Comparison of early developmental stages and adults of grass carp, Ctenopharyngodon idella, and hybrid carp (female grass carp × male bighead Aristichthys nobilis)

Hybrid carp and grass carp from the zygote to the prolarval stage and adults were compared to establish distinguishing characteristics. Zygote diameter, yolk sac and embryo length were not different between the fishes. Body pigmentation patterns showed distinct separation among the prolarvae of the hybrids and grass carp. Total and preanal myomeres were higher in grass carp larvae than in the hybrids. The grass carp and hybrid larvae also differed in 38% of the proportional morphometric characters.
Average meristic counts of adults were significantly higher in hybrids compared with grass carp. Adults of the two fishes showed significant differences in 63% of the proportional morphometric measurements. Gill raker counts showed no overlap and lateral line scales were smaller and more numerous in the hybrid adults.     

Functional Loss of Semaphorin 3C and/or Semaphorin 3D and Their Epistatic Interaction with Ret Are Critical to Hirschsprung Disease Liability

Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.     

Food intake and growth of hybrid carp (female grass carp, Ctenopharyngodon idella× male bighead, Aristichthys (Hypophthalmichthys) nobilis) fed on zooplankton and Chara

Hybrid carp were raised in 1651 indoor tanks. Two tanks were used in this study each stocked with five fish (average total length and weight = 235.7 mm; 116.1 g); one tank was provided with zooplankton and the other with zooplankton and Chara sp. The hybrids fed actively on zooplankton at an average daily rate of 39% and Chara at 2.6% by body weight. Growth was estimated as 0.36% (0.43 g) per day. The hybrids on the two food types showed no significant differences either in plankton consumption or growth rate.     

Studies in formulation and pharmacotechnical evaluation of controlled release transdermal delivery system of bupropion

The objective of the present study was to design and evaluate unilaminate transdermal adhesive matrix systems capable of diffusing bupropion base at a constant rate over an extended period of time as an alternative route of administration. Unilaminate transdermal adhesive matrices have been fabricated with different concentrations of Eudragit E as the adhesive and rate-controlling polymer. The in vitro release and epidermal flux through human cadaver skin were studied. The release of drug from the matrices obeyed zero order release kinetics (r ²=0.9810 to 0.9960). The delivery rate of bupropion ranged from 10.5 mg to 31.4 mg per day from a 3.14 cm² area of matrix. The relation between concentration of bupropion base in matrix and epidermal flux, concentration of drug in matrix, and epidermal adsorption of bupropion during diffusion follow hyperbolic fashion. Triethylcitrate (TEC) and dibutylphthalate (DBP) have no influence on the diffusion of bupropion through human cadaver skin when used as plasticizers. Incorporation of succinic acid in the adhesive matrix retarded diffusion due to the formation of rigid cross linking of the polymer, while propylene glycol and myristic acid, alone or in combination, significantly enhanced the flux of bupropion through human cadaver skin.     

Effects of temperature and stocking density on food consumption and growth of grass carp Ctenopharyngodon idella, Val.

Grass carp were raised in 227 1 indoor tanks at 12.8, 18.3, 23.9 and 29.4° C temperatures, each experimental condition received five fish. Five grass carp were also raised in a 1135 1 tank maintained at 29.4° C representing low stocking density (29.4° C-L). Food consumption, food conversion, and growth of carp were not different among the 18.3, 23.9, and 29.4° C experimental conditions with identical stocking density. The least growth and food consumption were noted for the fish at the 12.8° C temperature. The fish at the low stocking density had the best growth and maximum food consumption.     

The effects of acclimation on the salinity tolerance of grass carp, Ctenopharyngodon idella (Cuv. and Val.)

Grass carp acclimated to 3, 5, 7, and 9% salinities were tested for salinity tolerance. The upper tolerance limit (LS₅₀) for 3 and 5%, and 7% acclimated fish were 14.2 and 14.0%, respectively. Fish acclimated to 9% had a 50% mortality at an estimated salinity of 16.0%, for 24 h exposure. Weight losses for the 3 and 5% acclimated fish were significantly lower than for those acclimated to 9%.     

Protein-Protein Docking with Dynamic Residue Protonation States

Protein-protein interactions depend on a host of environmental factors. Local pH conditions influence the interactions through the protonation states of the ionizable residues that can change upon binding. In this work, we present a pH-sensitive docking approach, pHDock, that can sample side-chain protonation states of five ionizable residues (Asp, Glu, His, Tyr, Lys) on-the-fly during the docking simulation. pHDock produces successful local docking funnels in approximately half (79/161) the protein complexes, including 19 cases where standard RosettaDock fails. pHDock also performs better than the two control cases comprising docking at pH 7.0 or using fixed, predetermined protonation states. On average, the top-ranked pHDock structures have lower interface RMSDs and recover more native interface residue-residue contacts and hydrogen bonds compared to RosettaDock. Addition of backbone flexibility using a computationally-generated conformational ensemble further improves native contact and hydrogen bond recovery in the top-ranked structures. Although pHDock is designed to improve docking, it also successfully predicts a large pH-dependent binding affinity change in the Fc–FcRn complex, suggesting that it can be exploited to improve affinity predictions. The approaches in the study contribute to the goal of structural simulations of whole-cell protein-protein interactions including all the environmental factors, and they can be further expanded for pH-sensitive protein design.     

Rapid Calculation of Protein pK(a) Values Using Rosetta

We developed a Rosetta-based Monte Carlo method to calculate the pK(a) values of protein residues that commonly exhibit variable protonation states (Asp, Glu, Lys, His, and Tyr). We tested the technique by calculating pK(a) values for 264 residues from 34 proteins. The standard Rosetta score function, which is independent of any environmental conditions, failed to capture pK(a) shifts. After incorporating a Coulomb electrostatic potential and optimizing the solvation reference energies for pK(a) calculations, we employed a method that allowed side-chain flexibility and achieved a root mean-square deviation (RMSD) of 0.83 from experimental values (0.68 after discounting 11 predictions with an error over 2 pH units). Additional degrees of side-chain conformational freedom for the proximal residues facilitated the capture of charge-charge interactions in a few cases, resulting in an overall RMSD of 0.85 pH units. The addition of backbone flexibility increased the overall RMSD to 0.93 pH units but improved relative pK(a) predictions for proximal catalytic residues. The method also captures large pK(a) shifts of lysine and some glutamate point mutations in staphylococcal nuclease. Thus, a simple and fast method based on the Rosetta score function and limited conformational sampling produces pK(a) values that will be useful when rapid estimation is essential, such as in docking, design, and folding.