Clinical outcomes of transcarotid artery revascularization vs carotid endarterectomy from a large single-center experience.

BACKGROUND: Transcarotid artery revascularization (TCAR) has been practiced as an alternative for both carotid endarterectomy (CEA) and transfemoral carotid artery stenting, specifically in high-risk patients. More recently, the Centers for Medicare and Medicaid Services expanded coverage for TCAR in standard surgical risk patients if done within the Society for Vascular Surgery Vascular Quality Initiative TCAR surveillance project. A few registry studies (primarily from the Society for Vascular Surgery Vascular Quality Initiative) compared the early and up to 1-year outcomes of TCAR vs CEA or transfemoral carotid artery stenting. There is no large single-center study that reported late clinical outcomes. The present study compares intermediate clinical outcomes of TCAR vs CEA. METHODS: This study retrospectively analyzed collected data from TCAR surveillance project patients enrolled in our institution and compare it with CEA patients done by the same providers at the same time period. The primary outcome was combined perioperative stroke/death and late stroke/death. Secondary outcomes included combined stroke, death, and myocardial infarction, cranial nerve injury (CNI), and bleeding. Propensity matching was done to analyze outcome. Kaplan-Meier analysis was used to estimate freedom from stroke, stroke/death, and ≥50% and ≥80% restenosis. RESULTS: We analyzed 646 procedures (637 patients) (404 CEA, 242 TCAR). There was no significant difference in the indications for carotid intervention. However, TCAR patients had more high-risk criteria, including hypertension, coronary artery disease, congestive heart failure, and renal failure. There was no significant differences between CEA vs TCAR in 30-day perioperative stroke (1% vs 2%), stroke/death rate (1% vs 3%; P = .0849), or major hematomas (2% vs 2%). The rate of CNI was significantly different (5% for CEA vs 1% for TCAR; P = .0138). At late follow-up (2 years), the rate of stroke was 1% vs 4% (P = .0273), stroke/death 8% vs 15% (P = .008), ≥80 % restenosis 0.5% vs 3% (P = .0139) for CEA patients vs TCAR patients, respectively. After matching 242 CEAs and 242 TCARs, the perioperative stroke rate was 1% for CEA vs 2% for TCAR (P = .5037), the stroke/death rate was 2% vs 3% (P = .2423), and the CNI rate was 3% vs 1% (P = .127). At late follow-up, rates of stroke were 1% for CEA vs 4% for TCAR (P = .0615) and stroke/death were 8% vs 15% (P = .0345). The rate of ≥80% restenosis was 0.9% for CEA vs 3% for TCAR (P = .099). The rates of freedom from stroke at 6, 12, 18, and 24 months for CEA vs TCAR were 99%, 99%, 99%, and 99% vs 97%, 95%, 93% and 93%, respectively (P = .0806); stroke/death were 94%, 90%, 87%, and 86% vs 93%, 87%, 76%, and 75%, respectively (P = .0529); and ≥80% restenosis were 100%, 99%, 98%, and 98% vs 97%, 95%, 93%, and 93%, respectively (P = .1132). CONCLUSIONS: In a propensity-matched analysis, both CEA and TCAR have similar perioperative clinical outcomes. However, CEA was superior to TCAR for the rates of late stroke/death and had a somewhat lower rate of ≥80% restenosis at 2 years, but this difference was not statistically significant.

Multienzymatic Cascades and Nanomaterial Scaffolding-A Potential Way Forward for the Efficient Biosynthesis of Novel Chemical Products.

Synthetic biology is touted as the next industrial revolution as it promises access to greener biocatalytic syntheses to replace many industrial organic chemistries. Here, it is shown to what synthetic biology can offer in the form of multienzyme cascades for the synthesis of the most basic of new materials-chemicals, including especially designer chemical products and their analogs. Since achieving this is predicated on dramatically expanding the chemical space that enzymes access, such chemistry will probably be undertaken in cell-free or minimalist formats to overcome the inherent toxicity of non-natural substrates to living cells. Laying out relevant aspects that need to be considered in the design of multi-enzymatic cascades for these purposes is begun. Representative multienzymatic cascades are critically reviewed, which have been specifically developed for the synthesis of compounds that have either been made only by traditional organic synthesis along with those cascades utilized for novel compound syntheses. Lastly, an overview of strategies that look toward exploiting bio/nanomaterials for accessing channeling and other nanoscale materials phenomena in vitro to direct novel enzymatic biosynthesis and improve catalytic efficiency is provided. Finally, a perspective on what is needed for this field to develop in the short and long term is presented.

Bacterial Membrane Vesicles for In Vitro Catalysis.

The use of biological systems in manufacturing and medical applications has seen a dramatic rise in recent years as scientists and engineers have gained a greater understanding of both the strengths and limitations of biological systems. Biomanufacturing, or the use of biology for the production of biomolecules, chemical precursors, and others, is one particular area on the rise as enzymatic systems have been shown to be highly advantageous in limiting the need for harsh chemical processes and the formation of toxic products. Unfortunately, biological production of some products can be limited due to their toxic nature or reduced reaction efficiency due to competing metabolic pathways. In nature, microbes often secrete enzymes directly into the environment or encapsulate them within membrane vesicles to allow catalysis to occur outside the cell for the purpose of environmental conditioning, nutrient acquisition, or community interactions. Of particular interest to biotechnology applications, researchers have shown that membrane vesicle encapsulation often confers improved stability, solvent tolerance, and other benefits that are highly conducive to industrial manufacturing practices. While still an emerging field, this review will provide an introduction to biocatalysis and bacterial membrane vesicles, highlight the use of vesicles in catalytic processes in nature, describe successes of engineering vesicle/enzyme systems for biocatalysis, and end with a perspective on future directions, using selected examples to illustrate these systems' potential as an enabling tool for biotechnology and biomanufacturing.

Computationally Designed AMPs with Antibacterial and Antibiofilm Activity against MDR Acinetobacter baumannii.

The discovery of new antimicrobials is necessary to combat multidrug-resistant (MDR) bacteria, especially those that infect wounds and form prodigious biofilms, such as Acinetobacter baumannii. Antimicrobial peptides (AMPs) are a promising class of new therapeutics against drug-resistant bacteria, including gram-negatives. Here, we utilized a computational AMP design strategy combining database filtering technology plus positional analysis to design a series of novel peptides, named HRZN, designed to be active against A. baumannii. All of the HRZN peptides we synthesized exhibited antimicrobial activity against three MDR A. baumannii strains with HRZN-15 being the most active (MIC 4 µg/mL). This peptide also inhibited and eradicated biofilm of A. baumannii strain AB5075 at 8 and 16 µg/mL, which is highly effective. HRZN-15 permeabilized and depolarized the membrane of AB5075 rapidly, as demonstrated by the killing kinetics. HRZN 13 and 14 peptides had little to no hemolysis activity against human red blood cells, whereas HRZN-15, -16, and -17 peptides demonstrated more significant hemolytic activity. HRZN-15 also demonstrated toxicity to waxworms. Further modification of HRZN-15 could result in a new peptide with an improved toxicity profile. Overall, we successfully designed a set of new AMPs that demonstrated activity against MDR A. baumannii using a computational approach.

Different Strategies Affect Enzyme Packaging into Bacterial Outer Membrane Vesicles.

All Gram-negative bacteria are believed to produce outer membrane vesicles (OMVs), proteoliposomes shed from the outermost membrane. We previously separately engineered E. coli to produce and package two organophosphate (OP) hydrolyzing enzymes, phosphotriesterase (PTE) and diisopropylfluorophosphatase (DFPase), into secreted OMVs. From this work, we realized a need to thoroughly compare multiple packaging strategies to elicit design rules for this process, focused on (1) membrane anchors or periplasm-directing proteins (herein "anchors/directors") and (2) the linkers connecting these to the cargo enzyme; both may affect enzyme cargo activity. Herein, we assessed six anchors/directors to load PTE and DFPase into OMVs: four membrane anchors, namely, lipopeptide Lpp', SlyB, SLP, and OmpA, and two periplasm-directing proteins, namely, maltose-binding protein (MBP) and BtuF. To test the effect of linker length and rigidity, four different linkers were compared using the anchor Lpp'. Our results showed that PTE and DFPase were packaged with most anchors/directors to different degrees. For the Lpp' anchor, increased packaging and activity corresponded to increased linker length. Our findings demonstrate that the selection of anchors/directors and linkers can greatly influence the packaging and bioactivity of enzymes loaded into OMVs, and these findings have the potential to be utilized for packaging other enzymes into OMVs.

Long-term durability and clinical outcome of a prospective randomized trial comparing carotid endarterectomy with ACUSEAL polytetrafluoroethylene patching versus pericardial patching.

BACKGROUND: Several studies have shown the superiority of carotid endarterectomy (CEA) with patch closure over primary closure. However, no definite study has shown any significant differences in clinical outcome between various types of patches. Because more vascular surgeons have used pericardial patching recently, this study will analyze the late clinical outcome (≥10 years) of our previously reported prospective randomized trial comparing CEA with ACUSEAL (polytetrafluoroethylene) vs pericardial patching. METHODS: A total of 200 CEAs were randomized (1:1) to either Vascu-Guard pericardial patching or ACUSEAL patching. All patients had immediate duplex ultrasound imaging, which was repeated at 6 months and annually thereafter. Kaplan-Meier analysis was used to estimate rates of freedom from stroke, stroke-free survival, and rates of freedom from ≥50% and ≥80% restenosis. RESULTS: Overall demographic and clinical characteristics were somewhat similar with a mean follow-up of 80 months (range: 0-149 months). The rates of freedom from stroke were 97, 97, 97, 96, 93 for ACUSEAL vs 99, 98, 97, 97, 92 for pericardial patching (P = .1112) at 1, 2, 3, 5, and 10 years, respectively. Similarly, the rates of freedom from stroke/death were 94, 93, 90, 76, 50 for ACUSEAL vs 99, 96, 91, 78, 47 for pericardial patching (P = .8591). The rates of freedom from ≥50% restenosis were 98, 98, 96, 89, 79 for ACUSEAL vs 87, 83, 83, 81, 71 for pericardial patching (P = .0489). The rates of freedom from ≥80% restenosis were 99, 99, 99, 96, 85 for ACUSEAL vs 96, 96, 96, 93, 93 for pericardial patching (P = .9407). The overall survival rates were 95, 94, 91, 77, 51 for ACUSEAL vs 100, 98, 93, 79, 50 for pericardial patching (P = .9123). Other patch complications (eg, rupture, aneurysmal dilation, infection, etc) were similar. CONCLUSIONS: Both CEA with ACUSEAL (polytetrafluoroethylene) and pericardial patching are durable and have similar clinical outcomes at 10 years except that ACUSEAL patching has significantly better rates of freedom from ≥50% restenosis.

Thirty-Day Perioperative Clinical Outcomes of Transcarotid Artery Revascularization vs Carotid Endarterectomy in a Single-Center Experience.

BACKGROUND: Transcarotid artery revascularization (TCAR) has been proposed as a alternative to carotid endarterectomy (CEA) and transfemoral carotid artery stenting in high-risk patients. Recently Centers for Medicare and Medicaid Services expanded coverage for TCAR to include standard surgical risk patients within the Society of Vascular Surgery Vascular Quality Initiative TCAR Surveillance Project. Few single centers compared the clinical outcome of TCAR with CEA. This study compares 30-day perioperative clinical outcomes between TCAR and CEA. STUDY DESIGN: This is retrospective analysis of prospectively collected data from the TCAR Surveillance Project of TCAR patients enrolled in our institution and compared with CEAs done in the same time/with the same providers. The primary outcome was stroke and/or death. Secondary outcomes included stroke, death, MI, cranial nerve injury, bleeding, and others. Propensity matching was done to analyze outcomes. RESULTS: The study analyzed 501 patients (347 CEA, 154 TCAR). There were no significant differences in symptomatic status (43% for CEA vs 38% for TCAR, p = 0.303). TCAR had more patients with hypertension (p = 0.04), coronary artery disease (p = 0.028), and congestive heart failure (p = 0.039). The 30-day perioperative complication rates for CEA vs TCAR were as follows: stroke 1% vs 3% (p = 0.142), stroke/death 1% vs 3% (p = 0.185), MI 0.6% vs 0.7% (p = 1), death 0.6% vs 0% (p = 1), stroke/death/MI 2% vs 4% (p = 0.233), cranial nerve injury 4% vs 2% (p = 0.412), and major hematoma (requiring reintervention) 2% vs 3% (p = 1). After matching 154 CEA patients and 154 TCAR, 30-day perioperative complication rates were as follows: stroke 2% vs 3% (p = 0.723), stroke/death 3% vs 3% (p = 1), death 1.3% vs 0% (p = 0.498), MI 0.7% vs 0.7% (p = 1), and stroke/death/MI 3% vs 4% (p = 0.759). CONCLUSIONS: This study showed that using propensity match analysis, both CEA and TCAR have similar 30-day perioperative outcomes. Further long-term data are needed.

Modifications of Epitaxial Graphene on SiC for the Electrochemical Detection and Identification of Heavy Metal Salts in Seawater.

The electrochemical detection of heavy metal ions is reported using an inexpensive portable in-house built potentiostat and epitaxial graphene. Monolayer, hydrogen-intercalated quasi-freestanding bilayer, and multilayer epitaxial graphene were each tested as working electrodes before and after modification with an oxygen plasma etch to introduce oxygen chemical groups to the surface. The graphene samples were characterized using X-ray photoelectron spectroscopy, atomic force microscopy, Raman spectroscopy, and van der Pauw Hall measurements. Dose-response curves in seawater were evaluated with added trace levels of four heavy metal salts (CdCl2, CuSO4, HgCl2, and PbCl2), along with detection algorithms based on machine learning and library development for each form of graphene and its oxygen plasma modification. Oxygen plasma-modified, hydrogen-intercalated quasi-freestanding bilayer epitaxial graphene was found to perform best for correctly identifying heavy metals in seawater.

Packaging of Diisopropyl Fluorophosphatase (DFPase) in Bacterial Outer Membrane Vesicles Protects Its Activity at Extreme Temperature.

Enzymatic decontamination of organophosphate compounds offers a biofriendly pathway to the neutralization of highly dangerous compounds. Environmental dissemination of enzymes, however, is an ongoing problem considering the costly process of production and chemical modification for stability that can diminish catalytic activity. As a result, there is interest in the potential for enzymatic encapsulation in situ or into nascent bacterial membrane vesicles to improve catalytic stability across various environmental challenges associated with storage and field deployment. In this study, we have engineered bacterial outer membrane vesicles (OMVs) to encapsulate the diisopropyl fluorophosphatase (DFPase), an enzyme originally isolated from squid Loligo vulgaris and capable of hydrolyzing diisopropyl fluorophosphate (DFP) and other organophosphates compounds. Here we employed a recombinant lipopeptide anchor to direct recruitment of DFPase into OMVs, which were isolated from culture media and tested for catalytic activity against both diisopropyl fluorophosphate and paraoxon. Our encapsulation strategy prevented the loss of catalytic activity despite lyophilization, extended storage time (2 days), and extreme temperatures up to 80 °C. These data underscore the appeal of DFPase as a biodecontaminant of organophosphates as well as the potential for OMV packaging in stabilized field deployment applications.

Extracellular vesicle production in Gram-positive bacteria.

This is a highlight on the article 'Extracellular vesicle formation in Lactococcus lactis is stimulated by prophage-encoded holin-lysin system' by Yue Liu, Eddy Smid and Tjakko Abee.

PepVAE: Variational Autoencoder Framework for Antimicrobial Peptide Generation and Activity Prediction.

New methods for antimicrobial design are critical for combating pathogenic bacteria in the post-antibiotic era. Fortunately, competition within complex communities has led to the natural evolution of antimicrobial peptide (AMP) sequences that have promising bactericidal properties. Unfortunately, the identification, characterization, and production of AMPs can prove complex and time consuming. Here, we report a peptide generation framework, PepVAE, based around variational autoencoder (VAE) and antimicrobial activity prediction models for designing novel AMPs using only sequences and experimental minimum inhibitory concentration (MIC) data as input. Sampling from distinct regions of the learned latent space allows for controllable generation of new AMP sequences with minimal input parameters. Extensive analysis of the PepVAE-generated sequences paired with antimicrobial activity prediction models supports this modular design framework as a promising system for development of novel AMPs, demonstrating controlled production of AMPs with experimental validation of predicted antimicrobial activity.

Isolation and Characterization of Membrane Vesicles from Lactobacillus Species.

Throughout their life cycle, bacteria shed portions of their outermost membrane comprised of proteins, lipids, and a diversity of other biomolecules. These biological nanoparticles have been shown to have a range of highly diverse biological activities, including pathogenesis, community regulation, and cellular defense (among others). In recent publications, we have isolated and characterized membrane vesicles (MVs) from several species of Lactobacilli, microbes classified as commensals within the human gut microbiome ( Dean et al., 2019 and 2020). With increasing scientific understanding of host-microbe interactions, the gut-brain axis, and tailored probiotics for therapeutic or performance increasing applications, the protocols described herein will be useful to researchers developing new strategies for gut community engineering or the targeted delivery of bio-active molecules. Graphic abstract: Figure 1. Atomic force microscopic image of Lactobacillus casei ATCC 393 bacteria margins (white arrows) and membrane vesicles (black arrows).

Variational Autoencoder for Generation of Antimicrobial Peptides.

Over millennia, natural evolution has allowed for the emergence of countless biomolecules with highly specific roles within natural systems. As seen with peptides and proteins, often evolution produces molecules with a similar function but with variable amino acid composition and structure but diverging from a common ancestor, which can limit sequence diversity. Using antimicrobial peptides as a model biomolecule, we train a generative deep learning algorithm on a database of known antimicrobial peptides to generate novel peptide sequences with antimicrobial activity. Using a variational autoencoder, we are able to generate a latent space plot that can be surveyed for peptides with known properties and interpolated across a predictive vector between two defined points to identify novel peptides that show dose-responsive antimicrobial activity. These proof-of-concept studies demonstrate the potential for artificial intelligence-directed methods to generate new antimicrobial peptides and motivate their potential application toward peptide and protein design without the need for exhaustive screening of sequence libraries.

Multilayer Epitaxial Graphene on Silicon Carbide: A Stable Working Electrode for Seawater Samples Spiked with Environmental Contaminants.

The electrochemical response of multilayer epitaxial graphene electrodes on silicon carbide substrates was studied for use as an electrochemical sensor for seawater samples spiked with environmental contaminants using cyclic square wave voltammetry. Results indicate that these graphene working electrodes are more robust and have lower background current than either screen-printed carbon or edge-plane graphite in seawater. Identification algorithms developed using machine learning techniques are described for several heavy metals, herbicides, pesticides, and industrial compounds. Dose-response curves provide a basis for quantitative analysis.

Lactobacillus acidophilus Membrane Vesicles as a Vehicle of Bacteriocin Delivery.

Recent reports have shown that Gram-positive bacteria actively secrete spherical nanometer-sized proteoliposome membrane vesicles (MVs) into their surroundings. Though MVs are implicated in a broad range of biological functions, few studies have been conducted to examine their potential as delivery vehicles of antimicrobials. Here, we investigate the natural ability of Lactobacillus acidophilus MVs to carry and deliver bacteriocin peptides to the opportunistic pathogen, Lactobacillus delbrueckii. We demonstrate that upon treatment with lactacin B-inducing peptide, the proteome of the secreted MVs is enriched in putative bacteriocins encoded by the lab operon. Further, we show that purified MVs inhibit growth and compromise membrane integrity in L. delbrueckii, which is confirmed by confocal microscopy imaging and spectrophotometry. These results show that L. acidophilus MVs serve as conduits for antimicrobials to competing cells in the environment, suggesting a potential role for MVs in complex communities such as the gut microbiome. With the potential for controlling their payload through microbial engineering, MVs produced by L. acidophilus may be an interesting platform for effecting change in complex microbial communities or aiding in the development of new biomedical therapeutics.

Lipid-tagged single domain antibodies for improved enzyme-linked immunosorbent assays.

Anti-Staphylococcal Enterotoxin B single domain antibodies were engineered to include the N-terminal peptide sequence of the major outer membrane lipoprotein from Escherichia coli, which directs the N-terminal addition of lipid to the single domain antibody. We produced and purified two different single domain antibodies as well as a variant and dimer construct of one of the two, all with and without the added lipid. Their ability to function as the capture antibody in standard enzyme-linked immunosorbent assays were evaluated, finding that coating polystyrene microtiter plates with the lipid-tagged single domain antibodies gave a 3-fold improvement in the observed limit of detection. This increase was likely due to an increased amount of single domain antibody adsorbed to the microtiter plate, which translated to improved limits of detection of Staphylococcal Enterotoxin B over using the same single domain antibody sans lipid-tag. However, improved orientation may also play a role. Regardless of the mechanism, the biosynthetic lipid-tagging of single domain antibodies represent a facile modality that can enhance their ability to be utilized as immunoassay capture reagent as well as facilitate their incorporation into liposome targeting applications in the future.

Francisella novicida Two-Component System Response Regulator BfpR Modulates iglC Gene Expression, Antimicrobial Peptide Resistance, and Biofilm Production.

Response regulators are a critical part of the two-component system of gene expression regulation in bacteria, transferring a signal from a sensor kinase into DNA binding activity resulting in alteration of gene expression. In this study, we investigated a previously uncharacterized response regulator in Francisella novicida, FTN_1452 that we have named BfpR (Biofilm-regulating Francisella protein Regulator, FTN_1452). In contrast to another Francisella response regulator, QseB/PmrA, BfpR appears to be a negative regulator of biofilm production, and also a positive regulator of antimicrobial peptide resistance in this bacterium. The protein was crystallized and X-ray crystallography studies produced a 1.8 Å structure of the BfpR N-terminal receiver domain revealing interesting insight into its potential interaction with the sensor kinase. Structural analysis of BfpR places it in the OmpR/PhoP family of bacterial response regulators along with WalR and ResD. Proteomic and transcriptomic analyses suggest that BfpR overexpression affects expression of the critical Francisella virulence factor iglC, as well as other proteins in the bacterium. We demonstrate that mutation of bfpR is associated with an antimicrobial peptide resistance phenotype, a phenotype also associated with other response regulators, for the human cathelicidin peptide LL-37 and a sheep antimicrobial peptide SMAP-29. F. novicida with mutated bfpR replicated better than WT in intracellular infection assays in human-derived macrophages suggesting that the down-regulation of iglC expression in bfpR mutant may enable this intracellular replication to occur. Response regulators have been shown to play important roles in the regulation of bacterial biofilm production. We demonstrate that F. novicida biofilm formation was highly increased in the bfpR mutant, corresponding to altered glycogen synthesis. Waxworm infection experiments suggest a role of BfpR as a negative modulator of iglC expression with de-repression by Mg2+. In this study, we find that the response regulator BfpR may be a negative regulator of biofilm formation, and a positive regulator of antimicrobial peptide resistance in F. novicida.

Pressure cycling technology for challenging proteomic sample processing: application to barnacle adhesive.

Successful proteomic characterization of biological material depends on the development of robust sample processing methods. The acorn barnacle Amphibalanus amphitrite is a biofouling model for adhesive processes, but the identification of causative proteins involved has been hindered by their insoluble nature. Although effective, existing sample processing methods are labor and time intensive, slowing progress in this field. Here, a more efficient sample processing method is described which exploits pressure cycling technology (PCT) in combination with protein solvents. PCT aids in protein extraction and digestion for proteomics analysis. Barnacle adhesive proteins can be extracted and digested in the same tube using PCT, minimizing sample loss, increasing throughput to 16 concurrently processed samples, and decreasing sample processing time to under 8 hours. PCT methods produced similar proteomes in comparison to previous methods. Two solvents which were ineffective at extracting proteins from the adhesive at ambient pressure (urea and methanol) produced more protein identifications under pressure than highly polar hexafluoroisopropanol, leading to the identification and description of >40 novel proteins at the interface. Some of these have homology to proteins with elastomeric properties or domains involved with protein-protein interactions, while many have no sequence similarity to proteins in publicly available databases, highlighting the unique adherent processes evolved by barnacles. The methods described here can not only be used to further characterize barnacle adhesive to combat fouling, but may also be applied to other recalcitrant biological samples, including aggregative or fibrillar protein matrices produced during disease, where a lack of efficient sample processing methods has impeded advancement. Data are available via ProteomeXchange with identifier PXD012730.

Machine Learning Techniques for Chemical Identification Using Cyclic Square Wave Voltammetry.

Electroanalytical techniques are useful for detection and identification because the instrumentation is simple and can support a wide variety of assays. One example is cyclic square wave voltammetry (CSWV), a practical detection technique for different classes of compounds including explosives, herbicides/pesticides, industrial compounds, and heavy metals. A key barrier to the widespread application of CSWV for chemical identification is the necessity of a high performance, generalizable classification algorithm. Here, machine and deep learning models were developed for classifying samples based on voltammograms alone. The highest performing models were Long Short-Term Memory (LSTM) and Fully Convolutional Networks (FCNs), depending on the dataset against which performance was assessed. When compared to other algorithms, previously used for classification of CSWV and other similar data, our LSTM and FCN-based neural networks achieve higher sensitivity and specificity with the area under the curve values from receiver operating characteristic (ROC) analyses greater than 0.99 for several datasets. Class activation maps were paired with CSWV scans to assist in understanding the decision-making process of the networks, and their ability to utilize this information was examined. The best-performing models were then successfully applied to new or holdout experimental data. An automated method for processing CSWV data, training machine learning models, and evaluating their prediction performance is described, and the tools generated provide support for the identification of compounds using CSWV from samples in the field.

Response of Lactobacillus plantarum WCFS1 to the Gram-Negative Pathogen-Associated Quorum Sensing Molecule N-3-Oxododecanoyl Homoserine Lactone.

The bacterial quorum sensing phenomenon has been well studied since its discovery and has traditionally been considered to include signaling pathways recognized exclusively within either Gram-positive or Gram-negative bacteria. These groups of bacteria synthesize structurally distinct signaling molecules to mediate quorum sensing, where Gram-positive bacteria traditionally utilize small autoinducing peptides (AIPs) and Gram-negatives use small molecules such as acyl-homoserine lactones (AHLs). The structural differences between the types of signaling molecules have historically implied a lack of cross-talk among Gram-positive and Gram-negative quorum sensing systems. Recent investigations, however, have demonstrated the ability for AIPs and AHLs to be produced by non-canonical organisms, implying quorum sensing systems may be more universally recognized than previously hypothesized. With that in mind, our interests were piqued by the organisms Lactobacillus plantarum, a Gram-positive commensal probiotic known to participate in AIP-mediated quorum sensing, and Pseudomonas aeruginosa, a characterized Gram-negative pathogen whose virulence is in part controlled by AHL-mediated quorum sensing. Both health-related organisms are known to inhabit the human gut in various instances, both are characterized to elicit distinct effects on host immunity, and some studies hint at the putative ability of L. plantarum to degrade AHLs produced by P. aeruginosa. We therefore wanted to determine if L. plantarum cultures would respond to the addition of N-(3-oxododecanoyl)-L-homoserine lactone (3OC12) from P. aeruginosa by analyzing changes on both the transcriptome and proteome over time. Based on the observed upregulation of various two-component systems, response regulators, and native quorum sensing related genes, the resulting data provide evidence of an AHL recognition and response by L. plantarum.