Additive and epistatic effects influence spectral tuning in molluscan retinochrome opsin.

The relationship between genotype and phenotype is non-trivial because of the often complex molecular pathways that make it difficult to unambiguously relate phenotypes to specific genotypes. Photopigments, comprising an opsin apoprotein bound to a light-absorbing chromophore, present an opportunity to directly relate the amino acid sequence to an absorbance peak phenotype (λmax). We examined this relationship by conducting a series of site-directed mutagenesis experiments of retinochrome, a non-visual opsin, from two closely related species: the common bay scallop, Argopecten irradians, and the king scallop, Pecten maximus. Using protein folding models, we identified three amino acid sites of likely functional importance and expressed mutated retinochrome proteins in vitro. Our results show that the mutation of amino acids lining the opsin binding pocket is responsible for fine spectral tuning, or small changes in the λmax of these light-sensitive proteins. Mutations resulted in a blue or red shift as predicted, but with dissimilar magnitudes. Shifts ranged from a 16 nm blue shift to a 12 nm red shift from the wild-type λmax. These mutations do not show an additive effect, but rather suggest the presence of epistatic interactions. This work highlights the importance of binding pocket shape in the evolution of spectral tuning and builds on our ability to relate genotypic changes to phenotypes in an emerging model for opsin functional analysis.

Molecular phylogeny of the Pectinoidea (Bivalvia) indicates Propeamussiidae to be a non-monophyletic family with one clade sister to the scallops (Pectinidae).

Scallops (Pectinidae) are one of the most diverse families of bivalves and have been a model system in evolutionary biology. However, in order to understand phenotypic evolution, the Pectinidae needs to be placed in a deeper phylogenetic framework within the superfamily Pectinoidea. We reconstructed a molecular phylogeny for 60 species from four of the five extant families within the Pectinoidea using a five gene dataset (12S, 16S, 18S, 28S rRNAs and histone H3). Our analyses give consistent support for the non-monophyly of the Propeamussiidae, with a subset of species as the sister group to the Pectinidae, the Propeamussiidae type species as sister to the Spondylidae, and the majority of propeamussiid taxa sister to the Spondylidae + Pr. dalli. This topology represents a previously undescribed relationship of pectinoidean families. Our results suggest a single origin for eyes within the superfamily and likely multiple instances of loss for these characters. However, it is now evident that reconstructing the evolutionary relationships of Pectinoidea will require a more comprehensive taxonomic sampling of the Propeamussiidae sensu lato.

A high efficiency precision genome editing method with CRISPR in iPSCs.

The use of genetic engineering to generate point mutations in induced pluripotent stem cells (iPSCs) is essential for studying a specific genetic effect in an isogenic background. We demonstrate that a combination of p53 inhibition and pro-survival small molecules achieves a homologous recombination rate higher than 90% using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in human iPSCs. Our protocol reduces the effort and time required to create isogenic lines.

A laser Doppler instrument for in vivo measurements of blood flow in single renal arterioles.

A laser Doppler instrument has been developed to measure the blood flow in single vessels for the study of the dynamics of local control mechanisms. A commercial blood perfusion monitor, designed to measure blood perfusion in a vascular field containing many randomly oriented blood vessels, was modified to perform measurements of blood flow in a single arteriole. In vitro tests of the instrument revealed that the relationship between blood flow and Doppler shift was not a simple linear function. Causes of nonlinearity are revealed and proper use of the device avoids the problem. The device was applied to efferent arterioles that are visible on the surface of the rat kidney. An angiotensin converting enzyme inhibitor and graded doses of angiotensin II were used to perturb kidney blood flow. The induced changes in whole kidney blood flow, measured with an electromagnetic flow probe, and in single efferent arteriolar blood flow, measured with the new instrument, were correlated. An oscillation at approximately 0.035 Hz, previously described in the tubular pressure and attributed to a local feedback mechanism acting on arteriolar resistance, was found in the arteriolar blood flow. The new instrument is easy to use and provides temporal resolution not available with more conventional methods used for flow measurement in the microcirculation.

Method for characterization of adhesion properties of trace explosives in fingerprints and fingerprint simulations.

The near inevitable transfer of explosive particulate matter through fingerprints makes it possible to detect concealed explosives through surface sampling. Repeatable and well-characterized fingerprint simulation facilitates quantitative comparison between particulate sampling methods for subsequent detection of trace explosive residues. This study employs a simple, but reproducible sampling system to determine the accuracy of a fingerprint simulation. The sampling system uses a gas jet to entrain particles from a substrate and the resulting airborne particles are then aspirated onto a Teflon filter. A calibrated Barringer IonScan 400 ion mobility spectrometer was used to determine the mass of explosive material collected on the filter. The IonScan 400 was calibrated with known masses of 2,4,6-trinitrotoluene (TNT). The resulting calibration curve is in good agreement with that obtained by Garofolo et al. (1994) for an earlier model of the instrument. The collection efficiency of the sampling system was measured for three particle sizes (8.0. 10.0, and 13.0 microm) using spherical polystyrene particles laced with known quantities of TNT. Collection efficiency ranged from less than 1% for the larger particles to 5% for the smaller particles. Particle entrainment from the surface was monitored with dark field imaging of the remaining particles. The sampling system was then applied to two C4 test samples--a fingerprint transfer and a dry Teflon transfer. Over 100 ng of RDX was collected from the dry transfer sample, while less than 1 ng was collected from the fingerprint transfer. Possible explanations for this large difference are presented based on the system calibration.

Addressing sexuality in the elderly.

Many social myths and stigmas surround sexuality in the elderly and may account for society's and the medical profession's often ignoring sexuality in these older citizens. Rehabilitation nurses who deal with clients of any age with altered sexual needs should be especially sensitive to the fact that sexual assessment and counseling are very important elements in the rehabilitation process for the promotion of optimum functioning.