Quantitative analysis of DNA hybridization in a flowthrough microarray for molecular testing

Quantitative information about the nucleic acids hybridization reaction on microarrays is fundamental to designing optimized assays for molecular diagnostics. This study presents the kinetic, equilibrium, and thermodynamic analyses of DNA hybridization in a microarray system designed for fast molecular testing of pathogenic bacteria. Our microarray setup uses a porous, nylon membrane for probe immobilization and flowthrough incubation. The Langmuir model was used to determine the reaction rate constants of hybridization with antisense targets specific to Staphylococcus epidermidis and Staphylococcus aureus strains. The kinetic analysis revealed a sequence-dependent reaction rate, with association rate constants on the order of 10⁵ M⁻¹ s⁻¹ and dissociation rate constants of 10⁻⁴ s⁻¹. We found that by increasing the probe surface density from 10¹¹ to 10¹² molecules/cm², the hybridization rate and efficiency are suppressed while the melting temperature of the DNA duplex increases. The maximum fraction of hybridized capture probes at equilibrium did not exceed 50% for hybridization with antisense sequences and was below 6% for hybridization with long targets obtained from PCR. The van’t Hoff analysis of the temperature denaturation data showed that the DNA hybridization in our porous, flowthrough microarray is thermodynamically less favorable than the hybridization of the same sequences in solution.     

Adaptations in pulsatile insulin secretion, hepatic insulin clearance, and beta-cell mass to age-related insulin resistance in rats

In health insulin is secreted in discrete insulin secretory bursts from pancreatic beta-cells, collectively referred to as beta-cell mass. We sought to establish the relationship between beta-cell mass, insulin secretory-burst mass, and hepatic insulin clearance over a range of age-related insulin sensitivity in adult rats. To address this, we used a novel rat model with chronically implanted portal vein catheters in which we recently established the parameters to permit deconvolution of portal vein insulin concentration profiles to measure insulin secretion and resolve its pulsatile components. In the present study, we examined total and pulsatile insulin secretion, insulin sensitivity, hepatic insulin clearance, and beta-cell mass in 35 rats aged 2-12 mo. With aging, insulin sensitivity declined, but euglycemia was sustained by an adaptive increase in fasting and glucose-stimulated insulin secretion through the mechanism of a selective augmentation of insulin pulse mass. The latter was attributable to a closely related increase in beta-cell mass (r=0.8, P<0.001). Hepatic insulin clearance increased with increasing portal vein insulin pulse amplitude, damping the delivery of insulin in the systemic circulation. In consequence, the curvilinear relationship previously reported between insulin secretion and insulin sensitivity was extended to both insulin pulse mass and beta-cell mass vs. insulin sensitivity. These data support a central role of adaptive changes in beta-cell mass to permit appropriate insulin secretion in the setting of decreasing insulin sensitivity in the aging animal. They emphasize the cooperative role of pancreatic beta-cells and the liver in regulating the secretion and delivery of insulin to the systemic circulation.     

Tricyclic antidepressant amitriptyline alters sarcoplasmic reticulum calcium handling in ventricular myocytes

Tricyclic antidepressants such as amitriptyline (AMT) have been reported to have adverse side effects on cardiac performance. AMT effects on Ca handling in ventricular myocytes, however, are not well understood. Therefore, we investigated AMT action on sarcoplasmic reticulum (SR) Ca release in ventricular myocytes, ryanodine receptor (RyR) activity, and Ca uptake by SR microsomes. In permeabilized myocytes, AMT transiently increased free luminal Ca concentration ([Ca]) followed by marked depletion. AMT (10 microM) caused a rapid and a transient increase of Ca spark frequency, followed by a significant suppression of spark activity. The latter was associated with a decrease of Ca spark amplitude and SR Ca load to 87 and 60%, respectively. AMT (10 microM) completely abolished propagation of spontaneous Ca waves. Higher concentrations of AMT (0.1-1 mM) evoked SR Ca release reminiscent of the effect of caffeine (20 mM) and caused almost complete depletion of SR Ca content. Studies on single calsequestrin-free RyR channels revealed that AMT increased the mean open time and open probability (Po) in a dose-dependent fashion (dissociation constant = 4.2 microM). High concentrations of AMT (> 25 microM) evoked frequent long openings with Po reaching very high levels (> 0.70). In studies with cardiac SR microsomes, AMT slowed the rate of ATP-dependent Ca uptake. We conclude that AMT affects SR Ca handling in ventricular myocytes by multiple mechanisms, including direct stimulation of RyRs and inhibition of SR Ca uptake. These effects could contribute to AMT cardiotoxicity.     

IP3 receptor-dependent Ca2+ release modulates excitation-contraction coupling in rabbit ventricular myocytes

Inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-dependent Ca(2+) signaling exerts positive inotropic, but also arrhythmogenic, effects on excitation-contraction coupling (ECC) in the atrial myocardium. The role of IP(3)R-dependent sarcoplasmic reticulum (SR) Ca(2+) release in ECC in the ventricular myocardium remains controversial. Here we investigated the role of this signaling pathway during ECC in isolated rabbit ventricular myocytes. Immunoblotting of proteins from ventricular myocytes showed expression of both type 2 and type 3 IP(3)R at levels approximately 3.5-fold less than in atrial myocytes. In permeabilized myocytes, direct application of IP(3) (10 microM) produced a transient 21% increase in the frequency of Ca(2+) sparks (P < 0.05). This increase was accompanied by a 13% decrease in spark amplitude (P < 0.05) and a 7% decrease in SR Ca(2+) load (P < 0.05) and was inhibited by IP(3)R antagonists 2-aminoethoxydiphenylborate (2-APB; 20 microM) and heparin (0.5 mg/ml). In intact myocytes endothelin-1 (100 nM) was used to stimulate IP(3) production and caused a 38% (P < 0.05) increase in the amplitude of action potential-induced (0.5 Hz, field stimulation) Ca(2+) transients. This effect was abolished by the IP(3)R antagonist 2-APB (2 microM) or by using adenoviral expression of an IP(3) affinity trap that buffers cellular IP(3). Together, these data suggest that in rabbit ventricular myocytes IP(3)R-dependent Ca(2+) release has positive inotropic effects on ECC by facilitating Ca(2+) release through ryanodine receptor clusters.     

Geochemical signatures of benthic foraminiferal shells from a heat‐polluted shallow marine environment provide field evidence for growth and calcification under extreme warmth

Shallow marine calcifiers play an important role as marine ecosystem engineers and in the global carbon cycle. Understanding their response to warming is essential to evaluate the fate of marine ecosystems under global change scenarios. A rare opportunity to test the effect of warming acting on natural ecosystems is by investigation of heat‐polluted areas. Here, we study growth and calcification in benthic foraminifera that inhabit a thermally polluted coastal area in Israel, where they are exposed to elevated temperatures reaching up to ~42°C in summer. Live specimens of two known heat‐tolerant species Lachlanella sp. 1 and Pararotalia calcariformata were collected over a period of 1 year from two stations, representing thermally polluted and undisturbed (control) shallow hard bottom habitats. Single‐chamber element ratios of these specimens were obtained using laser ablation, and the Mg/Ca of the most recently grown final chambers were used to calculate their calcification temperatures. Our results provide the first direct field evidence that these foraminifera species not only persist at extreme warm temperatures but continue to calcify and grow. Species‐specific Mg/Ca thermometry indicates that P. calcariformata precipitate their shells at temperatures as high as 40°C and Lachlanella sp. 1 at least up to 36°C, but both species show a threshold for calcification at cold temperatures: calcification in P. calcariformata only occurred above 22°C and in Lachlanella sp. 1 above 15°C. Our observations from the heat‐polluted area indicate that under future warming scenarios, calcification in heat‐tolerant foraminifera species will not be inhibited during summer, but instead the temperature window for their calcification will be expanded throughout much of the year. The observed inhibition of calcification at low temperatures indicates that the role of heat‐tolerant foraminifera in carbonate production will most likely increase in future decades.     

A coarse-grained model for PCL: conformation,self-assembly of MePEG-b-PCL amphiphilic diblock copolymers

Poly-ε-caprolactone (PCL) is a biodegradable hydrophobic polyester that has been widely used in medical devices, tissue engineering and nanoparticle-based drug delivery. Coarse-grained molecular dynamics (CGMD) has been employed to study and gain insights into the conformational, structural and self-assembly behaviour of polymers, lipids and amphiphilic macromolecules. In this work, we developed a model for PCL within the framework of the MARTINI coarse-grained force field. The non-bonded interactions were based on the existing MARTINI bead types, while the bonded interactions were mapped onto a PCL rendition obtained from atomistic simulations. The model accurately reproduces the structural and dynamic properties of the PCL homopolymer and shows very reasonable temperature and solvent transferability. We also studied self-assembly of MePEG-b-PCL linear diblock copolymers using an existing MARTINI model for MePEG (Methoxy Polyethylene glycol), by analysing the critical micelle concentration (CMC), as well as the shape, size and morphology of the nano-polymeric micelles. We obtained excellent agreement of the CMC, while the size was under-predicted compared to experimental data. This robust model paves the way for CGMD modelling of PCL and serves as a starting point for future designs of PCL-related polymeric systems .     

Towards a molecular phylogeny ofApiaceae subfamilyApioideae: Additional information from nuclear ribosomal DNA ITS sequences

Evolutionary relationships among 116 representatives (80 genera) ofApiaceae (Umbelliferae) subfam.Apioideae were investigated by comparative sequencing of the two internal transcribed spacers of the 18S–26S nuclear ribosomal DNA repeat. The resultant phylogenies, inferred using maximum parsimony and neighbor-joining methods, clarified the relationships of several genera whose phylogenetic placements have heretofore been problematic. Comparisons between the phylogenies inferred and the distribution of several phytochemical (coumarins, flavonoids, and phenylpropenes) and morphological (stomates, pollen, and cotyledonary shape) characters were also made, revealing that many of these characters (like those morphological and anatomical characters of the fruit) are highly homoplastic. It is not surprising then that systems of classification ofApioideae based on these characters, particularly with regard to tribal and subtribal designations and relationships, are unsatisfactory. The results of recent serological investigations of the subfamily support several relationships proposed herein using molecular data.     

Improvements in proteomic metrics of low abundance proteins through proteome equalization using ProteoMiner prior to MudPIT

Ideally, shotgun proteomics would facilitate the identification of an entire proteome with 100% protein sequence coverage. In reality, the large dynamic range and complexity of cellular proteomes results in oversampling of abundant proteins, while peptides from low abundance proteins are undersampled or remain undetected. We tested the proteome equalization technology, ProteoMiner, in conjunction with Multidimensional Protein Identification Technology (MudPIT) to determine how the equalization of protein dynamic range could improve shotgun proteomics methods for the analysis of cellular proteomes. Our results suggest low abundance protein identifications were improved by two mechanisms: (1) depletion of high abundance proteins freed ion trap sampling space usually occupied by high abundance peptides and (2) enrichment of low abundance proteins increased the probability of sampling their corresponding more abundant peptides. Both mechanisms also contributed to dramatic increases in the quantity of peptides identified and the quality of MS/MS spectra acquired due to increases in precursor intensity of peptides from low abundance proteins. From our large data set of identified proteins, we categorized the dominant physicochemical factors that facilitate proteome equalization with a hexapeptide library. These results illustrate that equalization of the dynamic range of the cellular proteome is a promising methodology to improve low abundance protein identification confidence, reproducibility, and sequence coverage in shotgun proteomics experiments, opening a new avenue of research for improving proteome coverage.