Cartilage-Specific Overexpression of ERRγ Results in Chondrodysplasia and Reduced Chondrocyte Proliferation

While the role of estrogen receptor-related receptor alpha (ERRα) in chondrogenesis has been investigated, the involvement of ERR gamma (ERRγ) has not been determined. To assess the effect of increased ERRγ activity on cartilage development in vivo, we generated two transgenic (Tg) lines overexpressing ERRγ2 via a chondrocyte-specific promoter; the two lines exhibited ∼3 and ∼5 fold increased ERRγ2 protein expression respectively in E14.5 Tg versus wild type (WT) limbs. On postnatal day seven (P7), we observed a 4–10% reduction in the size of the craniofacial, axial and appendicular skeletons in Tg versus WT mice. The reduction in bone length was already present at birth and did not appear to involve bones that are derived via intramembranous bone formation as the bones of the calvaria, clavicle, and the mandible developed normally. Histological analysis of P7 growth plates revealed a reduction in the length of the Tg versus WT growth plate, the majority of which was attributable to a reduced proliferative zone. The reduced proliferative zone paralleled a decrease in the number of Ki67-positive proliferating cells, with no significant change in apoptosis, and was accompanied by large cell-free swaths of cartilage matrix, which extended through multiple zones of the growth plate. Using a bioinformatics approach, we identified known chondrogenesis-associated genes with at least one predicted ERR binding site in their proximal promoters, as well as cell cycle regulators known to be regulated by ERRγ. Of the genes identified, Col2al, Agg, Pth1r, and Cdkn1b (p27) were significantly upregulated, suggesting that ERRγ2 negatively regulates chondrocyte proliferation and positively regulates matrix synthesis to coordinate growth plate height and organization.     

Synthesis and Characterization of Dual-Functionalized Core-Shell Fluorescent Microspheres for Bioconjugation and Cellular Delivery

The efficient transport of micron-sized beads into cells, via a non-endocytosis mediated mechanism, has only recently been described. As such there is considerable scope for optimization and exploitation of this procedure to enable imaging and sensing applications to be realized. Herein, we report the design, synthesis and characterization of fluorescent microsphere-based cellular delivery agents that can also carry biological cargoes. These core-shell polymer microspheres possess two distinct chemical environments; the core is hydrophobic and can be labeled with fluorescent dye, to permit visual tracking of the microsphere during and after cellular delivery, whilst the outer shell renders the external surfaces of the microspheres hydrophilic, thus facilitating both bioconjugation and cellular compatibility. Cross-linked core particles were prepared in a dispersion polymerization reaction employing styrene, divinylbenzene and a thiol-functionalized co-monomer. These core particles were then shelled in a seeded emulsion polymerization reaction, employing styrene, divinylbenzene and methacrylic acid, to generate orthogonally functionalized core-shell microspheres which were internally labeled via the core thiol moieties through reaction with a thiol reactive dye (DY630-maleimide). Following internal labeling, bioconjugation of green fluorescent protein (GFP) to their carboxyl-functionalized surfaces was successfully accomplished using standard coupling protocols. The resultant dual-labeled microspheres were visualized by both of the fully resolvable fluorescence emissions of their cores (DY630) and shells (GFP). In vitro cellular uptake of these microspheres by HeLa cells was demonstrated conventionally by fluorescence-based flow cytometry, whilst MTT assays demonstrated that 92% of HeLa cells remained viable after uptake. Due to their size and surface functionalities, these far-red-labeled microspheres are ideal candidates for in vitro, cellular delivery of proteins.     

Roux-en-Y Gastric Bypass Increases Intravenous Ethanol Self-Administration in Dietary Obese Rats

Roux-en-Y gastric bypass surgery (RYGB) is an effective treatment for severe obesity. Clinical studies however have reported susceptibility to increased alcohol use after RYGB, and preclinical studies have shown increased alcohol intake in obese rats after RYGB. This could reflect a direct enhancement of alcohol’s rewarding effects in the brain or an indirect effect due to increased alcohol absorption after RGYB. To rule out the contribution that changes in alcohol absorption have on its rewarding effects, here we assessed the effects of RYGB on intravenously (IV) administered ethanol (1%). For this purpose, high fat (60% kcal from fat) diet-induced obese male Sprague Dawley rats were tested ∼2 months after RYGB or sham surgery (SHAM) using both fixed and progressive ratio schedules of reinforcement to evaluate if RGYB modified the reinforcing effects of IV ethanol. Compared to SHAM, RYGB rats made significantly more active spout responses to earn IV ethanol during the fixed ratio schedule, and achieved higher breakpoints during the progressive ratio schedule. Although additional studies are needed, our results provide preliminary evidence that RYGB increases the rewarding effects of alcohol independent of its effects on alcohol absorption.     

Etoposide Induces Nuclear Re-Localisation of AID

During B cell activation, the DNA lesions that initiate somatic hypermutation and class switch recombination are introduced by activation-induced cytidine deaminase (AID). AID is a highly mutagenic protein that is maintained in the cytoplasm at steady state, however AID is shuttled across the nuclear membrane and the protein transiently present in the nucleus appears sufficient for targeted alteration of immunoglobulin loci. AID has been implicated in epigenetic reprogramming in primordial germ cells and cell fusions and in induced pluripotent stem cells (iPS cells), however AID expression in non-B cells is very low. We hypothesised that epigenetic reprogramming would require a pathway that instigates prolonged nuclear residence of AID. Here we show that AID is completely re-localised to the nucleus during drug withdrawal following etoposide treatment, in the period in which double strand breaks (DSBs) are repaired. Re-localisation occurs 2-6 hours after etoposide treatment, and AID remains in the nucleus for 10 or more hours, during which time cells remain live and motile. Re-localisation is cell-cycle dependent and is only observed in G2. Analysis of DSB dynamics shows that AID is re-localised in response to etoposide treatment, however re-localisation occurs substantially after DSB formation and the levels of re-localisation do not correlate with γH2AX levels. We conclude that DSB formation initiates a slow-acting pathway which allows stable long-term nuclear localisation of AID, and that such a pathway may enable AID-induced DNA demethylation during epigenetic reprogramming.     

Monitoring of Mass Distribution Interventions for Trachoma in Plateau State,Nigeria

Mass drug administration (MDA) with antibiotics is a key component of the SAFE strategy for trachoma control. Guidelines recommend that where MDA is warranted the whole population be targeted with 80% considered the minimum acceptable coverage. In other countries, MDA is usually conducted by salaried Ministry of Health personnel (MOH). In Plateau State, Nigeria, the existing network of volunteer Community Directed Distributors (CDD) was used for the first trachoma MDA. We conducted a population-based cluster random survey (CRS) of MDA participation to determine the true coverage and compared this to coverage reported from CDD registers. We surveyed 1,791 people from 352 randomly selected households in 24 clusters in three districts in Plateau State in January 2011, following the implementation of MDA. Households were enumerated and all individuals present were asked about MDA participation. Household heads were questioned about household-level characteristics and predictors of participation. Individual responses were compared with the CDD registers. MDA coverage was estimated as 60.3% (95% CI 47.9–73.8%) by the survey compared with 75.8% from administrative program reports. CDD registration books for comparison with responses were available in 19 of the 24 clusters; there was a match for 658/682 (96%) of verifiable responses. CDD registers did not list 481 (41.3%) of the individuals surveyed. Gender and age were not associated with individual participation. Overall MDA coverage was lower than the minimum 80% target. The observed discrepancy between the administrative coverage estimate from program reports and the CRS was largely due to identification of communities missed by the MDA and not reported in the registers. CRS for evaluation of MDA provides a useful additional monitoring tool to CDD registers. These data support modification of distributor training and MDA delivery to increase coverage in subsequent rounds of MDA.     

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.     

Spatial Interactions between Successive Eye and Arm Movements: Signal Type Matters

Spatial interactions between consecutive movements are often attributed to inhibition of return (IOR), a phenomenon in which responses to previously signalled locations are slower than responses to unsignalled locations. In two experiments using peripheral target signals offset by 0°, 90°, or 180°, we show that consecutive saccadic (Experiment 1) and reaching (Experiment 3) responses exhibit a monotonic pattern of reaction times consistent with the currently established spatial distribution of IOR. In contrast, in two experiments with central target signals (i.e., arrowheads pointing at target locations), we find a non-monotonic pattern of reaction times for saccades (Experiment 2) and reaching movements (Experiment 4). The difference in the patterns of results observed demonstrates different behavioral effects that depend on signal type. The pattern of results observed for central stimuli are consistent with a model in which neural adaptation is occurring within motor networks encoding movement direction in a distributed manner.     

GFP Affects Human T Cell Activation and Cytokine Production following In Vitro Stimulation

There are many Green Fluorescent Proteins (GFPs) originating from diverse species that are invaluable to cell biologists today because of their ability to provide experimental visualization of protein expression. Since their initial discovery, they have been modified and improved to provide more stable variants with emission ranges spanning a wide array of colors. Due to their ease of expression both in-vitro and in-vivo, they are an attractive choice for use as markers in molecular biology. GFPs are generally assumed to have negligible effects on the cells to which they have been introduced. However, a growing number of reports indicate that this is not always the case. Consequently, because of GFP''s ubiquitous use, it is important to document the nature and extent of unintended effects. In this report, we find that GFP affects T cell activation, leading to defects in clustering, upregulation of the activation marker CD25 and IL-2 cytokine production following stimulation in human primary T cells that also express TurboGFP. We utilized a reporter assay which has been routinely used to assay the NF-κB pathway and found reduced NF-κB activitation in stimulated HEK293 and HeLa cells that were co-transfected with TurboGFP, suggesting that GFP interferes with signaling through the NF-κB pathway. These findings indicate that the utilization of GFP-tagged vectors may negatively impact in vitro experiments in T cells, emphasizing the critical importance of controls to identify any GFP-induced effects.