Complex Substrates Research Articles

Exponential and efficient target-catalyst rolling circle amplification for label-free and ultrasensitive fluorescent detection of miR-21 and p53 gene

MicroRNAs (miRNAs) and p53 gene can serve as valuable biomarkers for the diagnosis of a variety of cancers. Nevertheless, although the development of the DNA nanostructure on the detection of cancer-related biomarkers was initially demonstrated several years ago, the challenges of developing simpler, cheaper, and multi-level detection DNA biosensors persist. Herein, based on the rolling circle amplification (RCA) coupled with the target-triggered skill, we have developed a well-designed detecting platform. In this study, the dumbbell-shaped probes (DPP) could be cyclized and initiated through targets, thus beginning the target-catalyst RCA (tc-RCA) reaction, therefore engendering numerous dumbbell probe amplicons (DPA). Thereafter the probe primers (PP) mutually complementary to the loop of DPA was introduced, leading to the branch strand displacement reaction (B-SDA). SYBR Green I can effectively bind to the amplified double-stranded structures as a fluorescent reporter. Altering the target-binding sequence of the DPP, this biosensor can also be applied to detect different biomarkers. As a consequence, target miR-21 and p53 gene can be detected down to 0.65 fM and 2.04 fM respectively with a wide dynamic range. Moreover, we have also achieved the qualitative detection of interesting targets in cell lysates as well as the complex biological substrates and compared the results with reverse transcription quantitative PCR (RT-qPCR), thereby indicating the potential application in clinical diagnosis and biomedical research.

Substrate complexity affects the prevalence and interconnections of antibiotic, metal and biocide resistance genes, integron-integrase genes, human pathogens and virulence factors in anaerobic digestion

Anaerobic digestion (AD) is widely used to treat livestock manure that harbors diverse pollutants (resistance genes (ARGs), metal/biocide resistance genes (MBRGs), integron-integrase genes, human pathogens and pathogen virulence factors (VFs)). However, the interplays of these pollutants and the effects of substrate complexity on pollutants in AD are elusive. This study investigated the dynamics of these pollutants and bacterial communities during AD of swine manure, by metatranscriptomic sequencing and amplicon sequencing of 16 S rRNA and 16 S rRNA gene. The pollutant profiles and bacterial communities differed across AD processes, nevertheless with consistent dominance of ARGs of multi-drugs, tetracycline, aminoglycoside and rifamycin, MBRGs of multi-biocides, multi-metals, copper and arsenic, the integron-integrase gene intI1, potential pathogens of Escherichia coli, Streptococcus gallolyticus and Clostridium perfringens, VFs involved in pathogen adherence, and bacterial phyla of Firmicutes, Bacteroidetes and Proteobacteria. Reduced substrate complexity (replacing a part of swine manure, a complex substrate, with a simple substrate, apple waste or fructose) decreased the prevalence and stochastic turnover of ARGs and MBRGs. Network analyses revealed decreased interplays among pollutants under reduced substrate complexity. Our findings provide a mechanical understanding of diverse pollutants dynamics during AD, and reveal the importance of substrate complexity in controlling prevalence and interplays of pollutants.

Dynamic metabolic interactions and trophic roles of human gut microbes identified using a minimal microbiome exhibiting ecological properties

Microbe–microbe interactions in the human gut are influenced by host-derived glycans and diet. The high complexity of the gut microbiome poses a major challenge for unraveling the metabolic interactions and trophic roles of key microbes. Synthetic minimal microbiomes provide a pragmatic approach to investigate their ecology including metabolic interactions. Here, we rationally designed a synthetic microbiome termed Mucin and Diet based Minimal Microbiome (MDb-MM) by taking into account known physiological features of 16 key bacteria. We combined 16S rRNA gene-based composition analysis, metabolite measurements and metatranscriptomics to investigate community dynamics, stability, inter-species metabolic interactions and their trophic roles. The 16 species co-existed in the in vitro gut ecosystems containing a mixture of complex substrates representing dietary fibers and mucin. The triplicate MDb-MM’s followed the Taylor’s power law and exhibited strikingly similar ecological and metabolic patterns. The MDb-MM exhibited resistance and resilience to temporal perturbations as evidenced by the abundance and metabolic end products. Microbe-specific temporal dynamics in transcriptional niche overlap and trophic interaction network explained the observed co-existence in a competitive minimal microbiome. Overall, the present study provides crucial insights into the co-existence, metabolic niches and trophic roles of key intestinal microbes in a highly dynamic and competitive in vitro ecosystem.

Novel strategies towards efficient molecular biohydrogen production by dark fermentative mechanism: present progress and future perspective.

In the scenario of alarming increase in greenhouse and toxic gas emissions from the burning of conventional fuels, it is high time that the population drifts towards alternative fuel usage to obviate pollution. Hydrogen is an environment-friendly biofuel with high energy content. Several production methods exist to produce hydrogen, but the least energy intensive processes are the fermentative biohydrogen techniques. Dark fermentative biohydrogen production (DFBHP) is a value-added, less energy-consuming process to generate biohydrogen. In this process, biohydrogen can be produced from sugars as well as complex substrates that are generally considered as organic waste. Yet, the process is constrained by many factors such as low hydrogen yield, incomplete conversion of substrates, accumulation of volatile fatty acids which lead to the drop of the system pH resulting in hindered growth and hydrogen production by the bacteria. To circumvent these drawbacks, researchers have come up with several strategies that improve the yield of DFBHP process. These strategies can be classified as preliminary methodologies concerned with the process optimization and the latter that deals with pretreatment of substrate and seed sludge, bioaugmentation, co-culture of bacteria, supplementation of additives, bioreactor design considerations, metabolic engineering, nanotechnology, immobilization of bacteria, etc. This review sums up some of the improvement techniques that profoundly enhance the biohydrogen productivity in a DFBHP process.

Resource-aware whole-cell model of division of labour in a microbial consortium for complex-substrate degradation

BackgroundLow-cost sustainable feedstocks are essential for commercially viable biotechnologies. These feedstocks, often derived from plant or food waste, contain a multitude of different complex biomolecules which require multiple enzymes to hydrolyse and metabolise. Current standard biotechnology uses monocultures in which a single host expresses all the proteins required for the consolidated bioprocess. However, these hosts have limited capacity for expressing proteins before growth is impacted. This limitation may be overcome by utilising division of labour (DOL) in a consortium, where each member expresses a single protein of a longer degradation pathway.ResultsHere, we model a two-strain consortium, with one strain expressing an endohydrolase and a second strain expressing an exohydrolase, for cooperative degradation of a complex substrate. Our results suggest that there is a balance between increasing expression to enhance degradation versus the burden that higher expression causes. Once a threshold of burden is reached, the consortium will consistently perform better than an equivalent single-cell monoculture.ConclusionsWe demonstrate that resource-aware whole-cell models can be used to predict the benefits and limitations of using consortia systems to overcome burden. Our model predicts the region of expression where DOL would be beneficial for growth on starch, which will assist in making informed design choices for this, and other, complex-substrate degradation pathways.

High-resolution deposition of conductive and insulating materials at micrometer scale on complex substrates

Additive manufacturing transforms the landscape of modern microelectronics. Recent years have witnessed significant progress in the fabrication of 2D planar structures and free-standing 3D architectures. In this work, we present a much-needed intermediary approach: we introduce the Ultra-Precise Deposition (UPD) technology, a versatile platform for material deposition at micrometer scale on complex substrates. The versality of this approach is related to three aspects: material to be deposited (conductive or insulating), shape of the printed structures (lines, dots, arbitrary shapes), as well as type and shape of the substrate (rigid, flexible, hydrophilic, hydrophobic, substrates with pre-existing features). The process is based on the direct, maskless deposition of high-viscosity materials using narrow printing nozzles with the internal diameter in the range from 0.5 to 10 µm. For conductive structures we developed highly concentrated non-Newtonian pastes based on silver, copper, or gold nanoparticles. In this case, the feature size of the printed structures is in the range from 1 to 10 µm and their electrical conductivity is up to 40% of the bulk value, which is the record conductivity for metallic structures printed with spatial resolution below 10 µm. This result is the effect of the synergy between the printing process itself, formulation of the paste, and the proper sintering of the printed structures. We demonstrate a pathway to print such fine structures on complex substrates. We argue that this versatile and stable process paves the way for a widespread use of additive manufacturing for microfabrication.

Modelling and simulation of charge transport phenomena in graphene on SiO2 / Si substrate and graphene on complex oxide substrates

Graphene and silicon are two prominent lithium-ion battery anode materials that have recently received a lot of attention. In this paper we have modelled and simulated the charge transport phenomena in Graphene on Si / SiO2 and SrTiO3 substrates. The Graphene monolayer's interface with the SrTiO3 (111) surface is analyzed using ab initio density-functional measurements. Both charge and heat flows are produced in solids, at the same time when an electrochemical potential is available, bringing about novel properties. The band structure and the electron dissolution process decide the Seebeck coefficient and electrical conductivity. It has been discovered that the interaction of Graphene with SiTiO3 accommodates electronic properties, Seebeck coefficient, and electronic conductivity. For the Graphene / SrTiO3 interface, the best values for the Seebeck coefficient were calculated. All the findings of this work suggest that the Graphene-SrTiO3 (111) and Graphene-Si structure could exhibit interesting quantum transport behavior.

Enhanced Fermentative Hydrogen Production from Food Waste in Continuous Reactor after Butyric Acid Treatment

End-product accumulation during dark fermentation leads to process instability and hydrogen production inhibition. To overcome this constraint, microbial community adaptation to butyric acid can induce acid tolerance and thus enhance the hydrogen yields; however, adaptation and selection of appropriate microbial communities remains uncertain when dealing with complex substrates in a continuous fermentation mode. To address this question, a reactor fed in continuous mode with food waste (organic loading rate of 60 gVS·L·d−1; 12 h hydraulic retention time) was first stressed for 48 h with increasing concentrations of butyric acid (up to 8.7 g·L−1). Performances were compared with a control reactor (unstressed) for 13 days. During 6 days in a steady-state, the pre-stressed reactor produced 2.2 ± 0.2 LH2·L·d−1, which was 48% higher than in the control reactor (1.5 ± 0.2 LH2·L·d−1). The pretreatment also affected the metabolites’ distribution. The pre-stressed reactor presented a higher production of butyric acid (+44%) achieving up to 3.8 ± 0.3 g·L−1, a lower production of lactic acid (−56%), and an enhancement of substrate conversion (+9%). The performance improvement was attributed to the promotion of Clostridium guangxiense, a hydrogen -producer, with a relative abundance increasing from 22% in the unstressed reactor to 52% in the stressed reactor.

Fourier ptychographic microscope allows multi-scale monitoring of cells layout onto micropatterned substrates

Patterned microstructures are commonly used for investigating cells proliferation, guiding cell fate and promoting adhesion and differentiation. Analyzing the behavior of cells layered onto functionalized micropatterned substrates ideally requires large Field of View (FoV), depth of focus, and spatial resolution. Managing the trade-off among these features is pretentious with standard microscopy. Furthermore, patterned substrates have often very complex geometries. Thus, optical systems should be able to get rid of artefacts due to light scattering/diffraction from both the cells’ layer and the underneath structure whose pitches typically have dimensions at the visible wavelength scale. Moreover, the layers are seen as coupled by transmission imaging systems. Decoupling them is pivotal to understand their own properties and related dependences and for interpreting how the structure geometry, point-by-point, addresses the behavior and the functions of the living cells. Here we show the successful use of Fourier Ptychographic Microscopy (FPM) for investigating cell-substrate interaction on micropatterned substrates, solving in full the issue of the layers decoupling. In fact, we demonstrate that it is possible to extract paired but separate clear images of both layers, by computationally decoupling them in FPM reconstruction. In order to test the proposed modality, we chose fibroblast cells in adhesion on a complex substrate consisting of irregular micro-wrinkles on a thin layer of Au. We rely on a numerical multi-look approach, which is capable of restoring quantitative phase-contrast maps of the label-free cells unaffected by artefacts, over a 3.3 mm2 FoV with 0.5 µm resolution. Moreover, from one single FPM map, we demonstrate separate extraction of cells’ morphometry and the underneath wrinkled patterns. Two parameters characterizing the cell-substrate interaction are defined, showing correlation that paves the way to future exploitations of this processing protocol in the fields of mechanobiology and cells and tissue engineering.

Safety and feasibility of a novel multilectrode array catheter in mapping atrial and ventricular arrhythmias with high density: results from the multi-center OPTIMUM study

Abstract Funding Acknowledgements Type of funding sources: Private company. Main funding source(s): This study was funded by Biosense Webster, Inc. Background/Introduction The novel multielectrode array mapping catheter includes 48 electrodes symmetrically distributed across and along 6 splines (Picture), with an additional unipolar reference electrode to reduce far-field signals built in at the tip of the irrigation lumen. Preclinical studies demonstrated its ability to provide high density and high resolution mapping in complex substrates, but its usability and safety in patients with atrial or ventricular arrhythmias have not been investigated. Purpose To assess the safety and mapping performance of the catheter with the electroanatomical navigation system for mapping in the atria and ventricle in patients with a variety of complex arrhythmias. Methods The OPTIMUM study was a prospective, single arm study conducted at 3 European sites. Patients with scar-related atrial tachycardia (AT), PAF and persistent AF (PsAF), ventricular tachycardia (VT), or premature ventricular complexes (PVC) underwent pre-ablation mapping with the study catheter, and subsequent ablation according to institutional standard of care practice; the follow up period was 7 days. The primary safety endpoint was the incidence of device-related serious adverse events (SAE) within 7 days following the procedure. The primary effectiveness endpoint was the completion of protocol-required fast activation and electro anatomical pre-ablation mapping without resort to non-study mapping catheter(s), including fast anatomical mapping, mapping of substrate or any area of interest, and identification of conduction channel(s), gap(s) and critical isthmuses. General procedural characteristics as well as physician feedback (7-point Likert scale: 1 = poor and 7 = excellent) on various aspects of deployment, signal quality, and learning curve were also evaluated. Results Thirty-one patients (mean age: 59.8±14 years, 71.0% male) were enrolled and treated (9 scar-related AT, 12 PAF, and 1 PsAF, 6 VT, and 3 PVC). There was no (0/31) SAE reported, and the primary effectiveness endpoint was achieved in all subjects (31/31). Almost all operators rated highly (≥ 5) on noise reduction in bipolar maps (30/31) and short learning curve (27/31). Bipolar signals and catheter maneuverability were rated highly favorable (≥5) in the left and right atria and met-expectation-and-above (≥4) in the left and right ventricles. Conclusion(s) The first-in-human clinical data demonstrate the safety and effectiveness of the novel microelectrode array mapping catheter to efficiently map various atrial and ventricular complex arrhythmias with good signal quality. Physicians indicated high satisfaction with the learning curve, catheter maneuverability, tissue characterization in all cardiac chambers.

Pharmacological inhibition of SK-channels with AP14145 prevents atrial arrhythmogenic changes in a porcine model for obstructive respiratory events

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): This work was supported by the Novo Nordisk Foundation (Tandem Programme; #31634). Background Obstructive sleep apnea (OSA) creates a complex substrate for atrial fibrillation (AF), which is refractory to many clinically available pharmacological interventions. Purpose To investigate atrial antiarrhythmogenic properties and ventricular electrophysiological safety of small-conductance Ca2+ -activated K+ (SK)- channel inhibition in a porcine model for obstructive respiratory events. Methods In spontaneously breathing pigs, obstructive respiratory events were simulated by intermittent negative upper airway pressure (INAP) applied via a pressure device connected to the intubation tube. INAP was applied for 75 seconds, every 10 minutes, three times before and three times during infusion of the SK-channel inhibitor AP14145. Atrial effective refractory periods (AERP) were acquired before (Pre-INAP), during (INAP) and after (Post-) INAP. AF-inducibility was determined by a S1S2 atrial pacing protocol. For the purpose of drug safety, ventricular arrhythmicity was evaluated by heart rate adjusted QT-interval duration (QT-paced) and electromechanical window (EMW) calculation. Results During vehicle infusion, INAP transiently shortened AERP (Pre-INAP: 135±10 ms vs. Post-INAP 101±11 ms; p=0.008) and increased AF-inducibility. QT-paced prolonged during INAP (Pre-INAP 270±7 ms vs. INAP 275±7 ms; p=0.04) and EMW shortened progressively throughout INAP and Post-INAP (Pre-INAP 80±4 ms; INAP 59±6 ms, Post-INAP 46±10 ms). AP14145 prolonged baseline AERP, partially prevented INAP-induced AERP-shortening and reduced AF-susceptibility. AP14145 did neither alter QT-paced (Pre-AP14145 270±7 ms vs. AP14145 268±6 ms, p=0.83) nor INAP-induced QT-paced prolongation, but blunted Post-INAP associated EMW-shortening. Conclusion In a pig model for obstructive respiratory events, the SK-channel-inhibitor AP14145 prevented INAP-associated AERP-shortening and AF-susceptibility without impairing ventricular electrophysiology. Hence, SK-channels may represent a promising target for OSA-related AF.

Sympatho-vagal activation during obstructive respiratory events in pigs and blunted atrial arrhythmogenic effects by pharmacological IKACh-inhibition

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Novo nordisk foundation Background Obstructive sleep apnea (OSA) is associated with a sympatho-vagal activation which is suspected to create a complex substrate for atrial fibrillation (AF). Purpose In pigs, we investigated atrial arrhythmogenic consequences of simulated obstructive respiratory events by intermittent negative upper airway pressure (INAP) application and tested antiarrhythmic properties of an atria-specific IK,ACh-inhibitor. Methods In spontaneously breathing pigs, obstructive respiratory events were simulated by intermittent negative upper airway pressure (INAP) applied via a pressure device connected to the intubation tube. INAP was applied for 75 seconds, every 10 minutes, three times before (vehicle) and two times following infusion of an IK,ACh-inhibitor. P-wave, PQ-duration, atrial effective refractory periods (AERP) and left ventricular hemodynamic parameters (maximum upstroke velocity, minimal downslope velocity and systole duration) were acquired before (Pre-INAP), during (INAP) and after (Post-) INAP. AF-inducibility was determined by a S1S2 atrial pacing protocol. Results During vehicle infusion, INAP transiently shortened AERP (Pre-INAP: 147±8ms vs. Post-INAP 105±12 ms; p=0.021) and increased AF-inducibility (Pre-INAP: 10±6% vs. Post-INAP 57±15%; p=0.018). Whereas p-wave duration remained stable, PQ-duration prolonged (Pre-INAP: 130±6ms vs. Post-INAP 142±7ms; p=0.03). Left ventricular maximum upstroke velocity increased (Pre-INAP: 1990±118 mmHg/s vs. Post-INAP 3980±704 mmHg/s; p=0.04), minimal downslope velocity decreased and systole duration shortened (Pre-INAP: 236±6 ms vs. Post-INAP 202±12 ms; p=0.04). The IK,ACh-inhibitor increased AERP (vehicle 147±8ms vs. IK,ACh-inhibitor 171±9ms; p=0.05) and sufficiently prevented INAP-associated AERP-shortening (Pre-INAP: 171±9ms vs. Post-INAP 180±9ms; p=0.219) and PQ-duration prolongation (Pre-INAP: 116±8ms vs. Post-INAP 113±7ms; p=0.76). INAP-induced changes of left ventricular maximum upstroke/minimal downslope velocity and systole duration remained unaffected in the presence of the IK,ACh-inhibitor. Conclusion During obstructive respiratory events simulated by INAP in pigs, left ventricular hemodynamic changes demonstrated elevated sympathetic tone, while atrial electrophysiological parameters resemble elevated parasympathetic tone at the same time. This was associated with increased AF-susceptibility. Pharmacological IK,ACh-inhibition blunted INAP-induced impairment of atrial electrophysiology without affecting left ventricular hemodynamic changes. Hence, pharmacological IKACh-inhibition may constitute a promising treatment strategy in AF-patients with OSA.

New index of functional specificity to predict the redundancy of ecosystem functions in microbial communities.

An ecosystem function is suggested to be more sensitive to biodiversity loss (i.e. low functional redundancy) when focusing on specific-type functions than broad-type functions. Thus far, specific-type functions have been loosely defined as functions performed by a small number of species (facilitative species) or functions involved in utilizing complex substrates. However, quantitative examination of functional specificity remains underexplored. We quantified the functional redundancy of 33 ecosystem functions in a freshwater system from 76 prokaryotic community samples over 3 years. For each function, we used a sparse regression model to estimate the number of facilitative Amplicon Sequence Variants (ASVs) and to define taxon-based functional specificity. We also used Bertz structural complexity to determine substrate-based functional specificity. We found that functional redundancy increased with the taxon-based functional specificity, defined as the proportion of facilitative ASVs (= facilitative ASV richness/facilitative ASV richness + repressive ASV (ASVs reducing functioning) richness). When using substrate-based functional specificity, functional redundancy was influenced by Bertz complexity per se and by substrate acquisition mechanisms. Therefore, taxon-based functional specificity is a better predictive index for evaluating functional redundancy than substrate-based functional specificity. These findings provide a framework to quantitatively predict the consequences of diversity losses on ecosystem functioning.

Efficient modeling of organic adsorbates on oxygen-intercalated graphene on Ir(111)

Organic charge transfer complexes (CTCs) can be grown as thin films on intercalated graphene (Gr). Deciphering their precise film morphologies requires global ab initio structure search, where configurational sampling is computationally intractable unless we reconsider the model for the complex substrate. In this study, we employ charged freestanding Gr to approximate an intercalated Gr/O/Ir(111) substrate, without altering the adsoption properties of deposited molecules. We compare different methods of charging Gr and select the most appropriate substitute model for Gr/O/Ir(111) that maintains the adsorption properties of fluorinated tetracyanoquinodimethane (${\mathrm{F}}_{4}\mathrm{TCNQ}$) and tetrathiafulvalene (TTF), prototypical electron acceptor/donor molecules in CTCs. Next, we apply our model in the Bayesian optimization structure search method and density-functional theory to identify the stable structures of ${\mathrm{F}}_{4}\mathrm{TCNQ}$ and TTF on supported Gr. We find that both molecules physisorb to Gr in various configurations. The narrow range of adsorption energies indicates that the molecules may diffuse easily on the surface and molecule-molecule interactions likely have a central role in film formation. Our study shows that complex intercalated substrates may be approximated with charged freestanding Gr, which can facilitate exhaustive structure search of CTCs.

Compositional variability as a major hindering factor in continuous biohydrogen production from cassava starch wastewater: Possible solutions for complex substrates

Compositional variability as a major hindering factor in continuous biohydrogen production from cassava starch wastewater: Possible solutions for complex substrates

Optimizing volatile fatty acid production from municipal sludge: Linking microbial activity with reactor performance

Literature on optimum levels and combinations of bioreactor operating parameters to maximize volatile fatty acids (VFAs) via anaerobic digestion (AD) is still scant and contradictory. This study investigated the interactive impact of digester sludge retention time (SRT), temperature, and pH on optimizing VFAs production and linking reactor performance to acidogenic activities. VFAs production from municipal sludge was maximized in semi-continuous flow acid fermenters (AFs). Batch fermentation tests were utilized for the specific acidogenesis activity assays (SAdA) with a simpler substrate (glucose and acetic acid) and inocula from AFs. Fermenters operated at high pH of 8, low SRT of 2 days, and varying temperatures of 55 and 45 °C had the highest chemical oxygen demand (COD) solubilization and acidification yields. Similar fermenters had the highest VFAs fold increase (1.7–2.2) over the substrate, largest acetic acid contribution to total VFAs (52–58%), and maximum SAdA extent (368–461 mL H2/g VSS inoculum). Maximum hydrogen production rate indirectly represented substrate utilization rate, growth of the acidogens, and hence the specific activities of the acidogenic bacteria in the AFs. These results suggested that batch acidogenic assays could provide quantitative information regarding the presence and activities of cultures in continuously-fed AD utilizing more complex substrates, e.g. municipal sludge. A multiple linear regression model was derived successfully predicting VFAs fold increase of AFs with an R-squared value of 0.9999. The constructed contour plots predicted higher VFAs at relatively high fermenter pH (7.5–8.0), lower SRT (2–2.2 days), and lower temperatures (45–48 °C).

Preparation of Stable Superhydrophobic Coatings on Complex‐Shaped Substrates (Adv. Mater. Interfaces 13/2022)

Superhydrophobic Coatings on Complex Substrates In article number 2200095, Rong Zhang, Junping Zhang, and co-workers report preparation of stable superhydrophobic coatings on complex-shaped substrates. The coatings show excellent superhydrophobicity, superior impalement resistance and high stability. Moreover, the strategy is simple, ultrafast, scalable and low-cost.

Crystal Structures of an E1‐E2‐ubiquitin Thioester Mimetic Reveal Molecular Mechanisms of Transthioesterification

E1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1‐E2 transthioesterification. The molecular mechanisms of transthioesterification and the overall architecture of the E1‐E2‐Ub complex during catalysis are unknown. Here, we determined the structure of a covalently trapped E1‐E2‐ubiquitin thioester mimetic. Two distinct architectures of the complex are observed, one in which the Ub thioester (Ub(t)) contacts E1 in an open conformation and another in which Ub(t) instead contacts E2 in a dramatically different closed conformation. Altogether our structural and biochemical data suggest that these two conformational states represent snapshots of the E1‐E2‐Ub complex pre‐ and post‐thioester transfer, and are consistent with a model in which catalysis is enhanced by a Ub(t)‐mediated ‘affinity switch’ that drives the reaction forward by promoting productive complex formation or substrate release depending on the conformational state.

BS-400-21 MULTIPOLAR ELECTROGRAMS: A NOVEL ALGORITHM FOR ACCURATE ANNOTATION OF NEAR-FIELD POTENTIALS IN SCAR

Electrogram annotation in scar is challenging due to the presence of multicomponent potentials containing near and far field activities. While bipolar electrograms eliminate the mutual far field component enclosed between two closely spaced electrodes, a large far field component often remains. This may result in inconsistent voltage and time measurements. Multielectrode catheters provides an opportunity to improve far-field rejection by reducing the common components across multiple unipolar electrograms. To determine the effect of multiple unipolar signals decomposition method in the ability to reduce the far field component from the unipolar signal. In 6 swine with chronic infarction, the left ventricle was mapped using Carto 3® and multielectrode array catheter (OptrellTM, Biosense Webster). A novel algorithm utilizing Principal Component Analysis was used to estimate the common components of each unipolar signal with its neighboring electrodes, weighted with inverse proportion to their distance. The mutual component was subtracted from each unipolar signal for creating a multipolar electrogram as in Figure 1. The utility of multipolar electrograms in removing far-field activity from complex signals was compared with traditional bipolar electrograms and reported as median voltage amplitude with 25-75 interquartile range. In infarct border zones with complex multicomponent signals (n=76), multipolar electrograms produced a significantly lower far field signal amplitude in comparison to standard bipolar electrograms (0.45mV [IQR 0.3-0.9] vs 1.8mV [1.1-2.4], respectively P<0.001). Figure 1 shows an example of a complex multipotential signal recorded at infarct border zone as it represented by unipolar, bipolar and multipolar electrograms. Multipolar electrograms produced higher amplitude near field signals (2.9mV [IQR 2.2-3.6] vs 1.0 [0.6-1.8mV], respectively P<0.01) as shown in Figure 2. Multipolar electrograms produced by reduction of common components across multiple unipolar electrograms reduce far field potentials significantly greater than bipolar electrograms. This novel method may be particularly helpful for mapping complex substrates including scar.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Enhanced anaerobic treatment of synthetic protein-rich wastewater promoted by organic xerogels.

Carbon-based materials have been shown to enhance anaerobic digestion processes by promoting direct interspecies electron transfer in methanogenic consortia. However, little is known on their effects during the treatment of complex substrates, such as those derived from protein-rich wastewaters. Here, organic xerogels (OX) are tested, for the first time, as accelerators of the methanogenic activity of an anaerobic consortium treating a synthetic protein-rich wastewater. Three OX with distinct pore size distribution (10 and 1000nm for OX-10 and OX-1000, respectively) and structural conformation (graphene oxide integration into OX-10-GO polymeric matrix) were synthesized. OX-1000 promoted the highest methane production rate (5.21 mL/g*h, 13.5% increase with respect to the control incubated without OX) among the synthesized OX. Additionally, batch bioreactors amended with OX achieved higher chemical oxygen demand (COD) removal (up to 88%) as compared to the control, which only showed 50% of COD removal. Interestingly, amendment of bioreactors with OX also triggered the production of medium-chain fatty acids, including caprylate and caproate. Moreover, OX decreased the accumulation of ammonium, derived from proteins hydrolysis, partly explained by their adsorption capacities, and probably involving their electron-accepting capacity promoting anaerobic ammonium oxidation. This is the first time that OX were successfully applied as methanogenic accelerators for the anaerobic treatment of synthetic protein-rich wastewater, increasing the methane production rate and COD removal as well as triggering the production of medium chain fatty acids and attenuating the accumulation of ammonium. Therefore, OX are proposed as suitable materials to boost the efficiency of anaerobic systems to treat complex industrial wastewaters.