Respiratory Sinus Arrhythmia Versus Neck/Trapezius EMG and Incentive Inspirometry Biofeedback for Asthma: A Pilot Study

This pilot study compared biofeedback to increase respiratory sinus arrhythmia (RSA) with EMG and incentive inspirometry biofeedback in asthmatic adults. A three-group design (Waiting List Control n = 5, RSA biofeedback n = 6, and EMG biofeedback n = 6) was used. Six sessions of training were given in each of the biofeedback groups. In each of three testing sessions, five min. of respiratory resistance and EKG were obtained before and after a 20-min biofeedback session. Additional five-min epochs of data were collected at the beginning and end of the biofeedback period (or, in the control group, self-relaxation). Decreases in respiratory impedance occurred only in the RSA biofeedback group. Traub-Hering-Mayer (THM) waves (.03-.12 Hz) in heart period increased significantly in amplitude during RSA biofeedback. Subjects did not report significantly more relaxation during EMG or RSA biofeedback than during the control condition. However, decreases in pulmonary impedance, across groups, were associated with increases in relaxation. The results are consistent with Vaschillo's theory that RSA biofeedback exercises homeostatic autonomic reflex mechanisms through increasing the amplitude of cardiac oscillations. However, deep breathing during RSA biofeedback is a possible alternate explanation.     

Structural analysis of Francisella tularensis lipopolysaccharide.

The structure of the lipid A and core region of the lipopolysaccharide (LPS) from Francisella tularensis (ATCC 29684) was analysed using NMR, mass spectrometry and chemical methods. The LPS contains a beta-GlcN-(1-6)-GlcN lipid A backbone, but has a number of unusual structural features; it apparently has no substituent at O-1 of the reducing end GlcN residue in the lipid part in the major part of the population, no substituents at O-3 and O-4 of beta-GlcN, and no substituent at O-4 of the Kdo residue. The largest oligosaccharide, isolated after strong alkaline deacylation of NaBH4 reduced LPS had the following structure: where Delta-GalNA-(1-3)-beta-QuiNAc represents a modified fragment of the O-chain repeating unit. Two shorter oligosaccharides lacking the O-chain fragment were also identified. A minor amount of the disaccharide beta-GlcN-(1-6)-alpha-GlcN-1-P was isolated from the same reaction mixture, indicating the presence of free lipid A, unsubstituted by Kdo and with phosphate at the reducing end. The lipid A, isolated from the products of mild acid hydrolysis, had the structure 2-N-(3-O-acyl4-acyl2)-beta-GlcN-(1-6)-2-N-acyl1-3-O-acyl3-GlcN where acyl1, acyl2 and acyl3 are 3-hydroxyhexadecanoic or 3-hydroxyoctadecanoic acids, acyl4 is tetradecanoic or (minor) hexadecanoic acids. No phosphate substituents were found in this compound. OH-1 of the reducing end glucosamine, and OH-3 and OH-4 of the nonreducing end glucosamine residues were not substituted. LPS of F. tularensis exhibits unusual biological properties, including low endoxicity, which may be related to its unusual lipid A structure.     

Phycobilisomes from the mutant cyanobacterium Synechocystis sp. PCC 6803 missing chromophore domain of ApcE

Phycobilisome (PBS) is a giant photosynthetic antenna associated with the thylakoid membranes of cyanobacteria and red algae. PBS consists of two domains: central core and peripheral rods assembled of disc-shaped phycobiliprotein aggregates and linker polypeptides. The study of the PBS architecture is hindered due to the lack of the data on the structure of the large ApcE-linker also called L_CM. ApcE participates in the PBS core stabilization, PBS anchoring to the photosynthetic membrane, transfer of the light energy to chlorophyll, and, very probably, the interaction with the orange carotenoid protein (OCP) during the non-photochemical PBS quenching. We have constructed the cyanobacterium Synechocystis sp. PCC 6803 mutant lacking 235 N-terminal amino acids of the chromophorylated PBL_CM domain of ApcE. The altered fluorescence characteristics of the mutant PBSs indicate that the energy transfer to the terminal emitters within the mutant PBS is largely disturbed. The PBSs of the mutant become unable to attach to the thylakoid membrane, which correlates with the identified absence of the energy transfer from the PBSs to the photosystem II. At the same time, the energy transfer from the PBS to the photosystem I was registered in the mutant cells and seems to occur due to the small cylindrical CpcG2-PBSs formation in addition to the conventional PBSs. In contrast to the wild type Synechocystis, the OCP-mediated non-photochemical PBS quenching was not registered in the mutant cells. Thus, the PBL_CM domain takes part in formation of the OCP binding site in the PBS.     

Late Campanian to late Maastrichtian bryozoans encrusting on belemnite rostra from the Aktolagay Plateau in western Kazakhstan

Twenty species belonging to fifteen genera of cyclostome and cheilostome bryozoans encrusting belemnite rostra are described from the late Campanian to Maastrichtian of the Aktolagay Plateau, in western Kazakhstan. Due to the moderate to poor preservation of the material, only four cheilostome species are identified down to the species level: Wilbertopora? besoktiensis (Voigt, 1967), ‘Aechmellina’ stenostoma Voigt, 1930, and two new species, ‘Aechmellina’ viskovae and Cheethamia aktolagayensis. All remaining species are left in open nomenclature. Type material of Wilbertopora? besoktiensis from the early Maastrichtian of the Mangyshlak Peninsula in Kazakhstan, has been re-examined. Palaeobiogeographical and implications are discussed. Cheilostomes slightly dominated over cyclostomes in the Aktolagay Plateau fauna encrusting on belemnites in terms of diversity. The dominant colony forms observed were spots and sheets.     

Live‐cell CRISPR imaging in plants reveals dynamic telomere movements

Elucidating the spatiotemporal organization of the genome inside the nucleus is imperative to our understanding of the regulation of genes and non‐coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long‐standing gap between sequencing studies, which reveal genomic information, and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR associated protein 9 (Cas9). By fusing eGFP/mRuby2 to catalytically inactive versions of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR–dCas9, we reveal dynamic telomere movements of up to 2 μm over 30 min during interphase. Furthermore, we show that CRISPR–dCas9 can be combined with fluorescence‐labelled proteins to visualize DNA–protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for the imaging of multiple genomic loci in live plants cells. CRISPR imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells.     

Biophysical modeling of in vitro and in vivo processes underlying regulated photoprotective mechanism in cyanobacteria

Non-photochemical quenching (NPQ) is a mechanism responsible for high light tolerance in photosynthetic organisms. In cyanobacteria, NPQ is realized by the interplay between light-harvesting complexes, phycobilisomes (PBs), a light sensor and effector of NPQ, the photoactive orange carotenoid protein (OCP), and the fluorescence recovery protein (FRP). Here, we introduced a biophysical model, which takes into account the whole spectrum of interactions between PBs, OCP, and FRP and describes the experimental PBs fluorescence kinetics, unraveling interaction rate constants between the components involved and their relative concentrations in the cell. We took benefit from the possibility to reconstruct the photoprotection mechanism and its parts in vitro, where most of the parameters could be varied, to develop the model and then applied it to describe the NPQ kinetics in the Synechocystis sp. PCC 6803 mutant lacking photosystems. Our analyses revealed  that while an excess of the OCP over PBs is required to obtain substantial PBs fluorescence quenching in vitro, in vivo the OCP/PBs ratio is less than unity, due to higher local concentration of PBs, which was estimated as ~10^?5 M, compared to in vitro experiments. The analysis of PBs fluorescence recovery on the basis of the generalized model of enzymatic catalysis resulted in determination of the FRP concentration in vivo close to 10% of the OCP concentration. Finally, the possible role of the FRP oligomeric state alteration in the kinetics of PBs fluorescence was shown. This paper provides the most comprehensive model of the OCP-induced PBs fluorescence quenching to date and the results are important for better understanding of the regulatory molecular mechanisms underlying NPQ in cyanobacteria.

     

Characterization of the cell surface glycolipid from <Emphasis Type="Italic">Spirochaeta aurantia</Emphasis>

Spirochaeta aurantia is a free-living saprophytic spirochete that grows easily in simple laboratory media, and thus can be used as a model for the investigation of surface carbohydrate structures in spirochetae, which are normally not available in sufficient amounts. Freeze-substitution electron microscopy indicated the presence of a capsule-like material projecting from the surface of S. aurantia. Extraction of cells gave two major glycolipids, the one with a higher molecular mass glycolipid was designated large glycolipid A (LGLA). LGLA contained small amount of branched and unsaturated O-linked fatty acids, l-rhamnose, l-fucose, d-xylose, d-mannose, d-glucosamine, d-glycero-d-gluco-heptose (DDglcHep), d-glycero-d-manno-heptose (DDHep), and a novel branched tetradeoxydecose monosaccharide, which we proposed to call aurantose (Aur). The carbohydrate structure of LGLA was extremely complex and consisted of the repeating units built of 11 monosaccharides, arrangement of nine of them was determined as:

$\matrix {{\quad \quad \quad \quad \quad {\text{ - [ - 3 - }}\beta {\text{ - DDglcHep - }}3{\text{ - }}\beta {\text{ - D - GlcNAc - 2 - }}\beta {\text{ - D - Man - ] - }}}} \\ {{\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad |}} \\ {{\alpha {\text{ - Aur - 3 - }}\beta {\text{ - L - Rha - 4 - }}\beta {\text{ - D - Xyl - 4 - }}\alpha {\text{ - L - Fuc - 3 - }}\beta {\text{ - DDHep - 4}}}} \\ {{\quad \quad \quad \quad \quad \quad \quad \quad \quad |}} \\ {{\alpha {\text{ - L - Rha - 3}}}} \ $

which wasdeduced from the NMR and chemical data on the LGLA and its fragments, obtained by various degradations. Tentative position of two remaining sugars is proposed. LGLA was negative for gelation of Limulus amebocyte lysate, did not contain lipid A, and was unable to activate any known Toll-like receptors.

     

Fluorescence changes accompanying short-term light adaptations in photosystem I and photosystem II of the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 and phycobiliprotein-impaired mutants: State 1/State 2 transitions and carotenoid-induced quenching of phycobilisomes

The features of the two types of short-term light-adaptations of photosynthetic apparatus, State 1/State 2 transitions, and non-photochemical fluorescence quenching of phycobilisomes (PBS) by orange carotene-protein (OCP) were compared in the cyanobacterium Synechocystis sp. PCC 6803 wild type, CK pigment mutant lacking phycocyanin, and PAL mutant totally devoid of phycobiliproteins. The permanent presence of PBS-specific peaks in the in situ action spectra of photosystem I (PSI) and photosystem II (PSII), as well as in the 77 K fluorescence excitation spectra for chlorophyll emission at 690 nm (PSII) and 725 nm (PSI) showed that PBS are constitutive antenna complexes of both photosystems. The mutant strains compensated the lack of phycobiliproteins by higher PSII content and by intensification of photosynthetic linear electron transfer. The detectable changes of energy migration from PBS to the PSI and PSII in the Synechocystis wild type and the CK mutant in State 1 and State 2 according to the fluorescence excitation spectra measurements were not registered. The constant level of fluorescence emission of PSI during State 1/State 2 transitions and simultaneous increase of chlorophyll fluorescence emission of PSII in State 1 in Synechocystis PAL mutant allowed to propose that spillover is an unlikely mechanism of state transitions. Blue–green light absorbed by OCP diminished the rout of energy from PBS to PSI while energy migration from PBS to PSII was less influenced. Therefore, the main role of OCP-induced quenching of PBS is the limitation of PSI activity and cyclic electron transport under relatively high light conditions.