A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing

RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RETECD-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands

Architecture and regulation of a GDNF-GFRa1 synaptic adhesion assembly

Glial-cell line derived neurotrophic factor (GDNF) bound to its co-receptor GFRa1 stimulates the RET receptor tyrosine kinase, promoting neuronal survival and neuroprotection. The GDNF-GFRa1 complex also acts as a synaptic cell adhesion independently of RET. Here, we describe the structure of a decameric GDNF-GFRa1 assembly determined by crystallography and electron microscopy, revealing two GFRa1 pentamers bridged by five GDNF dimers. We reconsitituted the assembly in vitro between pairs of adhering liposomes and used cryo-electron tomography to visualize how the complex fulfils its membrane adhesion function. The GFRa1:GFRa1 pentameric interface was further validated both in vitro by native PAGE and in cellulo by cell-clustering and dendritic spine assays. Finally, we provide biochemical and cell-based evidence that RET and heparan sulfate cooperate to prevent assembly of the adhesion complex by competing for the adhesion interface. Our results provide a mechanistic framework to understand GDNF-driven cell adhesion, its relationship to trophic signalling, and the central role of GFRa1. Houghton, FM1, Adams SE^1, Ríos AS2, Masino, L3, Purkiss AG3, Briggs DC1, Ledda, F2, & McDonald N.Q.1,4

The efficacy of Salenvac, a Salmonella enterica subsp. Enterica serotype Enteritidis iron-restricted bacterin vaccine, in laying chickens

The protective effect of two vaccination regimes using Salenvac, a commercially available iron-restricted Salmonella enterica subsp. Enterica serotype Enteritidis PT4 bacterin vaccine, was verified in laying birds. Immunization was intramuscular at 1 day old and again at 4 weeks of age (V2), or at 1 day and 4 weeks with a third dose at 18 weeks of age (V3). Challenge S. Enteritidis (5 to 7.5) x 10(7) colony forming units) was given intravenously at 8, 17, 23, 30 and 59 weeks of age. For all age groups, both vaccination regimes reduced significantly the number of tissues and faecal samples that were culture positive for the challenge strain. For laying birds, fewer eggs (P < 0.001) were culture positive for S. Enteritidis after challenge from vaccinated laying birds ( 56/439 batches of eggs) than unvaccinated birds (99/252 batches). The data give compelling evidence that the vaccine is efficacious and may contribute to the reduction of layer infection and egg contamination

Bronchodilating effect of combined therapy with ipratropium bromide and ondansetron in patients with COPD.

The objectives of this study were to determine the effect of single and repeat dosing with oral ondansetron, a 5-HT3-specific receptor blocker, on the degree and duration of bronchodilation induced by inhaled ipratropium bromide in patients with COPD. Five clinics and university medical centers in four countries participated in the study; 47 patients with COPD were randomized to treatment; 44 completed all treatments. Patients had a baseline (pre-bronchodilator) FEV1>1L and post-bronchodilator (200mcg salbutamol) FEV1<90% of predicted, with FEV1 reversibility (to 80mcg inhaled ipratropium bromide and 400mcg salbutamol) of at least 12% or 200mL over baseline. The study was divided into two parts. In Part A, each patient received in a random order, four-way crossover manner, single doses of ondansetron placebo (oral) plus ipratropium bromide placebo (inhaled), ondansetron placebo plus ipratropium bromide 40mcg inhaled via MDI, ondansetron 24mg oral plus ipratropium bromide placebo and ondansetron 24mg plus ipratropium bromide 40mcg. In Part B, each patient received in a random order, two-way crossover manner, ipratropium bromide 40mcg tid via MDI plus ondansetron 8mg oral, qid, for 2 days; on day 3 patients received a single dose of ipratropium bromide 40mcg plus 8mg oral ondansetron. Alternatively, patients received ipratropium bromide via MDI and oral ondansetron placebo, as described above. Statistically significant differences in weighted mean FEV1 (0-6h), peak FEV1 and FEV1 determined 6h post-dose were noted comparing ipratropium bromide to placebo. Similar positive results were observed for sGaw and FVC. Addition of ondansetron to ipratropium bromide did not significantly modify values obtained with ipratropium alone. Ipratropium bromide induced a marked bronchodilation, compared to placebo. Addition of ondansetron (single or repeated doses) did not significantly increase the degree or duration of bronchodilation induced by ipratropium alone. sGaw was consistently more sensitive than FEV1 in measuring extent and duration of bronchodilation. AD - Department of Respiratory Diseases, Ghent University Hospital, B9000 Ghent, Belgium

Mês de parição, condição corporal e resposta ao protocolo de inseminação artificial em tempo fixo em vacas de corte primíparas Calving date, body condition score, and response to a timed artificial insemination protocol in first-calving beef cows

No experimento I, foi avaliada a alteração da condição corporal (CC) pré e pós-parto em 155 novilhas inseminadas para parir de setembro a dezembro. A CC foi avaliada mensalmente no pré e pós-parto, de junho a fevereiro. No experimento II, 538 vacas primíparas foram sincronizadas com o protocolo de inseminação artificial em tempo fixo (IATF) que usou estradiol junto ao dispositivo intravaginal de progesterona (CIDR®). As taxas de ciclicidade, sincronização e concepção foram avaliadas por ultra-som. No experimento I, os animais que pariram primeiro tiveram maior (P<0,001) redução na CC pós-parto. No experimento II, foi observado maior CC (P<0,0001) nos animais com menor número de dias pós-parto, maior (P<0,05) taxa de sincronização nas vacas de melhor CC e aumento (P<0,0001) na taxa de concepção proporcional ao aumento na CC (incremento médio na concepção de seis pontos percentuais para cada 0,25 ponto na CC). Não se deve antecipar a parição de novilhas de corte quando se pretende realizar IATF no início da estação de monta subseqüente.<br>In experiment I, it was evaluated the body condition score (BCS) change during pre and post-partum in 155 heifers, inseminated to calve from September to December. The BCS was monthly evaluated from June to February, during the pre and post-partum periods. In experiment II, 538 primiparous cows were synchronized with a timed artificial insemination (TAI) protocol which used estradiol associated with an intravaginal progesterone device (CIDR®). The cyclicity, synchronization, and conception rates were evaluated by ultrasound. In experiment I, the animals that calved earlier had higher (P<0.001) reduction on BCS. In experiment II, it was observed higher BCS (P<0.0001) in cows with lower days in post-partum, higher (P<0.05) synchronization rate in cows with a better BCS, and also an increase (P<0.0001) in conception rate as BCS got better (increase in six percentual points in conception for each increase of 0.25 in BCS). Beef heifers should not calve earlier when is planned to submitt these animals to TAI at the beginning of the next breeding season