Marine diatoms are important primary producers. Generally, considerable effort is needed to maintain many diatom cultures through periodic sub-inoculation and photoirradiation cultivation procedures. To reduce these endeavors and promote diatom research, this study established a simplified method for the cryopreservation of marine diatoms. Vegetative cells of Chaetoceros tenuissimus , Phaeodactylum tricornutum , and the other species were transferred into a ready-made cryoprotective reagent, CELLBANKER, and directly frozen at − 80 °C and/or in liquid nitrogen (at below − 180 °C) for over two days. Frozen samples were transferred into seawater-based media and cultivated under photoirradiation. Although C . tenuissimus cells were frozen for two years (756 days), certain vegetative cells appeared and grew satisfactorily. Two transgenic types and the wild-type of P . tricornutum were reactivated from the frozen state to maintain the transgenic features. Using the same procedure, we succeeded in cryopreserving Chaetoceros cf. ceratosporus . Our method will be helpful for briefly maintaining diatom cultures and preparing their backup without considerable effort.

Diadumene lineata is a colorful sea anemone with orange stripe tissue of the body column and plain tentacles with red lines. We subjected Diadumene lineata to expression cloning and obtained genes encoding orange (OFP: DiLiFP561) and red fluorescent proteins (RFPs: DiLiFP570 and DiLiFP571). These proteins formed obligatory tetramers. All three proteins showed bright fluorescence with the brightness of 58.3 mM −1 ·cm −1 (DiLiFP561), 43.9 mM −1 ·cm −1 (DiLiFP570), and 31.2 mM −1 ·cm −1 (DiLiFP571), which were equivalent to that of commonly used red fluorescent proteins. Amplitude-weighted average fluorescence lifetimes of DiLiFP561, DiLiFP570 and DiLiFP571 were determined as 3.7, 3.6 and 3.0 ns. We determined a crystal structure of DiLiFP570 at 1.63 Å resolution. The crystal structure of DiLiFP570 revealed that the chromophore has an extended π-conjugated structure similar to that of DsRed. Most of the amino acid residues surrounding the chromophore were common between DiLiFP570 and DiLiFP561, except M159 of DiLiFP570 (Lysine in DiLiFP561), which is located close to the chromophore hydroxyl group. Interestingly, a similar K-to-M substitution has been reported in a red-shifted variant of DsRed (mRFP1). It is a striking observation that the naturally evolved color-change variants are consistent with the mutation induced via protein engineering processes. The newly cloned proteins are promising as orange and red fluorescent markers for imaging with long fluorescence lifetime. Graphical abstract

The formation of intermediate filaments (IFs), a paradigmatic assembly system in biological macromolecules, depends on cations. Herein, to explore the combined effect of ionic strength and divalent cations, we used fluorescence microscopy and examined the in vitro effects of MgCl2, CaCl2, and SrCl2 on the KCl concentration-dependent growth of desmin IFs. Fluorescently-labeled desmin IF assembly initiated by KCl and 5 mM divalent cations led to the formation of single desmin IFs in the KCl concentration range of 25-50 mM. Addition of divalent cations resulted in increased fluorescence intensity in the filament images. KCl concentrations lower or higher than the aforementioned range resulted in the induction of networks of entangled IFs, which were visualized at high resolution via direct stochastic optical reconstruction microscopy. These findings provide insights into the versatility of the IF assembly mechanism and the optimization of fluorescence microscopy of single desmin IFs.

Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT)14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense. We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein “ember” from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 105 M−1·cm−1. The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers.

In the present study, mechanical phenomena on fractal agar gel were analyzed to understand the interfacial properties of hydrophilic biosurfaces. The evaluation of adhesion strength between the fractal agar gel surfaces showed that the fractal structure inhibits the adhesion between the agar gel surfaces. In addition, when the disintegration behavior of an agar gel block was observed between fractal agar gel substrates, the rough structure prevented the sliding of an agar gel block. These findings are useful for understanding the biological significance of rough structure on the biological surfaces.

AIMS:In atrial fibrillation (AF), abnormalities in Ca(2+) release contribute to arrhythmia generation and contractile dysfunction. We explore whether ryanodine receptor (RyR) cluster ultrastructure is altered and is associated with functional abnormalities in AF. METHODS AND RESULTS:Using high-resolution confocal microscopy (STED), we examined RyR cluster morphology in fixed atrial myocytes from sheep with persistent AF (N = 6) and control (Ctrl; N = 6) animals. RyR clusters on average contained 15 contiguous RyRs; this did not differ between AF and Ctrl. However, the distance between clusters was significantly reduced in AF (288 ± 12 vs. 376 ± 17 nm). When RyR clusters were grouped into Ca(2+) release units (CRUs), i.e. clusters separated by <150 nm, CRUs in AF had more clusters (3.43 ± 0.10 vs. 2.95 ± 0.02 in Ctrl), which were more dispersed. Furthermore, in AF cells, more RyR clusters were found between Z lines. In parallel experiments, Ca(2+) sparks were monitored in live permeabilized myocytes. In AF, myocytes had >50% higher spark frequency with increased spark time to peak (TTP) and duration, and a higher incidence of macrosparks. A computational model of the CRU was used to simulate the morphological alterations observed in AF cells. Increasing cluster fragmentation to the level observed in AF cells caused the observed changes, i.e. higher spark frequency, increased TTP and duration; RyR clusters dispersed between Z-lines increased the occurrence of macrosparks. CONCLUSION:In persistent AF, ultrastructural reorganization of RyR clusters within CRUs is associated with overactive Ca(2+) release, increasing the likelihood of propagating Ca(2+) release.

The lack of a suitable experimental setup and a limited number of in vitro investigations have impeded understanding of the factors that have important roles in altering the drag force acting on tiny microobjects in a fluidic medium. Here we investigated how the surface morphology affects the drag force acting on microparticles. The drag coefficient for micron-sized polymer-grafted polystyrene (PS) and negatively charged hard (silica) and soft (hydrogel) spheres were evaluated and compared with that of a PS particle. The drag coefficient of the PS particle was found to be comparable to that predicted according to the Stokes’ formula for a hard sphere. By contrast, polymer-grafted PS and negatively charged hard and soft spheres showed a considerable deviation from the Stokes’ formula. The drag coefficient decreased by ~60 and ~86% for the negatively charged hard and soft spheres, respectively, whereas it increased by ~20% for the polymer-grafted PS particles with respect to that of the PS particle. This work reveals the role of the surface morphology of small objects in altering the drag force acting on them; such knowledge may help elucidate the mechanism by which the surface morphology of aquatic microorganisms helps them to control the drag force, which consequently might facilitate the invention of new technologies for micromachines or microdevices. Drag force on micron-sized particles in an aqueous medium was determined and compared with that predicted by the Stokes’ law. Effect of a number of parameters such as surface morphology of particles, particle size, and so on on the drag force acting on the microparticles in an aqueous medium was systematically investigated. Surface morphology was found to strongly affect the drag force working on the microparticles. In addition surface charge, roughness, and physical nature of microparticles were also found involved in the alteration of the drag force.

In chronic atrial fibrillation (AF), abnormalities in Ca2+ release from RyR have been implicated as major factors contributing to arrhythmia and contractile dysfunction, but the relation to RyR organization remains unknown. Using STED microscopy we examined RyR cluster morphology in isolated atrial myocytes from sheep with persistent AF (N=6, 16-23 weeks of AF) and age matched control (Ctrl) animals; in parallel experiments we measured Ca2+ sparks in permeabilized myocytes. STED measurements revealed RyR clusters typically contained 15 contiguous RyR and on average this did not differ between AF and Ctrl. However, the nearest neighbor distance between clusters was reduced in AF. Grouping of clusters within 150 nm as functional Ca2+ release units (CRU) indicated that in AF these units exhibit increased fragmentation, with more clusters per functional unit. Measurement of Ca2+ sparks in permeabilized myocytes revealed a >50% increase in spark frequency and a higher prevalence of macrosparks. Spark time-to-peak (TTP) and duration were also increased, but width was reduced. Measurement of the intrinsic cellular buffer capacity showed this was reduced in AF. Using computational modeling it was found that the increased TTP and duration can be ascribed to the increased number of clusters per functional grouping in AF. The observed increased CRU fragmentation and reduction in Ca2+ buffering, can increased occurrence of sparks and macrosparks in AF. In conclusion, ultrastructural reorganization of RyR clusters within the functional units contributes to overactive Ca2+ release with increased chance of propagating events in AF.

This IUPAC Technical Report describes and compares the currently applied methods for measuring and analyzing time-resolved fluorescence traces using phase-modulation fluorometry as well as pulse fluorometry (direct emission decay measurements, single-photon timing, streak camera measurements, fluorescence upconversion, and optical Kerr gating). The paper starts with a brief description of the basic principles for time and frequency domain fluorescence spectroscopy. The fundamental equations are given, and recommendations for adequate use are emphasized. The up-to-date, commonly employed excitation sources and photodetectors are described in detail. The analysis of time-resolved fluorescence data is discussed. Attention is paid to possible artifacts, and remedies are presented on how to avoid them or to account for them. Finally, fluorescence lifetime standards for the nanosecond and picosecond timescales are collected.

The effects of silica particle addition on the wetting velocity on flat and fractal agar gel surfaces were analyzed along with the applicability of such particles for controlling the wetting dynamics of water. The contact angles (θD) of the colloidal dispersions obeyed the power law, i.e., θD∝t(-x), where t is time and x is a constant. Wetting was inhibited by the addition of a suitable amount of 20-nm-diameter silica particles. Specifically, the exponent x reached a minimum value for a silica composition of 0.1wt%. However, such inhibition effects were not observed upon the addition of silica particles with diameters of 100, 550, and, 1000nm. The mechanism of the inhibition of the liquid wetting on gel surfaces may be attributed to a slight increase in local viscosity around the contact line during wetting.

Background The syndrome of combined pulmonary fibrosis and emphysema (CPFE) is a recently described entity associating upper-lobe emphysema and lower-lobe fibrosis. We sought to evaluate differences in pulmonary function between CPFE patients with and without airflow obstruction. Subjects and methods Thirty-one CPFE patients were divided into two groups according to the presence or absence of irreversible airflow obstruction based on spirometry (forced expiratory volume in 1 second/forced vital capacity <70% following inhalation of a β2-agonist) as follows: CPFE patients with airflow obstruction (CPFE OB+ group, n=11), and CPFE patients without airflow obstruction (CPFE OB− group, n=20). Pulmonary function, including respiratory impedance evaluated using impulse oscillometry and dynamic hyperinflation following metronome-paced incremental hyperventilation, was retrospectively analyzed in comparison with that observed in 49 chronic obstructive pulmonary disease (COPD) patients (n=49). Results In imaging findings, low-attenuation-area scores on chest high-resolution computed tomography, representing the degree of emphysema, were significantly lower in the CPFE OB− group than in the CPFE OB+ and COPD groups. In contrast, the severity of pulmonary fibrosis was greater in the CPFE OB− group than in the CPFE OB+ group. In pulmonary function, lung hyperinflation was not apparent in the CPFE OB− group. Impairment of diffusion capacity was severe in both the CPFE OB− and CPFE OB+ groups. Impulse oscillometry showed that respiratory resistance was not apparent in the CPFE OB− group compared with the COPD group, and that easy collapsibility of small airways during expiration of tidal breath was not apparent in the CPFE OB+ group compared with the COPD group. Dynamic hyperinflation following metronome-paced incremental hyperventilation was significantly greater in the COPD group than in the CPFE OB− group, and also tended to be greater in the CPFE OB+ group than in the CPFE OB− group. Conclusion The mechanisms underlying impairment of physiological function may differ among CPFE OB+ patients, CPFE OB− patients, and COPD patients. CPFE is a heterogeneous disease, and may have distinct phenotypes physiologically and radiologically.