A scalable, fully automated process for construction of sequence-ready barcoded libraries for 454

We present an automated, high throughput library construction process for 454 technology. Sample handling errors and cross-contamination are minimized via end-to-end barcoding of plasticware, along with molecular DNA barcoding of constructs. Automation-friendly magnetic bead-based size selection and cleanup steps have been devised, eliminating major bottlenecks and significant sources of error. Using this methodology, one technician can create 96 sequence-ready 454 libraries in 2 days, a dramatic improvement over the standard method.     

Variation in the ribosomal internal transcribed spacers and 5.8S rDNA among five species of Acropora (Cnidaria; Scleractinia): patterns of variation consistent with reticulate evolution

The ITS sequences of Acropora spp. are the shortest so far identified in any metazoan and are among the shortest seen in eukaryotes; ITS1 was 70-80 bases, and ITS2 was 100-112 bases. The ITS sequences were also highly variable, but base composition and secondary structure prediction indicate that divergent sequence variants are unlikely to be pseudogenes. The pattern of variation was unusual in several other respects: (1) two distinct ITS2 types were detected in both A. hyacinthus and A. cytherea, species known to hybridize in vitro with high success rates, and a putative intermediate ITS2 form was also detected in A. cytherea; (2) A. valida was found to contain highly (29%) diverged ITS1 variants; and (3) A. longicyathus contained two distinct 5.8S rDNA types. These data are consistent with a reticulate evolutionary history for the genus Acropora.      

Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [γ-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [γ-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.     

Benzyloxy Derivatives of Triazine-based Coupling Reagents Designed for an Efficient Solid Phase Peptide Synthesis on Polystyrene Resin

Coupling reagents resembling the structure of Merrifield resin were designed and prepared from 2-chloro-4,6-dibenzyloxy-1,3,5-triazine and the different tertiary bases N-methylmorpholine, N-methylpiperidine, and DABCO. As previously observed for DMTMM, the appropriate N-(4,6-dibenzyloxy-1,3,5-triazin-2-yl) ammonium chloride salts were not suitable as efficient coupling reagents because of their low stability. On the other hand, the stability of the N-(4,6-dibenzyloxy-1,3,5-triazin-2-yl) ammonium tetrafluoroborates was suitable enough for prolonged storage and convenient application in SPPS. Moreover, we observed that the superactive intermediates formed during activation of Fmoc–Aib–OH with 4,6-dibenzyloxy-1,3,5-triazine-based coupling reagents lead to an increase in its concentration inside the polystyrene resin. Therefore, we hypothesize that this increase can enhance efficiency of 4,6-dibenzyloxy-1,3,5-triazine-based coupling reagents in SPPS.     

Continuous-time modeling of cell fate determination in Arabidopsis flowers

Background  

The genetic control of floral organ specification is currently being investigated by various approaches, both experimentally and through modeling. Models and simulations have mostly involved boolean or related methods, and so far a quantitative, continuous-time approach has not been explored.     

The Emerging Role of Copeptin

Direct measurement of the nonapeptide vasopressin has been limited by analyte instability ex vivo and in vivo rapid degradation, low serum concentrations requiring a sensitive assay and inherent secretory pulsatility. Copeptin is a 39 amino acid glycopeptide cleavage product of vasopressin synthesis with high stability, providing a marker of vasopressin secretion. Copeptin measurement has applications in diagnosis of diabetes insipidus and other diseases with altered vasopressin secretion. This review summarises our current understanding of serum copeptin measurement in diabetes insipidus and possible future applications of copeptin assays. As vasopressin is a stress hormone, there is emerging evidence on the use of copeptin for diagnosis and prognostication of disorders such as syndrome of inappropriate anti-diuretic hormone secretion, diabetes mellitus, critical illness, stroke, cardiovascular disease, respiratory disease, renal disease and thermal stress. Copeptin concentration measurement is likely to improve the diagnostic reliability of diabetes insipidus and, as a marker of stress, may have diagnostic or prognostic utility in specific clinical circumstances. Further studies are needed to determine if goal-directed therapy using plasma copeptin concentrations may improve patient outcomes.     

ANTHROPOMETRIC DETERMINANTS OF ROWING ERGOMETER PERFORMANCE IN PHYSICALLY INACTIVE COLLEGIATE FEMALES

The aim of the study was to evaluate anthropometric characteristics as determinants of 500 m rowing ergometer performance in physically inactive collegiate females. In this cross-sectional study, which included 196 collegiate females aged 19-23 years not participating in regular physical activities, body mass (BM), body height (BH), length of upper limbs (LA), length of lower limbs (LL), body mass index (BMI), slenderness index (SI), and the Choszcz-Podstawski index (CPI) were measured and a stepwise multiple regression analysis was performed. Participants performed 500 m maximal effort on a Concept II rowing ergometer. BM, BH, LA, LL, and the BMI, SI and CPI indices were found to be statistically significant determinants of 500 m performance. The best results (T) were achieved by females whose BH ranged from 170 to 180 cm, with LA and LL ranging from 75 to 80 cm and 85 to 90 cm, respectively. The best fitting statistical model was identified as: T = 11.6793 L_R – 0.1130 L_R² – 0.0589 L_N² + 29.2157 CPI² + 0.1370 LR·LN - 2.6926 LR·CPI – 211.7796. This study supports a need for additional studies focusing on understanding the importance of anthropometric differences in rowing ergometer performance, which could lead to establishing a better quality reference for evaluation of cardiorespiratory fitness tested using a rowing ergometer in collegiate females.