Regular measurement: A new method of biophysical experiment

A system of quantized relations between the value of the parameter (quantity) measured and the range of its experimental scattering has been revealed by analyzing the results of experiments on measuring the rates of various processes. Especially long series of stereotyped measurements under stable conditions (called regular measurements) are performed for determining the squared ratio of the standard deviation to the mean value of the quantity measured (D). Regular measurements of biochemical, chemical, and physical parameters show that the D value is related to the universal dimensionless constant D ₀, whose numerical value has been determined with considerable accuracy, and this relation is described by a simple rational equation. The simplicity of the relationship between D and D ₀ may be accounted for by the quantum behavior of the macroinstrument-object system manifested in the course of regular measurement.     

Accuracy and reproducibility of bending stiffness measurements by mechanical response tissue analysis in artificial human ulnas

Osteoporosis is characterized by reduced bone strength, but no FDA-approved medical device measures bone strength. Bone strength is strongly associated with bone stiffness, but no FDA-approved medical device measures bone stiffness either. Mechanical Response Tissue Analysis (MRTA) is a non-significant risk, non-invasive, radiation-free, vibration analysis technique for making immediate, direct functional measurements of the bending stiffness of long bones in humans in vivo. MRTA has been used for research purposes for more than 20 years, but little has been published about its accuracy. To begin to investigate its accuracy, we compared MRTA measurements of bending stiffness in 39 artificial human ulna bones to measurements made by Quasistatic Mechanical Testing (QMT). In the process, we also quantified the reproducibility (i.e., precision and repeatability) of both methods. MRTA precision (1.0±1.0%) and repeatability (3.1±3.1%) were not as high as those of QMT (0.2±0.2% and 1.3+1.7%, respectively; both p<10⁻⁴). The relationship between MRTA and QMT measurements of ulna bending stiffness was indistinguishable from the identity line (p=0.44) and paired measurements by the two methods agreed within a 95% confidence interval of ±5%. If such accuracy can be achieved on real human ulnas in situ, and if the ulna is representative of the appendicular skeleton, MRTA may prove clinically useful.     

Determination of dissociation constants of crystalline-alpha-chymotrypsin complexes

As a first step in investigations of the properties of crystalline enzymes, the binding of indole, N-formyl-l-phenylalanine, and N-formyl-l-p-iodophenylalanine to α-chymotrypsin crystals, and the binding of indole to tosyl-α-chymotrypsin crystals, has been studied. The methods used were spectrophotometric measurements of the concentration of indole in the supernatant, or measurements of the concentration of radioactively labeled indole in both the supernatant and the crystal. The dissociation constants of the specific binding site of the crystalline enzyme have been determined for indole and N-formyl-l-phenylalanine. It was found that indole does not bind to tosyl-α-chymotrypsin crystals and that N-formyl-p-iodophenylalanine does not bind to the substrate binding site of the crystalline enzyme.The information obtained from these simple equilibrium measurements is in agreement with X-ray diffraction studies. The approach is, therefore, capable of determining whether or not compounds bind to the active site of a crystalline enzyme, and whether the occupancy of this site is sufficient for structure determinations using X-ray diffraction methods.     

Measurement of breath-by-breath gas exchange during general anaesthesia using a single pneumotachograph

A breath-by-breath gas exchange measurement system using a single pneumotachograph suitable for use during general anaesthesia is described. The system's accuracy has been assessed by a combination of error sensitivity analysis, laboratory testing of the component measurements used to calculate gas exchange and measurements on volunteers and patients. The system is shown to have a mean accuracy of ± 2.6 ml breath⁻¹ for V_CO2 measurements, ± 7.12 ml breath⁻¹ for V_O2 and ± 5.55 ml breath⁻¹ for V_N2O measurement. The application of a lung gas stores correction using argon improved between breath variability by 50%.     

How small is the smallest? New record and remeasuring of Scydosella musawasensis Hall, 1999 (Coleoptera,Ptiliidae), the smallest known free-living insect

The smallest known beetle Scydosella musawasensis Hall is recorded for the second time. Precise measurements of its body size are given, and it is shown that the smallest examined representative of this species has a length of 325 µm.     

2,4-Diamino-3-methylbutanoic acid,a novel amino acid in root nodule hydrolysates from Lotus tenuis

The novel amino acid 2,4-diamino-3-methylbutanoic acid has been identified in nodules formed by two strains of Rhizobium bacteria on Lotus tenuis roots. Retention time measurements on a Chirasil-Val capillary column suggest it is present as the (2R,3S)-enantiomer. Several isomeric diamino acids were synthesized for comparative studies.     

Structural Evidence for Inter-Residue Hydrogen Bonding Observed for Cellobiose in Aqueous Solution

The structure of the disaccharide cellulose subunit cellobiose (4-O-β-D-glucopyranosyl-D-glucose) in solution has been determined via neutron diffraction with isotopic substitution (NDIS), computer modeling and nuclear magnetic resonance (NMR) spectroscopic studies. This study shows direct evidence for an intramolecular hydrogen bond between the reducing ring HO3 hydroxyl group and the non-reducing ring oxygen (O5′) that has been previously predicted by computation and NMR analysis. Moreover, this work shows that hydrogen bonding to the non-reducing ring O5′ oxygen is shared between water and the HO3 hydroxyl group with an average of 50% occupancy by each hydrogen-bond donor. The glycosidic torsion angles φ_H and ψ_H from the neutron diffraction-based model show a fairly tight distribution of angles around approximately 22^° and −40^°, respectively, in solution, consistent with the NMR measurements. Similarly, the hydroxymethyl torsional angles for both reducing and non-reducing rings are broadly consistent with the NMR measurements in this study, as well as with those from previous measurements for cellobiose in solution.     

Psychrometric Field Measurement of Water Potential Changes following Leaf Excision

In situ measurement of sudden leaf water potential changes has not been performed under field conditions. A laboratory investigation involving the measurement of leaf water potential prior to and 2 to 200 minutes after excision of citrus leaves (Citrus jambhiri) showed good linear correlation (r = 0.99) between in situ leaf psychrometer and Scholander pressure chamber measurements. Following this, a field investigation was conducted which involved psychrometric measurement prior to petiole excision and 1 minute after excision. Simultaneous pressure chamber measurements were performed on neighboring leaves prior to the time of excision and then on the psychrometer leaf about 2 minutes after excision. These data indicate that within the first 2 minutes after excision, psychrometer and pressure chamber measurements were linearly correlated (r = 0.97). Under high evaporative demand conditions, the rate of water potential decrease was between 250 and 700 kilopascals in the first minute after excision. These results show that the thermocouple psychrometer can be used as a dynamic and nondestructive field technique for monitoring leaf water potential.     

Speciation and Bioavailability Measurements of Environmental Plutonium Using Diffusion in Thin Films

The biological uptake of plutonium (Pu) in aquatic ecosystems is of particular concern since it is an alpha-particle emitter with long half-life which can potentially contribute to the exposure of biota and humans. The diffusive gradients in thin films technique is introduced here for in-situ measurements of Pu bioavailability and speciation. A diffusion cell constructed for laboratory experiments with Pu and the newly developed protocol make it possible to simulate the environmental behavior of Pu in model solutions of various chemical compositions. Adjustment of the oxidation states to Pu(IV) and Pu(V) described in this protocol is essential in order to investigate the complex redox chemistry of plutonium in the environment. The calibration of this technique and the results obtained in the laboratory experiments enable to develop a specific DGT device for in-situ Pu measurements in freshwaters. Accelerator-based mass-spectrometry measurements of Pu accumulated by DGTs in a karst spring allowed determining the bioavailability of Pu in a mineral freshwater environment. Application of this protocol for Pu measurements using DGT devices has a large potential to improve our understanding of the speciation and the biological transfer of Pu in aquatic ecosystems.     

The influence of seasonality in estimating mangrove leaf chlorophyll-a content from hyperspectral data

Mangrove photosynthetic activity and, consequently, physiological stress can be monitored indirectly using leaf chlorophyll-a (Chla) measurements. Recent studies have demonstrated the feasibility of mangrove leaf Chla content estimation from in situ hyperspectral vegetation indices (VI) but no such research has been conducted using data collected from contrasting seasons (i.e. dry and rainy). In this study, mangrove leaves were collected in a sub-tropical forest of the Mexican Pacific for Chla content determination and in situ hyperspectral measurements (450–1,000 nm). Specifically, we tested 35 VI to estimate Chla content based on a leaf sample of 360 collected from the same trees during both the dry and rainy seasons. The forest examined contained three species of mangrove (Rhizophora mangle, Avicennia germinans and Laguncularia racemosa) exhibiting various conditions of health (dwarf condition, tall and healthy). A principal component analysis, followed by linear regression analyses, were conducted in order to identify those VI that best predict mangrove leaf Chla content during the two seasons. The results indicate that VI derived from hyperspectral measurements collected during the dry season are better at estimating leaf Chla content than those collected during the rainy season. Among the 35 VI, the Vog1 (R₇₄₀/R₇₂₀) index was found to be the best predictor of mangrove leaf Chla content, resulting in R ² values of 0.80 and 0.68 for the dry and rainy season respectively. These results would suggest that for identifying variation in mangrove forest stress (i.e. health) in sub-tropical regions, hyperspectral measurements should be carried out during the dry season.     

3-D reconstruction of biological objects using underwater video technique and image processing

This paper describes a 3-D reconstruction method which allows accurate measurements of volume, surface area and other morphometric measurements of three-dimensional biological objects, without removing them from the sea. It represents a novel approach based on multiple views (eight resulted to be sufficient) from underwater video images and a new image processing procedure (MOD3D), whose application has met the basic requirements (i.e. to work on images recorded in turbid waters, with nonuniform lighting, to investigate large areas and in reasonable time, etc.) imposed when operating in the marine environment with simple, easy-to-use and nonprofessional equipment. It is a noninvasive, nondestructive and in the field fast method, thus suitable for sampling also at relevant depth, whose applicability has specifically been set up for a range of growth forms from massive to submassive and irregularly shaped. The accuracy of the method was assessed using models with three levels of 3-D complexity: simple, moderate and complex morphology. A high accuracy of volume measurements made through MOD3D image analysis software was achieved when compared with the laboratory water displacement method, which represents the most accurate method for volume measurement, with an overall mean percent error of about 1.7% (S.D. 2.2%). For all three levels of morphologic complexity, no significant differences (p>0.05) were found. Volume measurements obtained in field based on geometric approximation resulted rough, with significant differences from the MOD3D values (p<0.05). The geometric approximation was lower than MOD3D for simple and moderate morphology, and variable for complex morphology. For all three models, MOD3D values for surface area computation were consistently lower (mean error 13%) than the foil-wrapping values (p<0.05), due to overlap error when foil wrapping. Two applications were made with the bryozoan Pentapora fascialis and the coral Cladocora caespitosa to quantify carbonate standing stock and biomass of these two carbonate framework builders, whose importance has been recently recognised among the temperate sublittoral benthic species. Time required for the 3-D reconstruction method (about 3 h) makes it suitable for routine application particularly for relatively large area investigations, with irregularly shaped objects on rough substrate and several biological objects within the area.     

In Vivo Venous Assessment of Red Blood Cell Aggregate Sizes in Diabetic Patients with a Quantitative Cellular Ultrasound Imaging Method: Proof of Concept

Background

Diabetic patients present higher level of red blood cell (RBC) aggregation contributing to the development of vascular complications. While it has been suggested that this hematology/rheology parameter could bring additional prognostic information for the management of those patients, RBC aggregation screening is not included as a clinical practice. Most medical centers are not equipped to measure properly this parameter, although sedimentation tests can bring some indication. Here, we aimed at evaluating the feasibility of using ultrasound to assess in-vivo hyper-aggregation in type 2 diabetic patients.

Research design and methods

Seventeen diabetic patients and 15 control subjects underwent ultrasound measurements of RBC aggregation in both cephalic and great saphenous veins. Non-invasive in-vivo ultrasound measurements were performed using a newly developed cellular imaging technique, the structure factor size and attenuation estimator (SFSAE). Comparisons with an ex-vivo gold standard rheometry technique were done, along with measurements of pro-aggregating plasma molecule concentrations.

Results

In-vivo RBC aggregation was significantly higher in diabetic patients compared with controls for cephalic vein measurements, while a trend (p = 0.055) was noticed in the great saphenous vein. SFSAE measurements were correlated with gold standard in-vitro measures, fibrinogen and C-reactive protein plasma concentrations.

Conclusion

RBC aggregation can be measured in-vivo in diabetic patients using ultrasound. Prospective studies are needed to determine whether the SFSAE method could help clinicians in the early management of vascular complications in this patient population.     

An improved spectrophotometric assay for human plasma carboxypeptidase N

A continuous spectrophotometric assay for human plasma carboxypeptidase N utilizing furylacryloyl-alanyl-lysine is described. Synthesis was made by use of 9-(2-sulfo)fluorenylmethyloxycarbonyl (Sulfmoc) chloride as the N-?-amino-blocking group for lysine. The substrate has the advantage of containing a chromophore which allows difference measurements above 324 nm. The kinetic parameters K_m and $\text{K}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ have been determined for furylacryloyl-alanyl-lysine and -arginine. Difference measurements were related to micromoles of lysine or arginine released and were expressed as units.     

Calorimetric,density and circular dichroism studies of the reversible structural transition in Pf1 filamentous bacterial virus

The macromolecular structural transition of Pf1 filamentous bacterial virus detected by X-ray diffraction analysis has been studied in virus solutions by density, circular dichroism, and microcalorimetric measurements. The reversible structural change occurring between 5 °C and 25 °C has a calorimetrically determined transition enthalpy ΔH_t,cal of 14·5 ± 1.5 kJ (mol protein)^?1. The transition curves resulting from the density, circular dichroism, and calorimetric measurements have been analysed in terms of a two-state process to extract the van't Hoff enthalpy. Comparison of the effective transition enthalpy and the calorimetric ΔH_t,cal values gives about 26 protein subunits as the size of the co-operative unit. Parallel heat capacity and density measurements on fd virus show no such transition, in agreement with X-ray diffraction studies.     

Ion-Dependent Dynamics of DNA Ejections for Bacteriophage λ

We studied the control parameters that govern the dynamics of in vitro DNA ejection in bacteriophage λ. Previous work demonstrated that bacteriophage DNA is highly pressurized, and this pressure has been hypothesized to help drive DNA ejection. Ions influence this process by screening charges on DNA; however, a systematic variation of salt concentrations to explore these effects has not been undertaken. To study the nature of the forces driving DNA ejection, we performed in vitro measurements of DNA ejection in bulk and at the single-phage level. We present measurements on the dynamics of ejection and on the self-repulsion force driving ejection. We examine the role of ion concentration and identity in both measurements, and show that the charge of counterions is an important control parameter. These measurements show that the mobility of ejecting DNA is independent of ionic concentrations for a given amount of DNA in the capsid. We also present evidence that phage DNA forms loops during ejection, and confirm that this effect occurs using optical tweezers.     

Measurement of proton-linked transport activities in pyranine loaded chloroplast inner envelope vesicles

A method has been devised for loading chloroplast inner envelope vesicles prepared from pea (Pisum sativum L. var Progress No. 9) or spinach (Spinacia oleracea L.) with 8-hydroxypyrene-1,3,6-trisulfonate (pyranine), a membrane impermeant, fluorescent pH indicator. Two known proton-linked transport activities of the inner envelope, glycolate/H⁺ co-transport and phosphate/phosphoglycerate exchange have been shown to cause quenching of the internal pyranine fluorescence. This represents the first demonstration that these vesicles are sealed and competent for transport measurements. The technique, as it now stands, is essentially qualitative. It does, however, offer advantages over transport measurements with intact chloroplasts, for example compatibility with rapid mixing techniques and accessibility of the transport proteins to antibodies.     

Using FCS to accurately measure protein concentration in the presence of noise and photobleaching

Quantitative cell biology requires precise and accurate concentration measurements, resolved both in space and time. Fluorescence correlation spectroscopy (FCS) has been held as a promising technique to perform such measurements because the fluorescence fluctuations it relies on are directly dependent on the absolute number of fluorophores in the detection volume. However, the most interesting applications are in cells, where autofluorescence and confinement result in strong background noise and important levels of photobleaching. Both noise and photobleaching introduce systematic bias in FCS concentration measurements and need to be corrected for. Here, we propose to make use of the photobleaching inevitably occurring in confined environments to perform series of FCS measurements at different fluorophore concentration, which we show allows a precise in situ measurement of both background noise and molecular brightness. Such a measurement can then be used as a calibration to transform confocal intensity images into concentration maps. The power of this approach is first illustrated with in vitro measurements using different dye solutions, then its applicability for in vivo measurements is demonstrated in Drosophila embryos for a model nuclear protein and for two morphogens, Bicoid and Capicua.     

Probing the Conformation of FhaC with Small-Angle Neutron Scattering and Molecular Modeling

Probing the solution structure of membrane proteins represents a formidable challenge, particularly when using small-angle scattering. Detergent molecules often present residual scattering contributions even at their match point in small-angle neutron scattering (SANS) measurements. Here, we studied the conformation of FhaC, the outer-membrane, β-barrel transporter of the Bordetella pertussis filamentous hemagglutinin adhesin. SANS measurements were performed on homogeneous solutions of FhaC solubilized in n-octyl-d17-βD-glucoside and on a variant devoid of the α helix H1, which critically obstructs the FhaC pore, in two solvent conditions corresponding to the match points of the protein and the detergent, respectively. Protein-bound detergent amounted to 142 ± 10 mol/mol as determined by analytical ultracentrifugation. By using molecular modeling and starting from three distinct conformations of FhaC and its variant embedded in lipid bilayers, we generated ensembles of protein-detergent arrangement models with 120–160 detergent molecules. The scattered curves were back-calculated for each model and compared with experimental data. Good fits were obtained for relatively compact, connected detergent belts, which occasionally displayed small detergent-free patches on the outer surface of the β barrel. The combination of SANS and modeling clearly enabled us to infer the solution structure of FhaC, with H1 inside the pore as in the crystal structure. We believe that our strategy of combining explicit atomic detergent modeling with SANS measurements has significant potential for structural studies of other detergent-solubilized membrane proteins.     

Room-Temperature Distance Measurements of Immobilized Spin-Labeled Protein by DEER/PELDOR

Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (T_m) to be too short to perform DEER measurements at temperatures between ∼80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has longer T_m between 80 and 295 K in immobilized samples than conventional labels. Two of the spirocyclohexyl labels were attached to sites on T4 lysozyme introduced by site-directed spin labeling. Interspin distances up to ∼4 nm were measured by DEER at temperatures up to 160 K in water/glycerol glasses. In a glassy trehalose matrix the T_m for the doubly labeled T4 lysozyme was long enough to measure an interspin distance of 3.2 nm at 295 K, which could not be measured for the same protein labeled with the conventional 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-(methyl)methanethio-sulfonate label.     

Ratio of cut surface area to leaf sample volume for water potential measurements by thermocouple psychrometers

Evaporative losses from the cut edge of leaf samples are of considerable importance in measurements of leaf water potential using thermocouple psychrometers. The ratio of cut surface area to leaf sample volume (area to volume ratio) has been used to give an estimate of possible effects of evaporative loss in relation to sample size. A wide range of sample sizes with different area to volume ratios has been used. Our results using Glycine max L. Merr. cv Bragg indicate that leaf samples with area to volume values less than 0.2 square millimeter per cubic millimeter give psychrometric leaf water potential measurements that compare favorably with pressure chamber measurements.