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Mixed microorganism cultures are prevalent in the food industry. A variety of microbiological mixtures have been used in these unique fermenting processes to create distinctive flavor profiles and potential health benefits. Mixed cultures are typically not well characterized, which may be due to the lack of simple measurement tools. Image-based cytometry systems have been employed to automatically count bacteria or yeast cells. In this work, we aim to develop a novel image cytometry method to distinguish and enumerate mixed cultures of yeast and bacteria in beer products. Cellometer X2 from Nexcelom was used to count of Lactobacillus plantarum and Saccharomyces cerevisiae in mixed cultures using fluorescent dyes and size exclusion image analysis algorithm. Three experiments were performed for validation. (1) Yeast and bacteria monoculture titration, (2) mixed culture with various ratios, and (3) monitoring a Berliner Weisse mixed culture fermentation. All experiments were validated by comparing to manual counting of yeast and bacteria colony formation. They were highly comparable with ANOVA analysis showing p-value > 0.05. Overall, the novel image cytometry method was able to distinguish and count mixed cultures consistently and accurately, which may provide better characterization of mixed culture brewing applications and produce higher quality products.

Dysferlin is an integral membrane protein of the transverse tubules of skeletal muscle that is mutated or absent in Limb Girdle Muscular Dystrophy 2B and Miyoshi Myopathy. Here we examine the role of dysferlin’s seven C2 domains, C2A through C2G, in membrane repair and Ca(2+) release, as well as in targeting dysferlin to the transverse tubules of skeletal muscle. We report that deletion of either domains C2A or C2B inhibits membrane repair completely, whereas deletion of the C2C, C2D, C2E, C2F or C2G causes partial loss of membrane repair that is exacerbated in the absence of extracellular Ca(2+). Deletion of C2C, C2D, C2E, C2F or C2G also causes significant changes in Ca(2+) release, measured as the amplitude of the Ca(2+) transient before or after hypo-osmotic shock and the appearance of Ca(2+) waves. Most deletants accumulate in endoplasmic reticulum. Only the C2A domain can be deleted without affecting dysferlin trafficking to transverse tubules, but Dysf-ΔC2A fails to support normal Ca(2+) signaling after hypo-osmotic shock. Our data suggest that: (i) every C2 domain contributes to repair; (ii) all C2 domains except C2B regulate Ca(2+) signaling; (iii) TT localization is insufficient for normal Ca(2+) signaling; (iv) Ca(2+) dependence of repair is mediated by C2C through C2G. Thus, dysferlin’s C2 domains have distinct functions in Ca(2+) signaling and sarcolemmal membrane repair and may play distinct roles in skeletal muscle.

Electroporation, in particular with nanosecond pulses, is an efficient technique to generate nanometer-size membrane lesions without the use of toxins or other chemicals. The restoration of the membrane integrity takes minutes and is only partially dependent on [Ca

Chemotaxis, the directional motility of cells in response to spatial gradients of chemical cues, is a fundamental process behind a wide range of biological events, including the innate immune response and cancer metastasis. Recent advances in cell biology have shown that the protrusions that enable amoeboid cells to move are driven by the stochastic threshold crossings of an underlying excitable system. As a cell encounters a chemoattractant gradient, the size of this threshold is regulated spatially so that the crossings are biased towards the front of the cell. For efficient directional migration, cells must limit undesirable lateral and rear-directed protrusions. The inclusion of a control mechanism to suppress these unwanted firings would enhance chemotactic efficiency. It is known that absolute concentration robustness (ACR) exerts tight control over the mean and variance of species concentration. Here, we demonstrate how the coupling of the ACR mechanism to the cellular signaling machinery reduces the likelihood of threshold crossings in the excitable system. Moreover, we show that using the cell’s innate gradient sensing apparatus to direct the action of ACR to the rear, suppresses the lateral movement of the cells and that this results in improved chemotactic performance.

Neural oscillations are evident across cortex but their spatial structure is not well-explored. Are oscillations stationary or do they form “traveling waves”, i.e., spatially organized patterns whose peaks and troughs move sequentially across cortex? Here, we show that oscillations in the prefrontal cortex (PFC) organized as traveling waves in the theta (4-8Hz), alpha (8-12Hz) and beta (12-30Hz) bands. Some traveling waves were planar but most rotated. The waves were modulated during performance of a working memory task. During baseline conditions, waves flowed bidirectionally along a specific axis of orientation. Waves in different frequency bands could travel in different directions. During task performance, there was an increase in waves in one direction over the other, especially in the beta band.Author SummaryWe found that oscillations in the prefrontal cortex form “traveling waves”. Traveling waves are spatially extended patterns in which aligned peaks of activity move sequentially across the cortical surface. Some traveling waves were planar but most rotated. The prefrontal cortex is important for working memory. The traveling waves changed when monkeys performed a working memory task. There was an increase in waves in one direction over the other, especially in the beta band. Traveling waves can serve specific functions. For example, they help maintain network status and help control timing relationships between spikes. Given their functional advantages, a greater understanding of traveling waves should lead to a greater understanding of cortical function.

Electroporation, in particular with nanosecond pulses, is an efficient technique to generate nanometer-size membrane lesions without the use of toxins or other chemicals. The restoration of the membrane integrity takes minutes and is only partially dependent on [Ca2+]. We explored the impact of Ca2+ on the kinetics of membrane resealing by monitoring the entry of a YO-PRO-1 dye (YP) in BPAE and HEK cells. Ca2+ was promptly removed or added after the electric pulse (EP) by a fast-step perfusion. YP entry increased sharply after the EP and gradually slowed down following either a single- or a double-exponential function. In BPAE cells permeabilized by a single 300- or 600-ns EP at 14 kV/cm in a Ca2+-free medium, perfusion with 2 mM of external Ca2+ advanced the 90% resealing and reduced the dye uptake about twofold. Membrane restoration was accomplished by a combination of fast, Ca2+-independent resealing (τ = 13–15 s) and slow, Ca2+-dependent processes (τ ~70 s with Ca2+ and ~ 110 s or more without it). These time constants did not change when the membrane damage was doubled by increasing EP duration from 300 to 600 ns. However, injury by microsecond-range EP (300 and 600 μs) took longer to recover even when the membrane initially was less damaged, presumably because of the larger size of pores made in the membrane. Full membrane recovery was not prevented by blocking both extra- and intracellular Ca2+ (by loading cells with BAPTA or after Ca2+ depletion from the reticulum), suggesting the recruitment of unknown Ca2+-independent repair mechanisms.

Excitable waves evolve into standing patterns and lead to the various dynamic and static protrusions seen in migrating cells., The mechanisms regulating protrusions during amoeboid migration exhibit excitability. Theoretical studies have suggested the possible coexistence of traveling and standing waves in excitable systems. Here, we demonstrate the direct transformation of a traveling into a standing wave and establish conditions for the stability of this conversion. This theory combines excitable wave stopping and the emergence of a family of standing waves at zero velocity, without altering diffusion parameters. Experimentally, we show the existence of this phenomenon on the cell cortex of some Dictyostelium and mammalian mutant strains. We further predict a template that encompasses a spectrum of protrusive phenotypes, including pseudopodia and filopodia, through transitions between traveling and standing waves, allowing the cell to switch between excitability and bistability. Overall, this suggests that a previously-unidentified method of pattern formation, in which traveling waves spread, stop, and turn into standing waves that rearrange to form stable patterns, governs cell motility.

Infection is defined as invasion and colonization processes of pathogenic or harmful microorganisms on host cells. A diverse array of microorganisms such as bacteria, virus, fungi, and protozoa attack or infect host cell. They deploy enormous strategies to target or manipulate the host cell or to escape from host immunity. Virulence factors have been secreted as a either cell associated or secreted out of cells upon infection. Infection also depends on health of host cells. Pathogens secrete different types of toxins or enzymes or some molecules to destroy the immunity of the host cells. These are encoded by either chromosome or plasmid of host cells. Even drug resistant property is one of the tactics to destroy the antibiotics. Sometimes they modify themselves or mimic like host molecules upon infection and hence immune cells do not identify them. Nowadays these peculiar behaviors of microbial pathogen draw attention to many scientists worldwide. They not only affect humans, but they also infect other organisms that are economically important for human welfare. Some deadly diseases such as swine flu or dengue or other fatal diseases are really concern for human society. Every year these deadly diseases affect more than half of the people have been infected by these deadly diseases. In this section, we will discuss and shed the light on human pathogens and their disease characteristic. We will even discuss the virulence factors that aid the pathogen to progress the disease.

Significance Cell migration is central in physiological and pathological conditions such as immune response and cancer metastasis. The excitable network hypothesis can account for recent observations of propagating waves of signal transduction and cytoskeleton events as well as behaviors of migrating cells. However, the molecular feedback loops involved in these networks that bring about excitability are poorly understood. Here, we provide evidence for a positive-feedback loop based on a mutual inhibitory interaction between Ras and phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2]. Our results uncover an important role of PI(3,4)P2 in the regulation of Ras activity, which may extend well beyond cell migration., Signal transduction and cytoskeleton networks in a wide variety of cells display excitability, but the mechanisms are poorly understood. Here, we show that during random migration and in response to chemoattractants, cells maintain complementary spatial and temporal distributions of Ras activity and phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2]. In addition, depletion of PI(3,4)P2 by disruption of the 5-phosphatase, Dd5P4, or by recruitment of 4-phosphatase INPP4B to the plasma membrane, leads to elevated Ras activity, cell spreading, and altered migratory behavior. Furthermore, RasGAP2 and RapGAP3 bind to PI(3,4)P2, and the phenotypes of cells lacking these genes mimic those with low PI(3,4)P2 levels, providing a molecular mechanism. These findings suggest that Ras activity drives PI(3,4)P2 down, causing the PI(3,4)P2-binding GAPs to dissociate from the membrane, further activating Ras, completing a positive-feedback loop essential for excitability. Consistently, a computational model incorporating such a feedback loop in an excitable network model accurately simulates the dynamic distributions of active Ras and PI(3,4)P2 as well as cell migratory behavior. The mutually inhibitory Ras-PI(3,4)P2 mechanisms we uncovered here provide a framework for Ras regulation that may play a key role in many physiological processes.

Excitable systems are a class of dynamical systems that can generate self-sustaining waves of activity. These waves are known to manifest differently under diverse conditions, whereas some travel as planar or radial waves, and others evolve into rotating spirals. Excitable systems can also form stationary stable patterns through standing waves. Under certain conditions, these waves are also known to be reflected at no-flux boundaries. Here, we review the basic characteristics of these four entities: traveling, rotating, standing and reflected waves. By studying their mechanisms of formation, we show how through manipulation of three critical parameters: time-scale separation, space-scale separation and threshold, we can interchangeably control the formation of all the aforementioned wave types.

Summary The Ras/PI3K/extracellular signal-regulated kinases (ERK) signaling network plays fundamental roles in cell growth, survival, and migration and is frequently activated in cancer. Here, we show that the activities of the signaling network propagate as coordinated waves, biased by growth factor, which drive actin-based protrusions in human epithelial cells. The network exhibits hallmarks of biochemical excitability: the annihilation of oppositely directed waves, all-or-none responsiveness, and refractoriness. Abrupt perturbations to Ras, PI(4,5)P2, PI(3,4)P2, ERK, and TORC2 alter the threshold, observations that define positive and negative feedback loops within the network. Oncogenic transformation dramatically increases the wave activity, the frequency of ERK pulses, and the sensitivity to EGF stimuli. Wave activity was progressively enhanced across a series of increasingly metastatic breast cancer cell lines. The view that oncogenic transformation is a shift to a lower threshold of excitable Ras/PI3K/ERK network, caused by various combinations of genetic insults, can facilitate the assessment of cancer severity and effectiveness of interventions.

The tripartite motif (TRIM) gene family is a highly conserved group of E3 ubiquitin ligase proteins that can establish substrate specificity for the ubiquitin-proteasome complex and also have proteasome-independent functions. While several family members were studied previously, it is relatively recent that over 80 genes, based on sequence homology, were grouped to establish the TRIM gene family. Functional studies of various TRIM genes linked these proteins to modulation of inflammatory responses showing that they can contribute to a wide variety of disease states including cardiovascular, neurological and musculoskeletal diseases, as well as various forms of cancer. Given the fundamental role of the ubiquitin-proteasome complex in protein turnover and the importance of this regulation in most aspects of cellular physiology, it is not surprising that TRIM proteins display a wide spectrum of functions in a variety of cellular processes. This broad range of function and the highly conserved primary amino acid sequence of family members, particularly in the canonical TRIM E3 ubiquitin ligase domain, complicates the development of therapeutics that specifically target these proteins. A more comprehensive understanding of the structure and function of TRIM proteins will help guide therapeutic development for a number of different diseases. This review summarizes the structural organization of TRIM proteins, their domain architecture, common and unique post-translational modifications within the family, and potential binding partners and targets. Further discussion is provided on efforts to target TRIM proteins as therapeutic agents and how our increasing understanding of the nature of TRIM proteins can guide discovery of other therapeutics in the future.

The primary pathogen of the Great Barrier Reef sponge Rhopaloeides odorabile, recently identified as a novel strain (NW4327) of Pseudoalteromonas agarivorans, produced collagenase which degraded R. odorabile skeletal fibers. We now report the collagenase of P. agarivorans as a metalloprotease which required Ca2+ and Zn2+ as cofactors. The collagenase was a TonB dependent receptor (TBDR) having a carboxypeptidase regulatory like domain (CRLD) in the N-terminal along with an outer membrane (OM) channel superfamily domain. The genes for TBDR sub-components and collagenase formed one unified entity in the genome of P. agarivorans NW4327. This association of a collagenase with a TBDR distinguished it from all known functional collagenases till date and for the first time, established the enzymatic capability of TBDRs. Predicted TBDR model demonstrated only 15% identity with ferripyoverdin receptor and the CRLD displayed merely 24% identity with carboxypeptidase catalytic chain. Presence of signal peptide, lack of transmembrane helices, absence of N-terminal in the cytoplasmic side, extracellular localization and recovery from the culture supernatant implicated that the TBDR was secreted. Stronger binding of the collagenase with marine sponge type IV collagen than type I collagen, revealed through molecular docking, indicated higher specificity of the enzyme towards type IV collagen.

Sarcolemmal integrity is orchestrated through the interplay of preserving membrane strength and fast tracking the membrane repair process during an event of compromised membrane fragility. Several molecular players have been identified that act in a concerted fashion to maintain the barrier function of the muscle membrane. Substantial research findings in the field of muscle biology point out the importance of maintaining membrane integrity as a key contributory factor to cellular homeostasis. Innumerable data on the progression of membrane pathology associated with compromised muscle membrane integrity support targeting sarcolemmal integrity in skeletal and cardiac muscle as a model therapeutic strategy to alleviate some of the pathologic conditions. This review will discuss strategies that researchers have undertaken to compensate for an imbalance in sarcolemma membrane fragility and membrane repair to maintain muscle membrane integrity.

Limb girdle muscular dystrophy type 2B (LGMD2B) and other dysferlinopathies are degenerative muscle diseases that result from mutations in the dysferlin gene and have limited treatment options. The dysferlin protein has been linked to multiple cellular functions including a Ca2+-dependent membrane repair process that reseals disruptions in the sarcolemmal membrane. Recombinant human MG53 protein (rhMG53) can increase the membrane repair process in multiple cell types both in vitro and in vivo. Here, we tested whether rhMG53 protein can improve membrane repair in a dysferlin-deficient mouse model of LGMD2B (B6.129-Dysftm1Kcam/J). We found that rhMG53 can increase the integrity of the sarcolemmal membrane of isolated muscle fibers and whole muscles in a Ca2+-independent fashion when assayed by a multi-photon laser wounding assay. Intraperitoneal injection of rhMG53 into mice before acute eccentric treadmill exercise can decrease the release of intracellular enzymes from skeletal muscle and decrease the entry of immunoglobulin G and Evans blue dye into muscle fibers in vivo. These results indicate that short-term rhMG53 treatment can ameliorate one of the underlying defects in dysferlin-deficient muscle by increasing sarcolemmal membrane integrity. We also provide evidence that rhMG53 protein increases membrane integrity independently of the canonical dysferlin-mediated, Ca2+-dependent pathway known to be important for sarcolemmal membrane repair.

The diverse migratory modes displayed by different cell types are generally believed to be idiosyncratic. Here we show that the migratory behaviour of Dictyostelium was switched from amoeboid to keratocyte-like and oscillatory modes by synthetically decreasing phosphatidylinositol-4,5-bisphosphate levels or increasing Ras/Rap-related activities. The perturbations at these key nodes of an excitable signal transduction network initiated a causal chain of events: the threshold for network activation was lowered, the speed and range of propagating waves of signal transduction activity increased, actin-driven cellular protrusions expanded and, consequently, the cell migratory mode transitions ensued. Conversely, innately keratocyte-like and oscillatory cells were promptly converted to amoeboid by inhibition of Ras effectors with restoration of directed migration. We use computational analysis to explain how thresholds control cell migration and discuss the architecture of the signal transduction network that gives rise to excitability.

Angiotensin-converting enzyme inhibitors (ACEi) and mineralocorticoid receptor (MR) antagonists are FDA-approved drugs that inhibit the renin-angiotensin-aldosterone system (RAAS) and are used to treat heart failure. Combined treatment with the ACEi lisinopril and the nonspecific MR antagonist spironolactone surprisingly improves skeletal muscle, in addition to heart function and pathology in a Duchenne muscular dystrophy (DMD) mouse model. We recently demonstrated that MR is present in all limb and respiratory muscles and functions as a steroid hormone receptor in differentiated normal human skeletal muscle fibers. The goals of the current study were to begin to define cellular and molecular mechanisms mediating the skeletal muscle efficacy of RAAS inhibitor treatment. We also compared molecular changes resulting from RAAS inhibition with those resulting from the current DMD standard-of-care glucocorticoid treatment. Direct assessment of muscle membrane integrity demonstrated improvement in dystrophic mice treated with lisinopril and spironolactone compared with untreated mice. Short-term treatments of dystrophic mice with specific and nonspecific MR antagonists combined with lisinopril led to overlapping gene-expression profiles with beneficial regulation of metabolic processes and decreased inflammatory gene expression. Glucocorticoids increased apoptotic, proteolytic, and chemokine gene expression that was not changed by RAAS inhibitors in dystrophic mice. Microarray data identified potential genes that may underlie RAAS inhibitor treatment efficacy and the side effects of glucocorticoids. Direct effects of RAAS inhibitors on membrane integrity also contribute to improved pathology of dystrophic muscles. Together, these data will inform clinical development of MR antagonists for treating skeletal muscles in DMD.

Phosphatidylinositol-4,5-bisphosphate 3-kinases (PI3Ks) are regulatory enzymes involved in the generation of lipid species that modulate cellular signaling pathways through downstream effectors to influence a variety of cellular functions. Years of intensive study of PI3Ks have produced a significant body of literature in many areas, including that PI3K can mediate intracellular vesicular trafficking and through these actions contribute to a number of important physiological functions. This review focuses on the crucial roles that PI3K and AKT, a major downstream partner of PI3K, play in the regulation of vesicle trafficking during various forms of vesicular endocytosis and exocytosis.

We demonstrate through precise numerical simulations the possibility of flexible, thin-film solar cells, consisting of crystalline silicon, to achieve power conversion efficiency of 31%. Our optimized photonic crystal architecture consists of a 15 μm thick cell patterned with inverted micro-pyramids with lattice spacing comparable to the wavelength of near-infrared light, enabling strong wave-interference based light trapping and absorption. Unlike previous photonic crystal designs, photogenerated charge carrier flow is guided to a grid of interdigitated back contacts with optimized geometry to minimize Auger recombination losses due to lateral current flow. Front and back surface fields provided by optimized Gaussian doping profiles are shown to play a vital role in enhancing surface passivation. We carefully delineate the drop in power conversion efficiency when surface recombination velocities exceed 100 cm/s and the doping profiles deviate from prescribed values. These results are obtained by exact numerical simulation of Maxwell’s wave equations for light propagation throughout the cell architecture and a state-of-the-art model for charge carrier transport and Auger recombination.

Cellular protrusions are typically considered as distinct structures associated with specific regulators. However, we found that these regulators coordinately localize as propagating cortical waves, suggesting a common underlying mechanism. These molecular events fell into two excitable networks, the signal transduction network STEN and the cytoskeletal network CEN with different wave substructures. Computational studies using a coupled‐network model reproduced these features and showed that the morphology and kinetics of the waves depended on strengths of feedback loops. Chemically induced dimerization at multiple nodes produced distinct, coordinated alterations in patterns of other network components. Taken together, these studies indicate: STEN positive feedback is mediated by mutual inhibition between Ras/Rap and PIP2, while negative feedback depends on delayed PKB activation; PKBs link STEN to CEN; CEN includes positive feedback between Rac and F‐actin, and exerts fast positive and slow negative feedbacks to STEN. The alterations produced protrusions resembling filopodia, ruffles, pseudopodia, or lamellipodia, suggesting that these structures arise from a common regulatory mechanism and that the overall state of the STEN‐CEN system determines cellular morphology.

Chemotaxis, the directed motion of cells in response to chemical gradients, is important for a variety of biological processes ranging from embryogenesis to killing of pathogens. Increasing the speed and efficiency of directed migration is critical in such situations. We provide a control mechanism by which one can minimize the noise-driven firings at the back of the cell, enabling faster motion towards the front. We achieve this through a mechanism called absolute concentration robustness (ACR), which robustly maintains the steady-state concentration of intracellular biochemical species and, at the same time, provides control over the concentration variance. More particularly, by incorporating ACR, we develop a correspondence between the concentration mean and variance - both of which are independent of total concentrations. We show that by incorporating ACR into the back of a moving cell, we can create a mechanism to robustly control the noise variance at the back - thus limiting the deterring firings while the cell moves in the direction of the gradient.

The multiconfiguration time-dependent Hartree (MCTDH) approach has been employed to study the reaction dynamics of the He + HeH + reaction on the Panda–Sathyamurthy potential energy surface. The calculations have been performed for the reactants both in their ground and ro-vibrational excited states. Both centrifugal sudden (CS) and coupled channel (CC) studies have been performed for the He + HeH + reaction. The results have been compared with an earlier study performed using the split operator method, under CS approximation. The CC results show a distinct difference from the CS results. The observed differences for this system have been analyzed and explained in this study. The importance of the initial orientation of the reagents and the presence of preferred pathways of approach in the reaction dynamics of this system has been discussed.

Recent years have demonstrated that the actin cytoskeleton and other signaling elements in motile cells have many of the hallmarks of an excitable medium, including the presence of propagating waves, a refractory period, as well as a threshold for activation. Here we show how these behaviors can be explained by the presence of a signal transduction excitable network that integrates a number of signals and coordinates actin polymerization. In this model, spontaneous triggering of the excitable network accounts for the random migration of unstimulated cells. Moreover, internal and external signals both chemical and mechanical bias excitability spatially, thus providing a means by which cell motility is directed towards spatial cues. We also show how the model predicts that the set point of the excitable system can be altered by changing the threshold.

The power conversion efficiency of single-junction silicon solar cells has increased only by 1.5% despite extensive efforts over the past two decades. The current world-record efficiencies of silicon solar cells, within the 25%–26.7% range, fall well below the thermodynamic limit of 32.3%. We review the recent progress in photonic crystal light-trapping architectures poised to achieve 28%–31% conversion efficiency in flexible 3–20 μm-thick, single-junction crystalline-silicon solar cells. These photonic crystals utilize wave-interference based light-trapping, enabling solar absorption well beyond the Lambertian limit in the 300–1200 nm wavelength range. Using experimentally feasible doping profiles, carrier lifetimes, surface recombination velocities, and established Auger recombination losses, we review considerations leading to the prediction of 31% efficiency in a 15 μm-thick silicon photonic crystal cell with interdigitated back-contacts. This is beyond the conversion efficiency of any single-material photovoltaic device of any thickness.The power conversion efficiency of single-junction silicon solar cells has increased only by 1.5% despite extensive efforts over the past two decades. The current world-record efficiencies of silicon solar cells, within the 25%–26.7% range, fall well below the thermodynamic limit of 32.3%. We review the recent progress in photonic crystal light-trapping architectures poised to achieve 28%–31% conversion efficiency in flexible 3–20 μm-thick, single-junction crystalline-silicon solar cells. These photonic crystals utilize wave-interference based light-trapping, enabling solar absorption well beyond the Lambertian limit in the 300–1200 nm wavelength range. Using experimentally feasible doping profiles, carrier lifetimes, surface recombination velocities, and established Auger recombination losses, we review considerations leading to the prediction of 31% efficiency in a 15 μm-thick silicon photonic crystal cell with interdigitated back-contacts. This is beyond the conversion efficiency of any single-material...

A protease of the primary pathogen (Pseudoalteromonas agarivorans NW4327) of the disease affecting the Great Barrier Reef sponge Rhopaloeides odorabile was purified. Zymography demonstrated calcium-dependent collagenase and gelatinase activity of the purified protein. This metalloprotease was identified by matrix assisted laser desorption ionization time-of-flight mass spectrophotometry as a 52,509 Da U32 collagenase. Predicted tertiary structure of U32 collagenase (by Phyre2 fold recognition server) demonstrated 13% identity with known hydrolases establishing novelty of the enzyme. Molecular docking conceived two interacting loops of the collagenase that bound with collagen triple helices and two calcium ions remained centered between the loops. According to ConSurf multiple sequence alignment, the residues of loop1 of the collagenase were mostly conserved while variations among residues of loop2 were comparatively higher than loop1. Asp262, Glu263 of loop1 and Thr363, Lys364, Gln365 of loop2 participated in the interaction with Ca2+ and collagen. Root mean square deviation and root mean square fluctuation values signified higher stability of the collagen-Ca2+-collagenase complex and greater structural stability of the residues of the loops in the complex compared to apocollagenase. Observed properties of NW4327 U32 collagenase and its interaction with collagen were different from similar enzymes of thermophilic bacteria and terrestrial pathogens.

A flexible thin-film silicon solar cell with power conversion efficiency approaching 30% would be a game-changer for the photovoltaics industry. This dream has been considered unattainable, though, due to Si's indirect band gap. Also, silicon solar cells are typically thick, inflexible, and limited in efficiency by nonradiative charge-carrier losses in the large bulk volume of the cell. This research demonstrates how light trapping based on wave interference in photonic crystals could raise conversion efficiency to ~28%, over a large wavelength range of 300---1100 nm. This would set a new record for silicon-based photovoltaic technology.

The effect of proinflammatory cytokines on the expression and activity of soluble guanylyl cyclase (sGC) and cGMP-phosphodiesterases (PDEs) was determined in intestinal longitudinal smooth muscle. In control muscle cells, cGMP levels are regulated via activation of sGC and PDE5; the activity of the latter is regulated via feedback phosphorylation by cGMP-dependent protein kinase. In muscle cells isolated from muscle strips cultured with interleukin-1β (IL-1β) or tumor necrosis factor α (TNF-α) or obtained from the colon of TNBS (2,4,6-trinitrobenzene sulfonic acid)-treated mice, expression of inducible nitric oxide synthase (iNOS) was induced and sGC was S-nitrosylated, resulting in attenuation of nitric oxide (NO)-induced sGC activity and cGMP formation. The effect of cytokines on sGC S-nitrosylation and activity was blocked by the iNOS inhibitor 1400W [N-([3-(aminomethyl)phenyl]methyl)ethanimidamide dihydrochloride]. The effect of cytokines on cGMP levels measured in the absence of IBMX (3-isobutyl-1-methylxanthine), however, was partly reversed by 1400W or PDE1 inhibitor vinpocetine and completely reversed by a combination of 1400W and vinpocetine. Expression of PDE1A was induced and was accompanied by an increase in PDE1A activity in muscle cells isolated from muscle strips cultured with IL-1β or TNF-α or obtained from the colon of TNBS-treated mice; the effect of cytokines on PDE1 expression and activity was blocked by MG132 (benzyl N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]carbamate), an inhibitor of nuclear factor κB activity. NO-induced muscle relaxation was inhibited in longitudinal muscle cells isolated from muscle strips cultured with IL-1β or TNF-α or obtained from the colon of TNBS-treated mice, and this inhibition was completely reversed by the combination of both 1400W and vinpocetine. Inhibition of smooth muscle relaxation during inflammation reflects the combined effects of decreased sGC activity via S-nitrosylation and increased cGMP hydrolysis via PDE1 expression.

Spoof surface plasmon (SSP) on structured perfect electric conductor (PEC)–air interface is usually modeled by an effective plasma frequency in the long wavelength limit. In this limit, the effective permittivity model suggests a purely exponential decay of the field profile on both sides of the interface, drawing an analogy with a surface plasmon polariton (SPP)-like single mode (TM) propagation. However, most of the practical applications and experiments involving SSP fall in the regime where the feature-size of the structure is comparable to the wavelength. In this wavelength regime of practical interest, we show that in contrast to surface plasmon polariton (SPP), the propagation of the surface wave is hybrid involving both TM and TE modes. By combining angular spectrum representation and vector nature of the electromagnetic fields, we show that the field profile of SSP on patterned PEC can be described accurately by superposition of multiple evanescent orders caused by the diffraction from sub-wavelength features at the interface. This multimodal nature of the surface wave readily explains the anisotropic propagation observed earlier. Also, inclusion of at least one mode from higher order Brillouin zone is necessary for describing the near-field above the interface with reasonable accuracy.

Although directed migration of eukaryotic cells may have evolved to escape nutrient depletion, it has been adopted for an extensive range of physiological events during development and in the adult organism. The subversion of these movements results in disease, such as cancer. Mechanisms of propulsion and sensing are extremely diverse, but most eukaryotic cells move by extending actin-filled protrusions termed macropinosomes, pseudopodia, or lamellipodia or by extension of blebs. In addition to motility, directed migration involves polarity and directional sensing. The hundreds of gene products involved in these processes are organized into networks of parallel and interconnected pathways. Many of these components are activated or inhibited coordinately with stimulation and on each spontaneously extended protrusion. Moreover, these networks display hallmarks of excitability, including all-or-nothing responsiveness and wave propagation. Cellular protrusions result from signal transduction waves that propagate outwardly from an origin and drive cytoskeletal activity. The range of the propagating waves and hence the size of the protrusions can be altered by lowering or raising the threshold for network activation, with larger and wider protrusions favoring gliding or oscillatory behavior over amoeboid migration. Here, we evaluate the variety of models of excitable networks controlling directed migration and outline critical tests. We also discuss the utility of this emerging view in producing cell migration and in integrating the various extrinsic cues that direct migration.

Hydrogen sulfide (H2S), like nitric oxide (NO), causes smooth muscle relaxation, but unlike NO, does not stimulate soluble guanylyl cyclase (sGC) activity and generate cyclic guanosine 5′-monophosphate (cGMP). The aim of this study was to investigate the interplay between NO and H2S in colonic smooth muscle. In colonic smooth muscle from rabbit, mouse, and human, l-cysteine, substrate of cystathionine-γ-lyase (CSE), or NaHS, an H2S donor, inhibited phosphodiesterase 5 (PDE5) activity and augmented the increase in cGMP levels, IP3 receptor phosphorylation at Ser1756 (measured as a proxy for PKG activation), and muscle relaxation in response to NO donor S-nitrosoglutathione (GSNO), suggesting augmentation of cGMP/PKG pathway by H2S. The inhibitory effect of l-cysteine, but not NaHS, on PDE5 activity was blocked in cells transfected with CSE siRNA or treated with CSE inhibitor d,l-propargylglycine (dl-PPG), suggesting activation of CSE and generation of H2S in response to l-cysteine. H2S levels were increased in response to l-cysteine, and the effect of l-cysteine was augmented by GSNO in a cGMP-dependent protein kinase-sensitive manner, suggesting augmentation of CSE/H2S by cGMP/PKG pathway. As a result, GSNO-induced relaxation was inhibited by dl-PPG. In flat-sheet preparation of colon, l-cysteine augmented calcitonin gene-related peptide release in response to mucosal stimulation, and in intact segments, l-cysteine increased the velocity of pellet propulsion. These results demonstrate that in colonic smooth muscle, there is a novel interplay between NO and H2S. NO generates H2S via cGMP/PKG pathway, and H2S, in turn, inhibits PDE5 activity and augments NO-induced cGMP levels. In the intact colon, H2S promotes colonic transit. NEW & NOTEWORTHY Hydrogen sulfide (H2S) and nitric oxide (NO) are important regulators of gastrointestinal motility. The studies herein provide the cross talk between NO and H2S signaling to mediate smooth muscle relaxation and colonic transit. H2S inhibits phosphodiesterase 5 activity to augment cGMP levels in response to NO, which, in turn, via cGMP/PKG pathway, generates H2S. These studies suggest that interventions targeted at restoring NO and H2S homeostasis within the smooth muscle may provide novel therapeutic approaches to mitigate motility disorders.

Recent advances in the understanding of cell migration in amoeboid cells have demonstrated the existence of excitable dynamics in the regulation of the extension and retraction of pseudopodia. In particular, traveling waves of biochemical signaling and actin polymerization have been observed in a number of different cell types. These waves appear to be triggered at a point on the cell cortex and propagate along the perimeter of the cell, pushing the membrane outward to create pseudopods. These waves, however, do not always spread throughout the entire cell cortex. More often than not, they stop after traveling a certain distance along the membrane. In this paper, we look at the factors that create the traveling front, and influence the stopping of the wave. We show that the speed of the wave is controlled by the initial set point of the excitable system, as well as the existence of a critical point for each system, that governs the stopping of the wave.

Caveolae are specialized regions of the plasma membrane that concentrate receptors and associated signaling molecules critical in regulation of cellular response to transmitters and hormones. We have determined the effects of caveolin-1 (Cav-1) deletion, caveolin-1 siRNA, and caveolar disruption in mice on the signaling pathways that mediate contraction and relaxation in colonic smooth muscle and on the components of the peristaltic reflex in isolated tissue and propulsion in intact colonic segments. In Cav-1−/− mice, both relaxation and contraction were decreased in smooth muscle cells and muscle strips, as well as during both phases of the peristaltic reflex and colonic propulsion. The decrease in relaxation in response to the nitric oxide (NO) donor was accompanied by a decrease in cGMP levels and an increase in phosphodiesterase 5 (PDE5) activity. Relaxation by a PDE5-resistant cGMP analog was not affected in smooth muscle of Cav-1−/− mice, suggesting that inhibition of relaxation was due to augmentation of PDE5 activity. Similar effects on relaxation, PDE5 and cGMP were obtained in muscle cells upon disruption of caveolae by methyl-β-cyclodextrin or suppression of Cav-1. Sustained contraction mediated via inhibition of myosin light chain phosphatase (MLCP) activity is regulated by Rho kinase and PKC via phosphorylation of two endogenous inhibitors of MLCP: myosin phosphatase-targeting subunit (MYPT1) and 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17), respectively. The activity of both enzymes and phosphorylation of MYPT1 and CPI-17 were decreased in smooth muscle from Cav-1−/− mice. We conclude that the integrity of caveolae is essential for contractile and relaxant activity in colonic smooth muscle and the maintenance of neuromuscular function at organ level.