Use of the ARPE-19 cell line as a model of RPE polarity: basolateral secretion of FGF5

PURPOSE: To determine the polarity of fibroblast growth factor 5 (FGF5) secretions from retinal pigment epithelium (RPE) cells and to examine the viability and utility of the ARPE-19 cell line as a model for the study of RPE polarity. METHODS: Influenza infection and adenovirus-mediated gene transfer were used to deliver and express genes encoding influenza hemagglutinin (HA), p75-NTR (a neurotrophin receptor), low-density lipoprotein (LDL) receptor (LDLR), and FGF5 in confluent monolayers of ARPE-19 cells. The localization of HA, p75-NTR, and LDLR was determined by confocal microscopy. Domain selective biotinylation assays were used to quantitatively determine the polarities of p75-NTR and LDLR. The secretion of FGF5 into the apical and basal media of ARPE-19 cultures was examined by immunoblot analysis of conditioned media. RESULTS: Hemagglutinin and p75-NTR were found to be localized on the apical surface of infected and transduced ARPE-19 cells. In contrast, LDLR was associated preferentially with the basolateral membrane of ARPE-19 cells. Biotinylation studies indicated that 84% of p75-NTR was present on the apical surface, and 79% of LDLR was basolaterally polarized. Over the course of 6 hours, more than 90% of the total secreted FGF5 protein accumulated in the basolateral media. CONCLUSIONS: ARPE-19 cells exhibit a polarized distribution of cell surface markers when examined by either confocal microscopy or surface-labeling assays. This indicates that the ARPE-19 cell line is a valid model for studies of RPE cell polarity. FGF5, a secreted protein normally produced by RPE cells, is accumulated preferentially in the basal media after only 6 hours, suggesting that it is vectorially secreted from the basolateral surface of ARPE-19 cells.     

Role of intimin and bundle-forming pili in enteropathogenic Escherichia coli adhesion to pediatric intestinal tissue in vitro

Attaching and effacing (A/E) lesion formation is central to enteropathogenic Escherichia coli (EPEC) pathogenesis. In vitro experiments with human epithelial cell lines have implicated virulence plasmid-encoded bundle-forming pili (BFP) in initial binding and intimin in intimate attachment and A/E lesion formation. This study investigated the role of BFP and intimin in EPEC interactions with pediatric small intestinal biopsy tissue in in vitro organ culture. Organ culture infections (2 to 8 h) were performed with E2348/69 (a wild-type EPEC O127:H6 clinical isolate) and E2348/69 derivatives including CVD206 (eae deficient), CVD206(pCVD438) (eae-complemented CVD206), CVD206(pCVD438/01) (expressing intimin, which is nonfunctional due to a single amino acid substitution), JPN15 (spontaneous EPEC adherence factor virulence plasmid-cured E2348/69), and 31-6-1(1) (E2348/69 with a TnphoA insertion inactivation mutation in the virulence plasmid-encoded bfpA gene). Scanning and transmission electron microscopy revealed that after 8 h E2348/69 and CVD206 (pCVD438) (both Int+ BFP+) adhered to all specimens, causing A/E lesions with surrounding microvillous elongation. JPN15 and 31-6-1(1) (both Int+ BFP-) adhered and caused A/E lesions although bacteria adhered in "flat," two-dimensional groups. CVD206 and CVD206(pCVD438/01) (both Int- BFP+) did not adhere to any sample, and no pathological tissue changes were seen. Thus, in human intestinal organ culture, BFP do not appear to be involved in the initial stages of EPEC nonintimate adhesion but are implicated in the formation of complex, three-dimensional colonies via bacterium-bacterium interactions. Intimin appears to play an essential role in establishing colonization of EPEC on pediatric small intestinal tissue.     

Wireless Sensor Networks in IPv4/IPv6 Transition Scenarios

The wireless sensor networks (WSNs) concept was appeared in the middle of 90s and have been a subject under intensive research in the past few years. Several factors have contributed to this, but the potential for application of WSNs in almost every aspect of day-to-day life is the predominant one. This type of networks has been developed using proprietary solutions instead of standard solutions. More recently, the importance of standards motivated the use of IETF standards in WSNs, making the Internet integration easier. However, more efforts are necessary in order to provide a full integration. The WSNs use mainly IPv6 protocol, but the IPv4 is the predominant one in the Internet. As a consequence, IPv4 to IPv6 transition mechanisms must be provided to allow the interaction between all Internet connected devices independently of the supported IP version. It is also critical to provide a standard application interface to make easier the application development and independently of the hardware platform used. The RESTfull Web services can provide this standard interface. So, combine RESTfull Web services with IPv4 to IPv6 transition mechanisms can increase the WSN services dissemination. The transition mechanisms and the REST Web services are supported in the gateway in order to save the wireless sensor device resources’. The smartphone with Internet connectivity can also be the drive to the WSNs growth, because user-friendly applications can be used to retrieve and collect sensed data. This paper proposes a solution based on REST web services to permit the interaction between a mobile application and the IPv6 compliant WSN.     

Risk factors associated with lameness and hoof lesions in pasture-based dairy cattle systems in southeast Brazil

Cattle lameness is an important welfare concern that also has an economic impact on the dairy industry. It can be a significant problem among pasture-based herds. Our objectives were to identify cow- and herd-level factors related to lameness and hoof lesions in dairy cows grazing year-round in Minas Gerais, Brazil. We performed a cross-sectional study in 48 pasture-based dairy herds, visiting each farm in a single visit. We evaluated 2,262 cows for mobility score (0–3) and 392 cows for hoof lesions. We used a questionnaire and checklist to capture herd management data. All information obtained was used to build multivariable models. The factors associated with lameness were low body condition score, longer time spent in the corral, being kept in paddocks during the drought period, and poor hygiene. For hoof lesions, track features were the most significant factor in determining the likelihood of heel horn erosion, white line fissure, and sole hemorrhage—by more than 3 times. Different factors related to unhygienic conditions such as leg cleanliness, frequency of cleaning, and longer time spent in the corral were associated with infectious hoof lesions. Poor human–animal relationship was related to sole hemorrhage, but patient handling of cows on the track was a protective factor against interdigital hyperplasia. The results of this study suggest that improving hygiene conditions, track features, and cow handling can improve dairy cattle mobility scores in pasture-based farms under tropical conditions. These findings also represent a first step toward planning actions aimed at decreasing lameness and hoof lesions in the studied region.     

Anatomic correction of transposition of the great vessels

We present a new approach for anatomic correction of transposition of the great arteries. The two coronary arteries, with a piece of the aortic wall attached, are transposed to the posterior artery. The two aortic openings are closed with a patch. The aorta and pulmonary artery are transected, contraposed, ant then anastomosed. The interventricular septal defect is closed with a patch, through a right ventriculotomy approach, because the right ventricle is no longer part of the systemic circulation. Two patients, aged 3 months and 40 days weighing 4,200 and 3,700 grams, respectively, were operated upon with deep hypothermia and total circulatory arrest. There was good recovery from the operation, with normal cardiocirculatory conditions. Renal failure developed in the first patient, and she died on the third postoperative day. During this time the cardiocirculatory conditions were good. The second patient made an uneventful recovery. Hemodynamic studies 20 days after the operation showed complete correction of the malformation. Five and one-half months after the operation, he weighs 7,500 grams, and his development is very good. We believe that this operation will be reproducible by most cardiovascular septal defect and pulmonary hypertension.     

Rancinamycins I, II, III and IV structural studies

Rancinamycins are secondary metabolites produced by Streptomyces lincolnensis in a sulfur-depleted culture medium. The structures (except stereochemistry) of the main components of the rancinamycin-complex were determined by the use of IR, UV, PMR, and CMR spectra.