A Methodological Framework to Estimate the Site Fidelity of Tagged Animals Using Passive Acoustic Telemetry

The rapid expansion of the use of passive acoustic telemetry technologies has facilitated unprecedented opportunities for studying the behavior of marine organisms in their natural environment. This technological advance would greatly benefit from the parallel development of dedicated methodologies accounting for the variety of timescales involved in the remote detection of tagged animals related to instrumental, environmental and behavioral events. In this paper we propose a methodological framework for estimating the site fidelity (“residence times”) of acoustic tagged animals at different timescales, based on the survival analysis of continuous residence times recorded at multiple receivers. Our approach is validated through modeling and applied on two distinct datasets obtained from a small coastal pelagic species (bigeye scad, Selar crumenophthalmus) and a large, offshore pelagic species (yellowfin tuna, Thunnus albacares), which show very distinct spatial scales of behavior. The methodological framework proposed herein allows estimating the most appropriate temporal scale for processing passive acoustic telemetry data depending on the scientific question of interest. Our method provides residence times free of the bias inherent to environmental and instrumental noise that can be used to study the small scale behavior of acoustic tagged animals. At larger timescales, it can effectively identify residence times that encompass the diel behavioral excursions of fish out of the acoustic detection range. This study provides a systematic framework for the analysis of passive acoustic telemetry data that can be employed for the comparative study of different species and study sites. The same methodology can be used each time discrete records of animal detections of any nature are employed for estimating the site fidelity of an animal at different timescales.     

Localized Rejuvenation of a Crystal Mush Recorded in Zircon Temporal and Compositional Variation at the Lassen Volcanic Center,Northern California

Zircon ages and trace element compositions from recent silicic eruptions in the Lassen Volcanic Center (LVC) allow for an evaluation of the timing and conditions of rejuvenation (reheating and mobilization of crystals) within the LVC magmatic system. The LVC is the southernmost active Cascade volcano and, prior to the 1980 eruption of Mount St. Helens, was the site of the only eruption in the Cascade arc during the last century. The three most recent silicic eruptions from the LVC were very small to moderate-sized lava flows and domes of dacite (1915 and 27 ka eruptions of Lassen Peak) and rhyodacite (1.1 ka eruption of Chaos Crags). These eruptions produced mixed and mingled lavas that contain a diverse crystal cargo, including zircon. ²³⁸U-²³⁰Th model ages from interior and surface analyses of zircon reveal ages from ∼17 ka to secular equilibrium (>350 ka), with most zircon crystallizing during a period between ∼60–200 ka. These data support a model for localized rejuvenation of crystal mush beneath the LVC. This crystal mush evidently is the remnant of magmatism that ended ∼190 ka. Most zircon are thought to have been captured from “cold storage” in the crystal mush (670–725°C, Hf >10,000 ppm, Eu/Eu* 0.25–0.4) locally remobilized by intrusion of mafic magma. A smaller population of zircon (>730°C, Hf <10,000 ppm, Eu/Eu* >0.4) grew in, and are captured from, rejuvenation zones. These data suggest the dominant method to produce eruptible melt within the LVC is small-scale, local rejuvenation of the crystal mush accompanied by magma mixing and mingling. Based on zircon stability, the time required to heat, erupt and then cool to background conditions is relatively short, lasting a maximum of 10 s–1000 s years. Rejuvenation events in the LVC are ephemeral and permit eruption within an otherwise waning and cooling magmatic body.     

In vivo kinetics of Cajal body components

Cajal bodies (CBs) are subnuclear domains implicated in small nuclear ribonucleoprotein (snRNP) biogenesis. In most cell types, CBs coincide with nuclear gems, which contain the survival of motor neurons (SMN) complex, an essential snRNP assembly factor. Here, we analyze the exchange kinetics of multiple components of CBs and gems in living cells using photobleaching microscopy. We demonstrate differences in dissociation kinetics of CB constituents and relate them to their functions. Coilin and SMN complex members exhibit relatively long CB residence times, whereas components of snRNPs, small nucleolar RNPs, and factors shared with the nucleolus have significantly shorter residence times. Comparison of the dissociation kinetics of these shared proteins from either the nucleolus or the CB suggests the existence of compartment-specific retention mechanisms. The dynamic properties of several CB components do not depend on their interaction with coilin because their dissociation kinetics are unaltered in residual nuclear bodies of coilin knockout cells. Photobleaching and fluorescence resonance energy transfer experiments demonstrate that coilin and SMN can interact within CBs, but their interaction is not the major determinant of their residence times. These results suggest that CBs and gems are kinetically independent structures.     

痛风的临床病理特征分析并文献复习

目的:总结痛风临床病理特点。方法:回顾性分析1例痛风患者的生化机制、临床病理特征、刚果红染色、PAS染色特点、鉴别诊断要点,并复习相关文献。结果:患者主要临床表现为间断性多关节肿痛3年,加重伴发热3个月。体格检查发现患者有多发性皮下结节、多关节肿胀压痛。左腕、左肘皮下结节活检,经HE染色后光镜查见大量肉芽肿性病变,有的多核巨细胞内查见被吞噬的异物,有的病灶尚查见呈均质状物(尿酸盐结晶),其周围有较多异物巨细胞及纤维结缔组织包绕呈结节状,在结节的周边纤维血管周围可查见残留分化成熟的淋巴细胞及少数嗜酸性粒细胞。刚果红、PAS染色均为阴性。偏光显微镜下,刚果红未查见绿色强折光晶体,但见多量略呈淡黄色具有强折光性的晶体呈棒状或梭形。结论:痛风在刚果红染色偏光显微镜下观察呈淡黄色梭形或针状结晶,具有强折光性晶体,但这是否是痛风在刚果红染色的特征尚有待于进一步研究。     

NMR observation of individual molecules of hydration water bound to DNA duplexes: direct evidence for a spine of hydration water present in aqueous solution.

The residence times of individual hydration water molecules in the major and minor grooves of DNA were measured by nuclear magnetic resonance (NMR) spectroscopy in aqueous solutions of d-(CGCGAATTCGCG)2 and d-(AAAAATTTTT)2. The experimental observations were nuclear Overhauser effects (NOE) between water protons and the protons of the DNA. The positive sign of NOEs with the thymine methyl groups shows that the residence times of the hydration water molecules near these protons in the major groove of the DNA must be shorter than about 500 ps, which coincides with the behavior of surface hydration water in peptides and proteins. Negative NOEs were observed with the hydrogen atoms in position 2 of adenine in both duplexes studied. This indicates that a 'spine of hydration' in the minor groove, as observed by X-ray diffraction in DNA crystals, is present also in solution, with residence times significantly longer than 1 ns. Such residence times are reminiscent of 'interior' hydration water molecules in globular proteins, which are an integral part of the molecular architecture both in solution and in crystals.     

Control of nucleation in the crystallization of lysozyme.

This work investigates the influence of storage of lysozyme in solution on its crystallization. The crystallization of hen egg-white lysozyme exhibits a storage effect (aging) that depends on the length of time the lysozyme solution is stored, after dissolving from freeze-dried powder, before being brought to crystallization conditions. The number of crystals obtained increases, while their size decreases, as the solution ages. Observations suggest that this effect is due to the presence of fungi that multiply in the stored protein solution. This aging effect was used to control nucleation and determine the number and size of lysozyme crystals to be formed in a given sample.     

The Role of Quartz in the Origin of Optical Activity on Earth

A thorough analysis of literature data on distribution of rightand left quartz in many locations on the surface of Earthindicates that quartz enantiomorph crystals are distributed inequal amounts in all locations. Therefore optically activequartz crystals of one or the other enantiomorph could not serveas the source of homochirality in the evolution of biosphere.Hence the calculation of a PVED based on published `small excessof left quartz crystals' on Earth lacks a sound physical basis.     

Markovian and non-Markovian protein sequence evolution: aggregated Markov process models

Over the years, there have been claims that evolution proceeds according to systematically different processes over different timescales and that protein evolution behaves in a non-Markovian manner. On the other hand, Markov models are fundamental to many applications in evolutionary studies. Apparent non-Markovian or time-dependent behavior has been attributed to influence of the genetic code at short timescales and dominance of physicochemical properties of the amino acids at long timescales. However, any long time period is simply the accumulation of many short time periods, and it remains unclear why evolution should appear to act systematically differently across the range of timescales studied. We show that the observed time-dependent behavior can be explained qualitatively by modeling protein sequence evolution as an aggregated Markov process (AMP): a time-homogeneous Markovian substitution model observed only at the level of the amino acids encoded by the protein-coding DNA sequence. The study of AMPs sheds new light on the relationship between amino acid-level and codon-level models of sequence evolution, and our results suggest that protein evolution should be modeled at the codon level rather than using amino acid substitution models.     

Ferromanganese phases and the chemistry of contemporary seawater

We report on a correlation between oceanic residence times of chemical elements and their enrichment in marine ferromanganese deposits. When a logarithmic plot of oceanic residence time vs. atomic number is placed above a similar plot of enrichment factor vs. atomic number, the plots exhibit a clear mirror image relationship. We suggest that this relationship is not accidental, but rather implies an important role for ferromanganese phases in the regulation of seawater chemistry, especially the residence times of the minor elements. The formation of ferromanganese phases, as nodules, crusts, particles and coatings on pelagic clays, is dependent upon oxic seawater conditions similar to those existing today. The absence of oxic conditions throughout much of geologic time insures that ferromanganese minerals would have been rare or nonexistent sedimentary deposits. If these phases are as important as contemporary residence time correlations suggest, the minor element chemistry of earlier oceans must have been radically different from that of the present. Moreover, in times of deep water stagnation over the Phanerozic, conditions would have been thermodynamically unsuitable for ferromanganese mineral formation and hence suitable for the accumulation of high concentrations of certain toxic metals. We suggest that metal poisoning resulting from deep water turnover may have contributed to some of the great marine extinctions occurring over Phanerozoic Time.     

Effect of gallbladder hypomotility on cholesterol crystallization and growth in CCK-deficient mice

We investigated the effect of gallbladder hypomotility on cholesterol crystallization and growth during the early stage of gallstone formation in CCK knockout mice. Contrary to wild-type mice, fasting gallbladder volumes were enlarged and the response of gallbladder emptying to a high-fat meal was impaired in knockout mice on chow or the lithogenic diet. In the lithogenic state, large amounts of mucin gel and liquid crystals as well as arc-like and tubular crystals formed first, followed by rapid formation of classic parallelogram-shaped cholesterol monohydrate crystals in knockout mice. Furthermore, three patterns of crystal growth habits were observed: proportional enlargement, spiral dislocation growth, and twin crystal growth, all enlarging solid cholesterol crystals. At day 15 on the lithogenic diet, 75% of knockout mice formed gallstones. However, wild-type mice formed very little mucin gel, liquid, and solid crystals, and gallstones were not observed. We conclude that lack of CCK induces gallbladder hypomotility that prolongs the residence time of excess cholesterol in the gallbladder, leading to rapid crystallization and precipitation of solid cholesterol crystals. Moreover, during the early stage of gallstone formation, there are two pathways of liquid and polymorph anhydrous crystals evolving to monohydrate crystals and three modes for cholesterol crystal growth.     

The inhibition of hemoglobin C crystallization by hemoglobin F

We have reported that circulating CC erythrocytes containing HbO2 C crystals exhibit little or no Hb F suggesting that Hb F may inhibit the crystallization of Hb C. We report now that Hb F inhibits in vitro crystallization of HbO2 and HbCO C when compared to the effect of Hb A in a wide range of mixture proportions. For example, while HbCO C solutions form tetragonal C crystals within 25 min, no crystals form within 2 h with 30% Hb F, whereas 550 crystals/mm3 form with 30% Hb A. Furthermore, an increase in the percent of Hb A is correlated with a greater number of orthorhombic crystal formation rather than the tetragonal morphology observed with 100% Hb C. We also report that Hb A2 (containing delta chains that exhibit 10 sequence differences with beta chains) and Hb Lepore Boston-Washington (a fusion mutant of delta and beta chains that contains only six of these differences) both inhibit Hb C crystallization. By comparing the sequences of the three inhibitory hemoglobins, we conclude that position Gln-87 in the gamma chains is, at least partially, the cause of the inhibitory effect of Hb F on the crystallization of Hb C.     

Preparation of Macroporous Epitaxial Quartz Films on Silicon by Chemical Solution Deposition

This work describes the detailed protocol for preparing piezoelectric macroporous epitaxial quartz films on silicon(100) substrates. This is a three-step process based on the preparation of a sol in a one-pot synthesis which is followed by the deposition of a gel film on Si(100) substrates by evaporation induced self-assembly using the dip-coating technique and ends with a thermal treatment of the material to induce the gel crystallization and the growth of the quartz film. The formation of a silica gel is based on the reaction of a tetraethyl orthosilicate and water, catalyzed by HCl, in ethanol. However, the solution contains two additional components that are essential for preparing mesoporous epitaxial quartz films from these silica gels dip-coated on Si. Alkaline earth ions, like Sr²⁺ act as glass melting agents that facilitate the crystallization of silica and in combination with cetyl trimethylammonium bromide (CTAB) amphiphilic template form a phase separation responsible of the macroporosity of the films. The good matching between the quartz and silicon cell parameters is also essential in the stabilization of quartz over other SiO₂ polymorphs and is at the origin of the epitaxial growth.     

Measurement of fluid viscosity at microliter volumes using quartz impedance analysis

The purpose of this work was to measure viscosity of fluids at low microliter volumes by means of quartz crystal impedance analysis. To achieve this, a novel setup was designed that allowed for measurement of viscosity at volumes of 8 to 10 μL. The technique was based on the principle of electromechanical coupling of piezoelectric quartz crystals. The arrangement was simple with measurement times ranging from 2 to 3 minutes. The crystal setup assembly did not impose any unwanted initial stress on the unloaded quartz crystal. Quartz crystals of 5- and 10-MHz fundamental frequency were calibrated with glycerol-water mixtures of known density and viscosity prior to viscosity measurements. True frequency shifts, for the purpose of this work, were determined followed by viscosity measurement of aqueous solutions of sucrose, urea, PEG-400, glucose, and ethylene glycol at 25°C±0.5°C. The measured viscosities were found to be reproducible and consistent with the values reported in the literature. Minor inconsistencies in the measured resistance and frequency shifts did not affect the results significantly, and were found to be experimental in origin rather than due to electrode surface roughness. Besides, as expected for a viscoelastic fluid, PEG 8000 solutions, the calculated viscosities were found to be less than the reported values due to frequency dependence of storage and loss modulus components of complex viscosity. From the results, it can be concluded that the present setup can provide accurate assessment of viscosity of Newtonian fluids and also shows potential for analyzing non-Newtonian fluids at low microliter volumes.     

Crystallization of biological macromolecules from flash frozen samples on the Russian Space Station Mir

One hundred eighty-three flash frozen, liquid-liquid diffusion and batch method protein and virus crystallization samples were launched aboard the Space Shuttle Discovery on June 27 (STS-71) and transferred to the Russian Space Station Mir on July 1, 1995. They were returned to earth November 20, 1995 (STS-74). Subsequent examination showed that of the 19 types of proteins and viruses investigated, 17 were crystallized during the period on Mir. The experiment demonstrates the utility of this very simple and inexpensive approach for the crystallization of biological macromolecules in space over extended time periods. The distribution of crystals among the three types of containers used indicated small samples yielded results equal or better than larger samples and that long diffusion path lengths were clearly better. Distribution of crystals within the container tubes showed a striking gradient of quality and size that indicated long, narrow tubes yield superior crystals, as predicted from other work based on crystallization in capillaries.     

On the origin of surface imposed anisotropic growth of salicylic and acetylsalicylic acids crystals during droplet evaporation

In this paper droplet evaporative crystallization of salicylic acid (SA) and acetylsalicylic acid (ASA) crystals on different surfaces, such as glass, polyvinyl alcohol (PVA), and paraffin was studied. The obtained crystals were analyzed using powder X-ray diffraction (PXRD) technique. In order to better understand the effect of the surface on evaporative crystallization, crystals deposited on glass were scraped off. Moreover, evaporative crystallization of a large volume of solution was performed. As we found, paraffin which is non-polar surface promotes formation of crystals morphologically similar to those obtained via bulk evaporative crystallization. On the other hand, when crystallization is carried out on the polar surfaces (glass and PVA), there is a significant orientation effect. This phenomenon is manifested by the reduction of the number of peaks in PXRD spectrum recorded for deposited on the surface crystals. Noteworthy, reduction of PXRD signals is not observed for powder samples obtained after scraping crystals off the glass. In order to explain the mechanism of carboxylic crystals growth on the polar surfaces, quantum-chemical computations were performed. It has been found that crystal faces of the strongest orientation effect can be characterized by the highest surface densities of intermolecular interactions energy (IIE). In case of SA and ASA crystals formed on the polar surfaces the most dominant faces are characterized by the highest adhesive and cohesive properties. This suggests that the selection rules of the orientation effect comes directly from surface IIE densities.     

Immobilization of an amino acid racemase for application in crystallization‐based chiral resolutions of asparagine monohydrate

Integration of racemization and a resolution process is an attractive way to overcome yield limitations in the production of pure chiral molecules. Preferential crystallization and other crystallization‐based techniques usually produce low enantiomeric excess in solution, which is a constraint for coupling with racemization. We developed an enzymatic fixed bed reactor that can potentially overcome these unfavorable conditions and improve the overall yield of preferential crystallization. Enzyme immobilization strategies were investigated on covalent‐binding supports. The amino acid racemase immobilized in Purolite ECR 8309F with a load of 35 mg‐enzyme/g‐support showed highest specific activity (approx. 500 U/g‐support) and no loss in activity in reusability tests. Effects of substrate inhibition observed for the free enzyme were overcome after immobilization. A packed bed reactor with the immobilized racemase showed good performance in steady state operation processing low enantiomeric excess inlet. Kinetic parameters from batch reactor experiments can be successfully used for prediction of packed bed reactor performance. Full conversions could be achieved for residence times above 1.1 min. The results suggest the potential of the prepared racemase reactor to be combined with preferential crystallization to improve resolution of asparagine enantiomers.     

Molecular-level thermodynamic and kinetic parameters for the self-assembly of apoferritin molecules into crystals

The self-assembly of apoferritin molecules into crystals is a suitable model for protein crystallization and aggregation; these processes underlie several biological and biomedical phenomena, as well as for protein and virus self-assembly. We use the atomic force microscope in situ, during the crystallization of apoferritin to visualize and quantify at the molecular level the processes responsible for crystal growth. To evaluate the governing thermodynamic parameters, we image the configuration of the incorporation sites, "kinks", on the surface of a growing crystal. We show that the kinks are due to thermal fluctuations of the molecules at the crystal-solution interface. This allows evaluation of the free energy of the intermolecular bond phi=3.0 k(B)T=7.3 kJ/mol. The crystallization free energy, extracted from the protein solubility, is -42 kJ/mol. Published determinations of the second virial coefficient and the protein solubility between 0 and 40 degrees C revealed that the enthalpy of crystallization is close to zero. Analyses based on these three values suggest that the main component in the crystallization driving force is the entropy gain of the water molecules bound to the protein molecules in solution and released upon crystallization. Furthermore, monitoring the incorporation of individual molecules in to the kinks, we determine the characteristic frequency of attachment of individual molecules at one set of conditions. This allows a correlation between the mesoscopic kinetic coefficient for growth and the molecular-level thermodynamic and kinetic parameters determined here. We found that step growth velocity, scaled by the molecular size, equals the product of the kink density and attachment frequency, i.e. the latter pair are the molecular-level parameters for self-assembly of the molecules into crystals.     

Systematic Improvement of Protein Crystals by Determining the Supersolubility Curves of Phase Diagrams

A systematic approach for improving protein crystals by growing them in the metastable zone using the vapor diffusion technique is described. This is a simple technique for optimization of crystallization conditions. Screening around known conditions is performed to establish a working phase diagram for the crystallization of the protein. Dilutions of the crystallization drops across the supersolubility curve into the metastable zone are then carried out as follows: the coverslips holding the hanging drops are transferred, after being incubated for some time at conditions normally giving many small crystals, over reservoirs at concentrations which normally yield clear drops. Fewer, much larger crystals are obtained when the incubation times are optimized, compared with conventional crystallization at similar conditions. This systematic approach has led to the structure determination of the light-harvesting protein C-phycocyanin to the highest-ever resolution of 1.45 Å.     

Evidence for dynamics in proteins as a mechanism for ligand dissociation

Signal transduction, regulatory processes and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times (1/k(off)) should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into k(off) involving a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase whose binding affinities vary by six orders of magnitude. NMR relaxation-dispersion experiments revealed a common set of residues near the binding site that undergo a concerted millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with the binding affinity K(i) and k(off), suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although the forward rate of conformational exchange, k(conf,forward), is faster than k(off), the use of the ligand series allowed for connections to be drawn between kinetic events on different timescales.     

Imaging of protein crystals with two-photon microscopy

Second-order nonlinear optical imaging of chiral crystals (SONICC), which portrays second-harmonic generation (SHG) by noncentrosymmetric crystals, is emerging as a powerful imaging technique for protein crystals in media opaque to visible light because of its high signal-to-noise ratio. Here we report the incorporation of both SONICC and two-photon excited fluorescence (TPEF) into one imaging system that allows visualization of crystals as small as ~10 μm in their longest dimension. Using this system, we then documented an inverse correlation between the level of symmetry in examined crystals and the intensity of their SHG. Moreover, because of blue-green TPEF exhibited by most tested protein crystals, we also could identify and image SHG-silent protein crystals. Our experimental data suggest that the TPEF in protein crystals is mainly caused by the oxidation of tryptophan residues. Additionally, we found that unspecific fluorescent dyes are able to bind to lysozyme crystals and enhance their detection by TPEF. We finally confirmed that the observed fluorescence was generated by a two-photon rather than a three-photon process. The capability for imaging small protein crystals in turbid or opaque media with nondamaging infrared light in a single system makes the combination of SHG and intrinsic visible TPEF a powerful tool for nondestructive protein crystal identification and characterization during crystallization trials.