAMP-mediated proton extrusion (pHi recovery) by A-IC in outer medullary collecting ducts measured using the ratiometric probe BCECF. These results, and our prior data showing that the bicarbonate-stimulated soluble adenylyl cyclase (sAC) is highly expressed in kidney intercalated cells, support the idea that cAMP generated either by sAC, or by activation of other signaling pathways, is part of the signal transduction mechanism involved in acid-base sensing and V-ATPase membrane trafficking in kidney intercalated cells

Subcellular distribution of phosphorylated AQP2 in MDCK cells grown in 3D culture and in Brattleboro rat kidney.

<p>Antibodies recognizing AQP2 phosphorylated at S256, S261, S264 or S269 highlighted the subcellular localization of phosphorylated AQP2 in MDCK-AQP2 cysts (A-H). The staining pattern of differentially phosphorylated AQP2 in the cyst is reminiscent of that observed in Brattleboro rat kidneys (I-P). Prior to stimulation, cysts were pre-incubated in serum free medium for 120 min. Comparing the distribution of phosphorylated AQP2 in cyst versus kidney tissue, pS256 is found in the cytosol and apical membrane in both the cyst and kidney tissue without VP treatment (A, I) Total AQP2 staining of the tissue in panel I can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131719#pone.0131719.g003" target="_blank">Fig 3B</a>. After stimulation with VP (E,M), the redistribution of pS256 AQP2 to the apical membrane is pronounced in the kidney tissue, but is not significantly different in the cyst tissue (quantification in panel R). VP stimulation resulted in a reduction of both apical and cytosolic pS261 fluorescence intensity in cyst and kidney tissue (B vs. F, J vs. N). Under stimulated conditions, some non-specific labeling of the nuclei by the p261 antibody can be observed (F). pS264 staining signal translocated from the cytosolic compartment (C,K) to the apical membrane (G, O) after VP stimulation in both cyst and kidney tissue (H, P). In both the cyst, and the rat tissue pS269 staining was only observed following stimulation, and solely located on the apical membrane (H,P). Bar = 10 μm. (Q) Differential phosphorylation of AQP2 at various serine residues after FK stimulation was detected by western blot in AQP2 expressing MDCK cells grown as a monolayer culture. The bar graph represents quantification of western blots (means ± SE, N = 3 experiments): total AQP2 and phosphorylation antibody results are presented as relative to loading control. No significant difference was observed between non-stimulated and VP/FK simulated conditions for AQP2 pS256 or pS264. On the other hand, stimulation resulted in a significant (asterisk) decrease in pS261 (p = .002) and a significant increase in pS269 (p = .04). (R) Quantitative assessment of cyst immunofluorescence images reveal significant (P < = 0.05) increases in apical, relative to intracellular, pS264 (N = 4 cysts) and pS269 (N = 8 cysts) AQP2 staining following AVP stimulation. No significant difference in apical, relative to intracellular pS261 (N = 5 cysts) fluorescence is observed due to concomitant decreases (relative to treatment) in both apical and intracellular pS261 staining.</p

Regulated trafficking of AQP2 is intact in MDCK cysts.

<p>AQP2 trafficking in MDCK-AQP2 cysts is intact and staining patterns for total AQP2 are comparable to those observed in Brattleboro, and normal rat kidney. (A) MDCK-AQP2 cysts were incubated in serum free medium for 120 minutes. Addition of AVP, FK, or CPT-cAMP to the medium for 40 minutes resulting in apical membrane accumulation of AQP2 (arrows). Bar = 10 μm. (D) Asterisk denotes that significantly (P < = 0.05) more apical, but not basolateral staining of AQP2, relative to intracellular AQP2, was observed following stimulation with AVP, FK or CPT-cAMP. N = 5 cysts (NT, non-treated), N = 12 cysts (AVP/FK/CPT-cAMP stimulated). The data for AVP, FK and cAMP-treated cysts were pooled together because we saw no statistically significant difference in Apical/Internal total AQP2 or Basolateral/Internal total AQP2 between the treatment modalities. (B) In the Brattleboro rat kidney AQP2 was located mainly in the subapical region while apical membrane accumulation of AQP2 was seen after treatment with dDAVP for 3 days. Bar = 10 μm. (C) Similarly, in a tissue slice culture from normal rat kidney, incubation in medium without VP resulted in AQP2 in the cytosol and subapical region. dDAVP treatment for 20 minutes resulted in AQP2 translocation to the apical membrane, with AQP2 still detectable in the cytosol. (E) In transmission electron micrographs, AQP2 in the MDCK-AQP2 cyst is labeled with 15nm gold particles. AQP2 gold particles distributed diffusely throughout the cytosol under baseline, non-stimulated conditions (left panel) while AQP2 accumulated on the apical membrane after VP stimulation (right panel), but not on the basolateral membrane (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131719#pone.0131719.s002" target="_blank">S2 Fig</a>). Bars = 500 nm</p

Ultrastructure of MDCK-AQP2 cysts.

<p>The ultrastructure of the cyst closely resembles the polarized architecture of renal tubular epithelium in animal kidney. (A) A differential interference contrast image of a whole cyst. The lumen is clearly visible despite some remnants of cells that were shed in the lumen during lumen formation. The basal membrane is in contact with the Matrigel substrate. Scale = 10 μm. (B) In a TEM cross section of a single cell from a MDCK-AQP2 cyst, microvilli can be seen on the apical membrane facing the lumen (L) while the nucleus (N) is oriented toward the basal membrane. Scale = 2 μm. (C) At the apical domain, neighboring cells interact through tight junctions (TJ) and desmosomes (D) and the presence of clathrin-coated pits (arrow) indicate active endocytosis in the apical and subapical region. Scale = 500 nm. (D) Similarly, clathrin coated vesicles are observed at the basal and lateral membranes of the cyst cells (arrows). Scale = 500 nm. In all images (M) indicates mitochondria.</p