Cellulosomes-structure and ultrastructure

The cellulosome is a macromolecular machine, whose components interact in a synergistic manner to catalyze the efficient degradation of cellulose. The cellulosome complex is composed of numerous kinds of cellulases and related enzyme subunits, which are assembled into the complex by virtue of a unique type of scaffolding subunit (scaffoldin). Each of the cellulosomal subunits consists of a multiple set of modules, two classes of which (dockerin domains on the enzymes and cohesin domains on scaffoldin) govern the incorporation of the enzymatic subunits into the cellulosome complex. Another scaffoldin module-the cellulose-binding domain-is responsible for binding to the substrate. Some cellulosomes appear to be tethered to the cell envelope via similarly intricate, multiple-domain anchoring proteins. The assemblage is organized into dynamic polycellulosomal organelles, which adorn the cell surface. The cellulosome dictates both the binding of the cell to the substrate and its extracellular decomposition to soluble sugars, which are then taken up and assimilated by normal cellular processes.     

Network Events on Multiple Space and Time Scales in Cultured Neural Networks and in a Stochastic Rate Model

Cortical networks, in-vitro as well as in-vivo, can spontaneously generate a variety of collective dynamical events such as network spikes, UP and DOWN states, global oscillations, and avalanches. Though each of them has been variously recognized in previous works as expression of the excitability of the cortical tissue and the associated nonlinear dynamics, a unified picture of the determinant factors (dynamical and architectural) is desirable and not yet available. Progress has also been partially hindered by the use of a variety of statistical measures to define the network events of interest. We propose here a common probabilistic definition of network events that, applied to the firing activity of cultured neural networks, highlights the co-occurrence of network spikes, power-law distributed avalanches, and exponentially distributed ‘quasi-orbits’, which offer a third type of collective behavior. A rate model, including synaptic excitation and inhibition with no imposed topology, synaptic short-term depression, and finite-size noise, accounts for all these different, coexisting phenomena. We find that their emergence is largely regulated by the proximity to an oscillatory instability of the dynamics, where the non-linear excitable behavior leads to a self-amplification of activity fluctuations over a wide range of scales in space and time. In this sense, the cultured network dynamics is compatible with an excitation-inhibition balance corresponding to a slightly sub-critical regime. Finally, we propose and test a method to infer the characteristic time of the fatigue process, from the observed time course of the network’s firing rate. Unlike the model, possessing a single fatigue mechanism, the cultured network appears to show multiple time scales, signalling the possible coexistence of different fatigue mechanisms.     

Optical characteristics and parameters of the plasma of a barrier discharge excited in a mixture of mercury dibromide vapor with nitrogen and helium

Results are presented from experimental and theoretical studies of the optical characteristics and parameters of the plasma of an atmospheric-pressure barrier discharge excited in a HgBr₂: N₂: He mixture, which was used as the working medium of a small-size (with a radiation area of 8 cm²) exciplex gas-discharge radiation source. The mean radiation power of 87 mW was achieved at the radiation wavelength λ_max = 502 nm. The electron energy distribution function, the transport characteristics, the specific energy lost in the processes involving electrons, the electron temperature and density, and the rate constants of elastic and inelastic electron scattering by the components of the working mixture were calculated as functions of the reduced field E/N. The plasma of a discharge excited in a HgBr₂: N₂: He mixture can be used as the working medium of a small-size blue-green radiation source. Such a source can find application in biotechnology, photonics, and medicine and can also be used to manufacture gas-discharge display panels.     

Novel Technique for Quantifying Adhesion of Metarhizium anisopliae Conidia to the Tick Cuticle

The present study describes an accurate quantitative method for quantifying the adherence of conidia to the arthropod cuticle and the dynamics of conidial germination on the host. The method was developed using conidia of Metarhizium anisopliae var. anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae) and engorged Rhipicephalus annulatus (Say) (Arachnida: Ixodidae) females and was also verified for M. anisopliae var. acridum Driver et Milner (Hypocreales: Clavicipitaceae) and Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) larvae. This novel method is based on using an organic solvent (dichloromethane [DCM]) to remove the adhered conidia from the tick cuticle, suspending the conidia in a detergent solution, and then counting them using a hemocytometer. To confirm the efficacy of the method, scanning electron microscopy (SEM) was used to observe the conidial adherence to and removal from the tick cuticle. As the concentration of conidia in the suspension increased, there were correlating increases in both the number of conidia adhering to engorged female R. annulatus and tick mortality. However, no correlation was observed between a tick''s susceptibility to fungal infection and the amount of adhered conidia. These findings support the commonly accepted understanding of the nature of the adhesion process. The mechanism enabling the removal of the adhered conidia from the host cuticle is discussed.The entomopathogenic fungus Metarhizium anisopliae var. anisopliae (Metschn.) Sorokîn (1883) infects a broad range of arthropod hosts and can be used as a biopesticide against different insect and tick species (8, 22, 35, 36). The adhesion of the conidia of entomopathogenic fungi to the host cuticle is the initial stage of the pathogenic process and includes both passive and active events (5, 10). The hydrophobic epicuticular lipid layer plays an important role during both the attachment process and the germination of the conidia on the surface of the host (15, 19). According to Boucias et al. (7), the attachment of conidia to the host cuticle is based on nonspecific hydrophobic and electrostatic forces. The conidia of most entomopathogenic fungi, including M. anisopliae, have an outer cell layer made up of rodlets (6). The hydrophobins, specific proteins present in the rodlet layer, mediate the passive adhesion of conidia to hydrophobic surfaces, such as the cuticles of arthropods (16, 45, 46). However, as germination commences, the hydrophobins are replaced by an adhesion-like protein, Mad1, which promotes tighter and more-specific adhesion between the conidia and the host (44). Many factors may affect the adhesion and persistence of conidia on the host cuticle (i.e., characteristics of the pathogen, including its virulence [2, 18, 48], conditions under which the pathogen is cultured [17], type of spores [7, 16], topographical and chemical properties of the host cuticle [9, 38, 42], host surface hydrophobicity [15, 23], host behavior [31, 33], and environmental conditions [33]). Conidia of M. anisopliae have shown an affinity to cuticular regions containing setae or spines (7, 38) and to highly hydrophobic cuticle regions, such as mosquitoes'' siphon tubes (23) and intersegmental folds (43). Sites with higher numbers of adhered conidia varied among host species. However, in general, the membranous intersegmental regions were often particularly attractive sites for conidial attachment (26). Variation in the distribution of conidia across different anatomical regions has also been noted in studies of several tick species inoculated with entomopathogenic fungi (3, 21, 22). An evaluation of the attachment of Beauveria bassiana conidia to three tick species, Dermacentor variabilis, Rhipicephalus sanguineus, and Ixodes scapularis, demonstrated that the distribution patterns of the different conidia on the ticks'' bodies were not uniform (22). The density of the conidia and their germination varied dramatically across different anatomical regions of Amblyomma maculatum and A. americanum that had been inoculated with B. bassiana (21). Arruda et al. (3) demonstrated that mass adhesion of M. anisopliae conidia to engorged Boophilus microplus females occurs predominantly on ticks'' legs, suggesting its association with the presence of setae.There are a few approaches for assessing the adhesion of conidia to the host cuticle that are based on direct observation of the conidia on the arthropod cuticle. They involve examining a few areas on the surface of an arthropod by means of scanning electron microscopy (SEM) (11, 15, 30), transmission electron microscopy (TEM) (4), or fluorescence microscopy following vital staining of the conidia (2, 28, 29, 37). These methods are expensive, time-consuming, and relatively inaccurate due to the uneven distribution of conidia on the host surface.In this work, we describe a quantitative method for determining the total amount of conidia that have adhered to a whole host cuticle. This method is based on removing adhered conidia from the tick cuticle using an organic solvent, separating the conidia from the extract by centrifugation, resuspending the conidia in a detergent solution, and then counting the conidia in a hemocytometer. The efficacy of the method was evaluated by comparing the results of this procedure with those of a supplementary examination of conidial removal using SEM.The term “adhered” is often used to define conidia in different states: washed or unwashed after inoculation, present on the host cuticle immediately after inoculation, or kept for several hours (1, 2, 38). In this paper, the term “adhered conidia” refers to conidia that remained on the cuticle after washing by vortexing the inoculated and dried host in an aqueous solution of Triton X-100 and rinsing of the material under tap water.     

Dual restriction enzyme digest of cationic-gold-coated DNA scaffolds

DNA strands coated with AuNPs were cleaved by restriction enzymes while in solution or on a surface. Enzymatic activity was verified by gel electrophoresis prior to surface analysis. Cleavage results suggest that enzymes can recognize the AuNP-coated strands while on the surfaces, though specificity in digestion has not yet been verified. Development allows for advances in site specific localization of components using biological media.     

Rab1b interacts with GBF1 and modulates both ARF1 dynamics and COPI association

Assembly of the cytosolic coat protein I (COPI) complex at the ER-Golgi interface is directed by the ADP ribosylation factor1 (Arf1) and its guanine nucleotide exchange factor (GBF1). Rab1b GTPase modulates COPI recruitment, but the molecular mechanism underlying this action remains unclear. Our data reveal that in vivo expression of the GTP-restricted Rab1b mutant (Rab1Q67L) increased the association of GBF1 and COPI to peripheral structures localized at the ER exit sites (ERES) interface. Active Rab1b also stabilized Arf1 on Golgi membranes. Furthermore, we characterized GBF1 as a new Rab1b effector, and showed that its N-terminal domain was involved in this interaction. Rab1b small interfering RNA oligonucleotide assays suggested that Rab1b was required for GBF1 membrane association. To further understand how Rab1b functions in ER-to-Golgi transport, we analyzed GFP-Rab1b dynamics in HeLa cells. Time-lapse microscopy indicated that the majority of the Rab1b-labeled punctuated structures are relatively short-lived with limited-range movements. FRAP of Golgi GFP-Rab1bwt showed rapid recovery (t(1/2) 120 s) with minimal dependence on microtubules. Our data support a model where Rab1b-GTP induces GBF1 recruitment at the ERES interface and at the Golgi complex where it is required for COPII/COPI exchange or COPI vesicle formation, respectively.     

Continuous delivery of D-luciferin by implanted micro-osmotic pumps enables true real-time bioluminescence imaging of luciferase activity in vivo

Bioluminescence imaging (BLI) of luciferase reporters in small animal models offers an attractive approach to monitor regulation of gene expression, signal transduction, and protein-protein interactions, as well as following tumor progression, cell engraftment, infectious pathogens, and target-specific drug action. Conventional BLI can be repeated within the same animal after bolus reinjections of a bioluminescent substrate. However, intervals between image acquisitions are governed by substrate pharmacokinetics and excretion, therefore restricting temporal resolution of reinjection protocols to the order of hours, limiting analyses of processes in vivo with short time constants. To eliminate these constraints, we examined use of implanted micro-osmotic pumps for continuous, long-term delivery of bioluminescent substrates. Pump-assisted d-luciferin delivery enabled BLI for > or = 7 days from a variety of luciferase reporters. Pumps allowed direct repetitive imaging at < 5-minute intervals of the pharmacodynamics of proteasome- and IKK-inhibiting drugs in mice bearing tumors stably expressing ubiquitin-firefly luciferase or IkappaBalpha-firefly luciferase fusion reporters. Circadian oscillations in the olfactory bulbs of transgenic rats expressing firefly luciferase under the control of the period1 promoter also were temporally resolved over the course of several days. We conclude that implanted pumps provide reliable, prolonged substrate delivery for high temporal resolution BLI, traversing complications of repetitive substrate injections.     

Antimicrobial benzodiazepine‐based short cationic peptidomimetics

Antimicrobial peptides (AMPs) appear to be good candidates for the development of new antibiotic drugs. We describe here the synthesis of peptidomimetic compounds that are based on a benzodiazepine scaffold flanked with positively charged and hydrophobic amino acids. These compounds mimic the essential properties of cationic AMPs. The new design possesses the benzodiazepine scaffold that is comprised of two glycine amino acids and which confers flexibility and aromatic hydrophobic ‘back’, and two arms used for further synthesis on solid phase for incorporation of charged and hydrophobic amino acids. This approach allowed us a better understanding of the influence of these features on the antimicrobial activity and selectivity. A novel compound was discovered which has MICs of 12.5 µg/ml against Staphylococcus aureus and 25 µg/ml against Escherichia coli, similar to the well‐known antimicrobial peptide MSI‐78. In contrast to MSI‐78, the above mentioned compound has lower lytic effect against mammalian red blood cells. These peptidomimetic compounds will pave the way for future design of potent synthetic mimics of AMPs for therapeutic and biomedical applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Aromatic aldehyde and hydrazine activated peptide coated quantum dots for easy bioconjugation and live cell imaging

We present a robust scheme for preparation of semiconductor quantum dots (QDs) and cognate partners in a conjugation ready format. Our approach is based on bis-aryl hydrazone bond formation mediated by aromatic aldehyde and hydrazinonicotinate acetone hydrazone (HyNic) activated peptide coated quantum dots. We demonstrate controlled preparation of antibody--QD bioconjugates for specific targeting of endogenous epidermal growth factor receptors in breast cancer cells and for single QD tracking of transmembrane proteins via an extracellular epitope. The same approach was also used for optical mapping of RNA polymerases bound to combed genomic DNA in vitro.