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Environmental challenges and development require plants to reallocate resources between primary and specialized metabolites to survive. Genome-scale metabolic models, which map carbon flux through metabolic pathways, are a valuable tool in the study of tradeoffs that arise at this interface. Due to annotation gaps, models that characterize all the enzymatic steps in individual specialized pathways and their linkages to each other and to central carbon metabolism are difficult to construct. Recent studies have successfully curated subsystems of specialized metabolism and characterized the interfaces where flux is diverted to the precursors of glucosinolates, terpenes, and anthocyanins. Although advances in metabolite profiling can help to constrain models at this interface, quantitative analysis remains challenging because of the different timescales on which specialized metabolites from constitutive and reactive pathways accumulate., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2022. Published by Elsevier Ltd.)

It has been shown that short-chain fatty acids (SCFAs) produced by the gut microbiome are of importance to host tissue health; however, measuring such compounds in biological samples is often limited to using hours to days old fecal and blood plasma samples. Organ-on-a-chip models have been created to simplify the complexity but struggle to reproduce the full biology of the gut specifically. We recently reported a tissue-in-a-chip gut model that incorporates gut explanted tissue into a microfluidic device. The system maintains a biologically relevant oxygen gradient and tissue ex vivo for days at a time, but minimal characterization of biological activity was reported. Herein, we use 1 H-NMR to analyze the SCFA content of tissue media effluents from gut explants cultured in the recently developed microfluidic organotypic device (MOD). 1 H-NMR can identify key SCFAs in the complex samples with minimal sample preparation. Our findings show that maintaining physiologically relevant oxygen conditions, something often missing from many other culture systems, significantly impacts the SCFA profile. Additionally, we noted the changes in SCFAs with culture time and potential variability between SCFA levels in male and female mouse tissue explants cultured in the MOD system based on 1 H-NMR spectral profiles.

Urinary tract infections (UTIs) are one of the most common infections across the world and can lead to serious complications such as sepsis if not treated in a timely manner. Uropathogenic Escherichia coli account for 75% of all UTIs. Early diagnosis is crucial to help control UTIs, but current culturing methods are expensive and time-consuming and lack sensitivity. The existing point-of-care methods fall short because they rely on indirect detection from elevated nitrates in urine rather than detecting the actual bacteria causing the infection. Magnetophoresis is a powerful method used to separate and/or isolate cells of interest from complex matrices for analysis. However, magnetophoresis typically requires complex and expensive instrumentation to control flow in microfluidic devices. Coupling magnetophoresis with microfluidic paper-based analytical devices (μPADs) enables pump-free flow control and simple and low-cost operation. Early magnetophoresis μPADs showed detection limits competitive with traditional methods but higher than targets for clinical use. Here, we demonstrate magnetophoresis using hybrid μPADs that rely on capillary action in hydrophilic polyethylene terephthalate channels combined with paper pumps. We were able to detect E. coli with a calculated limit of detection of 2.40 × 10 2 colony-forming units per mL.

Among lateral flow immunoassay (LFIA) platforms, enzyme-based LFIAs provide signal amplification to improve sensitivity. However, most enzyme-based LFIAs require multiple timed steps, complicating their utility in point-of-care testing (POCT). Here, we report a microfluidic interface for LFIAs that automates sample, buffer, and reagent addition, greatly simplifying operation while achieving the high analytical stringency associated with more complex assays. The microfluidic interface also maintains the low cost and small footprint of standard LFIAs. The platform is fabricated from a combination of polyester film, double-sided adhesive tape, and nitrocellulose, and fits in the palm of your hand. All reagents are dried on the nitrocellulose to facilitate sequential reagent delivery, and the sample is used as the wash buffer to minimize steps. After the sample addition, a user simply waits 15 min for a colorimetric result. This manuscript discusses the development and optimization of the channel geometry to achieve a simple step enzyme amplified immunoassay. As a proof-of-concept target, we selected lipoarabinomannan (LAM), a WHO identified urinary biomarker of active tuberculosis, to demonstrate the device feasibility and reliability. The results revealed that the device successfully detected LAM in phosphate buffer (PBS) as well as spiked urine samples within 15 min after sample loading. The minimum concentration of color change was achieved at 25 ng mL -1 .

All nine presently known European species of the Apertochrysa prasina group (formerly the Pseudomallada prasinus group) are diagnosed. Separate keys for the identification of living and preserved specimens of each sex are presented. For males, the furwing trait (increased numbers of setae on costal crossveins) is often crucial for identification, especially in preserved specimens. In some cases, females of the newly recognized prasinoid species (Ap1, Ap2, Ap3) are impossible to distinguish without live coloration. This is largely why the three new species are difficult to assign to one of the many old (often female) type specimens of former prasinoid species or varieties, which had been synonymized with Chrysopa prasina in the past.

A microfluidic paper-based thermoplastic electrode (TPE) array has been developed for point-of-care detection of Na + and K + ions using a custom-made portable potentiometer. TPEs were fabricated using polystyrene as the binder and two different types of graphite to compare the electrode performance. The newly designed TPE array embedded in a polymethyl methacrylate chip consists of two working electrodes modified with carbon black nanomaterial and an ion-selective membrane, and an all-solid-state reference electrode modified with Ag/AgCl ink and poly(butyl methacrylate-co-methyl methacrylate) membrane via drop-casting. Ion-selective membrane compositions and conditioning steps were optimized. Under optimized conditions, ion-selective TPEs demonstrated fast response time (4 s) and good stability. The TPE array demonstrated a Nernstian behavior for K + with a sensitivity of 59.2 ± 0.2 mV decade -1 and near-Nernstian response for Na + with a sensitivity of 54.0 ± 1.1 mV decade -1 in the range 10 -1  - 10 -4  M and 1 -  10 -3  M, respectively. The detection limits were 1 × 10 -5  M and 1 × 10 -4  M for K + and Na + , respectively. In addition, a K + and Na + selective microfluidic paper-based analytical device (µPAD) was applied to artificial serum analysis and found in good agreement with average recoveries of 101.3% and 99.7%, respectively, suggesting that the developed ISE array is suitable for detection of sodium and potassium in complex matrix., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Point-of-care (POC) methods currently available for detecting SARS-CoV-2 infections still lack accuracy. Here, we report the development of a highly sensitive electrochemical immunoassay capable of quantitatively detecting the presence of the SARS-CoV-2 virus in patient nasopharyngeal samples using stencil-printed carbon electrodes (SPCEs) functionalized with capture antibodies targeting the SARS-CoV-2 nucleocapsid protein (N protein). Samples are added to the electrode surface, followed by horseradish peroxidase (HRP)-conjugated detection antibodies also targeting the SARS-CoV-2 N protein. The concentration of the virus in samples is quantified using chronoamperometry in the presence of 3,3'5,5'-tetramethylbenzidine. Limits of detection equivalent to less than 50 plaque forming units/mL (PFU/mL) were determined with virus sample volumes of 20 μL. No cross-reactivity was detected with the influenza virus and other coronavirus N proteins. Patient nasopharyngeal samples were tested as part of a proof-of-concept clinical study where samples were also tested using the gold-standard real-time quantitative polymerase chain reaction (RT-qPCR) method. Preliminary results from a data set of 22 samples demonstrated a clinical specificity of 100% ( n = 9 negative samples according to RT-qPCR) and a clinical sensitivity of 70% for samples with RT-PCR cycle threshold (Ct) values under 30 ( n = 10) and 100% for samples with Ct values under 25 ( n = 5), which complies with the World Health Organization (WHO) criteria for POC COVID-19 diagnostic tests. Our functionalized SPCEs were also validated against standard plaque assays, and very good agreement was found between both methods ( R 2 = 0.9993, n = 6), suggesting that our assay could be used to assess patient infectivity. The assay currently takes 70 min from sampling to results.

Exposure to natural rubber latex (NRL) can result in sensitivity to NRL protein with resulting allergic reactions. Low-cost, portable, simple, sensitive analytical tools for NRL protein measurements are needed for rapid and accurate assessments of allergenic risks at the point-of-care (POC) instead of using traditional methods that require large and expensive instruments, long-time analysis, and complex sample preparation steps. Here, an electrochemical paper-based analytical device (ePAD) is presented by combining sample preparation and electrochemical detection within a single device to offer a one-step NRL analysis. The lack of antibodies and/or enzymes against NRL makes POC analysis difficult. In this work, detection is based on electrochemical measurement of the remaining Cu after in situ protein complexation instead of more complex biological assays for the first time. Graphene screen-printed electrodes modified with 1,10-phenanthroline and Nafion were used in the ePAD to improve Cu signal 18-fold relative to unmodified carbon screen-printed electrodes. The optimum parameters including 1,10-phenanthroline concentration, reaction time between Cu and protein, and the starting Cu concentration were 5% w/v, 1 min, and 600 μg mL -1 , respectively. In addition to short analysis time (4 min), the system selectivity indicated no other interfering species affecting protein detection. The proposed ePAD achieved an LOD of 3.0 mg dL -1 and a linear range of 10.0-200.0 mg dL -1 . Finally, the proposed sensors were applied for NRL protein detection and the results were not significantly different from the traditional Lowry method at 95% confidence.

This work presents an all-in-one origami paper-based electrochemical platform for simple and inexpensive l-cysteine (Cys) detection using Cys as a monomer for modifying electrode surfaces. The proposed method combines the steps of electropolymerization and detection into a single device to offer a highly convenient method for the end-user. In comparison, the sensitivity toward Cys detection is a significantly increased using this modified electrode. The developed device provided a linear concentration range of 10-800 μM with a limit of detection of 5.5 μM. For application, the device was successfully applied to detect Cys in different food products such as wheat flour, bread, and cake with satisfactory results, yielding excellent intra-day and inter-day relative standard deviations (1.5-4.9%) and recoveries (84.2-110.8%). This discovery is important from the viewpoint of the development of Cys detection in other applications in the future., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Motivation: Nutrient and contaminant behavior in the subsurface are governed by multiple coupled hydrobiogeochemical processes which occur across different temporal and spatial scales. Accurate description of macroscopic system behavior requires accounting for the effects of microscopic and especially microbial processes. Microbial processes mediate precipitation and dissolution and change aqueous geochemistry, all of which impacts macroscopic system behavior. As 'omics data describing microbial processes is increasingly affordable and available, novel methods for using this data quickly and effectively for improved ecosystem models are needed., Results: We propose a workflow ('Omics to Reactive Transport-ORT) for utilizing metagenomic and environmental data to describe the effect of microbiological processes in macroscopic reactive transport models. This workflow utilizes and couples two open-source software packages: KBase (a software platform for systems biology) and PFLOTRAN (a reactive transport modeling code). We describe the architecture of ORT and demonstrate an implementation using metagenomic and geochemical data from a river system. Our demonstration uses microbiological drivers of nitrification and denitrification to predict nitrogen cycling patterns which agree with those provided with generalized stoichiometries. While our example uses data from a single measurement, our workflow can be applied to spatiotemporal metagenomic datasets to allow for iterative coupling between KBase and PFLOTRAN., Availability and Implementation: Interactive models available at https://pflotranmodeling.paf.subsurfaceinsights.com/pflotran-simple-model/. Microbiological data available at NCBI via BioProject ID PRJNA576070. ORT Python code available at https://github.com/subsurfaceinsights/ort-kbase-to-pflotran. KBase narrative available at https://narrative.kbase.us/narrative/71260 or static narrative (no login required) at https://kbase.us/n/71260/258., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The importance of aromaticity vs. hydrophobicity of the central hydrophobic core (CHC, residues 17-20) in governing fibril formation in Aβ(1-42) has been the focus of an ongoing debate in the literature., Introduction: Mutations in the CHC (especially at Phe19 and Phe20) have been used to examine the relative impact of hydrophobicity and aromaticity on the degree of aggregation of Aβ(1-42). However, the results have not been conclusive., Methods: Partial least squares (PLS) modeling of aggregation rates, using reduced properties of a series of position 19 mutants, was employed to identify the physicochemical properties that had the greatest impact on the extent of aggregation., Results: The PLS models indicate that hydrophobicity at position 19 of Aβ(1-42) appears to be the primary and dominant factor in controlling Aβ(1-42) aggregation, with aromaticity having little effect., Conclusion: This study illustrates the value of using reduced properties of amino acids in conjunction with PLS modeling to investigate mutational effects in peptides and proteins, as the reduced properties can capture in a quantitative manner the different physicochemical properties of the amino acid side chains. In this particular study, hydrophobicity at position 19 was determined to be the dominant property controlling aggregation, while size, charge, and aromaticity had little impact., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Spontaneous reactions between metabolites are often neglected in favor of emphasizing enzyme-catalyzed chemistry because spontaneous reaction rates are assumed to be insignificant under physiological conditions. However, synthetic biology and engineering efforts can raise natural metabolites' levels or introduce unnatural ones, so that previously innocuous or nonexistent spontaneous reactions become an issue. Problems arise when spontaneous reaction rates exceed the capacity of a platform organism to dispose of toxic or chemically active reaction products. While various reliable sources list competing or toxic enzymatic pathways' side-reactions, no corresponding compilation of spontaneous side-reactions exists, nor is it possible to predict their occurrence. We addressed this deficiency by creating the Chemical Damage (CD)-MINE resource. First, we used literature data to construct a comprehensive database of metabolite reactions that occur spontaneously in physiological conditions. We then leveraged this data to construct 148 reaction rules describing the known spontaneous chemistry in a substrate-generic way. We applied these rules to all compounds in the ModelSEED database, predicting 180,891 spontaneous reactions. The resulting (CD)-MINE is available at https://minedatabase.mcs.anl.gov/cdmine/#/home and through developer tools. We also demonstrate how damage-prone intermediates and end products are widely distributed among metabolic pathways, and how predicting spontaneous chemical damage helps rationalize toxicity and carbon loss using examples from published pathways to commercial products. We explain how analyzing damage-prone areas in metabolism helps design effective engineering strategies. Finally, we use the CD-MINE toolset to predict the formation of the novel damage product N-carbamoyl proline, and present mass spectrometric evidence for its presence in Escherichia coli., (Copyright © 2022 International Metabolic Engineering Society. All rights reserved.)

Huanglongbing (HLB), or citrus greening disease, has significantly decreased citrus production all over the world. The disease management currently depends on the efficient application and adequate distribution of insecticides to reduce the density of the disease vector, the Asian citrus psyllid. Here, we use a novel fluorescent-based method to evaluate insecticide distribution in an HLB-infected citrus grove in Florida. Specifically, we evaluated six different locations within citrus trees, the top and bottom sides of leaves, the effect of application approach (tractor versus airplane), and different application rates. We found that despite the insecticide distribution being highly variable among the different locations within a tree, the top of the leaves received an average increase of 21 times more than the bottom of the leaves. Application by tractor also resulted in a 4- to 87-fold increase in insecticide coverage compared with aerial application, depending on the location in the tree and side of the leaf. When taken to context with the type of insecticide that is applied (systemic vs. contact), these results can be used to optimize a pest management strategy to effectively target psyllids and other pests while minimizing the time and money spent on insecticide application and reducing risk to the environment.

The DOE Systems Biology Knowledgebase (KBase) platform offers a range of powerful tools for the reconstruction, refinement, and analysis of genome-scale metabolic models built from microbial isolate genomes. In this chapter, we describe and demonstrate these tools in action with an analysis of isoprene production in the Bacillus subtilis DSM genome. Two different methods are applied to build initial metabolic models for the DSM genome, then the models are gapfilled in three different growth conditions. Next, flux balance analysis (FBA) and flux variability analysis (FVA) techniques are applied to both study the growth of these models in minimal media and classify reactions within each model based on essentiality and functionality. The models are applied with the FBA method to predict essential genes, which are then compared to an updated list of essential genes obtained for B. subtilis 168, a very similar strain to the DSM isolate. The models are also applied to simulate Biolog growth conditions, and these results are compared with Biolog data collected for B. subtilis 168. Finally, the DSM metabolic models are applied to explore the pathways and genes responsible for producing isoprene in this strain. These studies demonstrate the accuracy and utility of models generated from the KBase pipelines, as well as exploring the tools available for analyzing these models., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

A method for lead (Pb) detection in soil is presented. Pb is a dangerous environmental pollutant that is present in soils, posing a health risk to millions of people worldwide, and regular monitoring of Pb contamination in soils is essential to public health. Many sensitive methods for detection of heavy metals in solid matrices exist, but they cannot be performed on-site because they are costly (>$30 per sample), require trained personnel, and many classical sample preparation methods are not safe to bring into the field. We describe an alternative process, combining a safer sample preparation method with electrochemical analysis. The process requires minimal training, making it an attractive overall method for regular environmental screening of Pb in soils. Extract obtained from the soil is pH adjusted and analyzed using a stencil-printed carbon electrode and square wave anodic stripping voltammetry. In this work, a study of 15 neighborhood soils examining the concentration of Pb present post-extraction was performed to demonstrate the method. The limit of detection for the electrochemical analysis was calculated to be 16 ppb-well below the United States Environmental Protection Agency's action limit for Pb in soils (400 mg kg -1 or 4000 ppb)-and third party inductively coupled plasma-optical emission spectroscopy analysis validated the results obtained in this study to within ±17% on average.

While asymmetrical flow field-flow fractionation (AF4) has been widely used for separation of high molecular weight species and even particles, its ability to resolve lower molecular weight species has rarely been explored. Over the course of many projects, we have discovered that AF4 can be an effective analytical method for separating peptides from oligomers and higher molecular weight aggregates. The methodology can be used even for peptides as small as 2 kD in molecular weight. Using multi-angle laser light scattering (MALLS) detection, accurate masses of the parent peptide can be obtained, provided accurate extinction coefficients are provided. It was shown that AF4 can be stability-indicating, suggesting that AF4-MALLS may be a suitable alternative to the use of SEC to monitor the aggregation of peptides., (Copyright © 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Enzymes have in vivo life spans. Analysis of life spans, i.e., lifetime totals of catalytic turnovers, suggests that nonsurvivable collateral chemical damage from the very reactions that enzymes catalyze is a common but underdiagnosed cause of enzyme death. Analysis also implies that many enzymes are moderately deficient in that their active-site regions are not naturally as hardened against such collateral damage as they could be, leaving room for improvement by rational design or directed evolution. Enzyme life span might also be improved by engineering systems that repair otherwise fatal active-site damage, of which a handful are known and more are inferred to exist. Unfortunately, the data needed to design and execute such improvements are lacking: there are too few measurements of in vivo life span, and existing information about the extent, nature, and mechanisms of active-site damage and repair during normal enzyme operation is too scarce, anecdotal, and speculative to act on. Fortunately, advances in proteomics, metabolomics, cheminformatics, comparative genomics, and structural biochemistry now empower a systematic, data-driven approach for identifying, predicting, and validating instances of active-site damage and its repair. These capabilities would be practically useful in enzyme redesign and improvement of in-use stability and could change our thinking about which enzymes die young in vivo , and why.

Rapid and inexpensive serological tests for SARS-CoV-2 antibodies are needed to conduct population-level seroprevalence surveillance studies and can improve diagnostic reliability when used in combination with viral tests. Here, we report a novel low-cost electrochemical capillary-flow device to quantify IgG antibodies targeting SARS-CoV-2 nucleocapsid proteins (anti-N antibody) down to 5 ng/mL in low-volume (10 μL) human whole blood samples in under 20 min. No sample preparation is needed as the device integrates a blood-filtration membrane for on-board plasma extraction. The device is made of stacked layers of a hydrophilic polyester and double-sided adhesive films, which create a passive microfluidic circuit that automates the steps of an enzyme-linked immunosorbent assay (ELISA). The sample and reagents are sequentially delivered to a nitrocellulose membrane that is modified with a recombinant SARS-CoV-2 nucleocapsid protein. When present in the sample, anti-N antibodies are captured on the nitrocellulose membrane and detected via chronoamperometry performed on a screen-printed carbon electrode. As a result of this quantitative electrochemical readout, no result interpretation is required, making the device ideal for point-of-care (POC) use by non-trained users. Moreover, we show that the device can be coupled to a near-field communication potentiostat operated from a smartphone, confirming its true POC potential. The novelty of this work resides in the integration of sensitive electrochemical detection with capillary-flow immunoassay, providing accuracy at the point of care. This novel electrochemical capillary-flow device has the potential to aid the diagnosis of infectious diseases at the point of care.

For electrochemical immunosensors, inexpensive electrodes with fast redox kinetics, and simple stable methods of electrode functionalization are vital. However, many inexpensive and easy to fabricate electrodes suffer from poor redox kinetics, and functionalization can often be difficult and/or unstable. Diazonium tosylates are particularly stable soluble salts that can be useful for electrode functionalization. Recently developed thermoplastic electrodes (TPEs) have been inexpensive, moldable, and highly electroactive carbon composite materials. Herein, the synthesis and grafting of diazonium tosylate salts were optimized for modification of TPEs and used to develop the first TPE immunosensors. With diazonium tosylates, TPEs were amine functionalized either directly through grafting of p -aminophenyl diazonium salt or indirectly through grafting p -nitrophenyl diazonium salt followed by electrochemical reduction to an amine. Diazonium tosylates were synthesized in situ as a paste in 6 min. Once the reaction paste was spread over the electrodes, near monolayer coverage (1.0 ± 0.2 nmol cm -2 ) was achieved for p -nitrophenyl diazonium salt within 5 min. Amine functionalized electrodes were conjugated to C-reactive protein (CRP) antibodies. Antibody-modified TPEs were applied for the sensitive detection of CRP, a biomarker of cardiovascular disease using electrochemical enzyme-linked immunosorbent assays (ELISA). LODs were determined to be 2 ng mL -1 in buffer, with high selectivity against interfering species for both functionalization methods. The direct p -aminophenyl modification method had the highest sensitivity to CRP and was further tested in spiked serum with an LOD of 10 ng mL -1 . This low-cost and robust TPE immunosensor platform can be easily adapted for other analytes and multiplexed detection.

The importance of user-friendly, inexpensive, sensitive, and selective detection of viruses has been highlighted again due to the recent Coronavirus disease 2019 (COVID-19) pandemic. Among the analytical tools, paper-based devices (PADs) have become a leading alternative for point-of-care (POC) testing. In this review, we discuss the recent development strategies and applications in nucleic acid-based, antibody/antigen-based and other affinity-based PADs using optical and electrochemical detection methods for sensing viruses. In addition, advantages and drawbacks of presented PADs are identified. Current state and insights towards future perspectives are presented regarding developing POC diagnosis platform for COVID-19. This review considers state-of-the-art technologies for further development and improvement in PADs performance for virus detection., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Elsevier B.V. All rights reserved.)

Microfluidic paper-based analytical devices (μPADs) have garnered significant interest as a promising analytical platform in the past decade. Compared with traditional microfluidics, μPADs present unique advantages, such as easy fabrication using established patterning methods, economical cost, ability to drive and manipulate flow without equipment, and capability of storing reagents for various applications. This Review aims to provide a comprehensive review of the field, highlighting fabrication methods available to date with their respective advantages and drawbacks, device designs and modifications to accommodate different assay needs, detection strategies, and the growing applications of μPADs. Finally, we discuss how the field needs to continue moving forward to realize its full potential.

The World Health Organization (WHO) calls for the development of a rapid, biomarker-based, non-sputum test capable of detecting all forms of tuberculosis (TB) at the point-of-care to enable immediate treatment initiation. Lipoarabinomannan (LAM) is the only WHO-endorsed TB biomarker that can be detected in urine, an easily collected sample matrix. For obtaining optimal sensitivity, we and others have shown that some form of sample pretreatment is necessary to remove background from patient urine samples. A number of systems are paper-based often destined for resource limited settings. Our current work presents incorporation of one such sample pretreatment, proteinase K (ProK) immobilized on paper (IPK) and test its performance in comparison to standard proteinase K (SPK) treatment that involves addition and deactivation at high temperature prior to performing a capture ELISA. Herein, a simple and economical method was developed for using ProK immobilized strips to pretreat urine samples. Simplification and cost reduction of the proposed pretreatment strip were achieved by using Whatman no.1 paper and by minimizing the concentration of ProK (an expensive but necessary reagent) used to pretreat the clinical samples prior to ELISA. To test the applicability of IPK, capture ELISA was carried out on either LAM-spiked urine or the clinical samples after pretreatment with ProK at 400 μg/mL for 30 minutes at room temperature. The optimal conditions and stability of the IPK were tested and validation was performed on a set of 25 previously analyzed archived clinical urine samples with known TB and HIV status. The results of IPK and SPK treated samples were in agreement showing that the urine LAM test currently under development has the potential to reach adult and pediatric patients regardless of HIV status or site of infection, and to facilitate global TB control to improve assay performance and ultimately treatment outcomes., Competing Interests: The authors have declared that no competing interests exist.

α 1 -Acid glycoprotein (AGP) is a glycoprotein present in serum, which is associated with the modulation of the immune system in response to stress or injuries, and a biomarker for inflammatory diseases and cancers. Here, we propose a pump-free microfluidic device for the electrochemical determination of AGP. The microfluidic device utilizes capillary-driven flow and a passive mixing system to label the AGP with the Os (VI) complex (an electrochemical tag) inside the main channel, before delivering the products to the electrode surface. Furthermore, thanks to the resulting geometry, all the analytical steps can be carried out inside the device: labeling, washing, and detection by adsorptive transfer stripping square wave voltammetry. The microfluidic device exhibited a linear range from 500 to 2000 mg L -1 ( R 2 = 0.990) and adequate limit of detection (LOD = 231 mg L -1 ). Commercial serum samples were analyzed to demonstrate the success of the method, yielding recoveries around 83%. Due to its simplicity, low sample consumption, low cost, short analysis time, disposability, and portability, the proposed method can serve as a point-of-care/need testing device for AGP.

The albumin-to-alkaline phosphatase ratio (AAPR) has been a cancer prognostic indicator. This paper presents the concept of a dual-color change distance-based paper device (dPAD) for albumin (Alb) and alkaline phosphatase (ALP) detection to evaluate this cancer prognostic index. Whereas Alb interacts with the bromocresol green (BCG) indicator to form a bluish-green complex, ALP hydrolyzes l-ascorbic acid-2-phosphate (AAP) to produce ascorbic acid (AA), which reacts with KIO 3 to generate I 2 and I - . I 2 /I - reacts with silver hexagonal nanoprisms (purple color) in the presence of Cu 2+ , resulting in a color change from purple to colorless. The distance of the color change from yellow to the bluish-green and purple to colorless correlates to Alb and ALP concentration, respectively. The angle index for the AAPR is then defined by drawing a straight line that connects the tops of the two changed band lengths in the detection area. The highest bluish-green color band length on the Alb region is the midpoint, which is the position set of the protractor at 0°, and the angle is measured using a simple protractor. The results indicate that an AAPR below 0.57 will have an angle greater than 40° and correlates with a risk factor for lung cancer. The naked-eye detection limits for Alb and ALP were found to be 0.8 g/L and 5 U/L ( n = 10), respectively. The practical application of the developed dPAD was successfully demonstrated by Alb and ALP analysis in human serum and validated against standard methods. The proposed method does not require incubation conditions for the ALP assay, which strongly reduces the overall analysis steps and time. Moreover, our device provides a low-cost, simple, sensitive, selective, accurate, and precise determination of the AAPR.

Per- and polyfluoroalkyl substances (PFAS) are a class of compounds that have become environmental contaminants of emerging concern. They are highly persistent, toxic, bioaccumulative, and ubiquitous which makes them important to detect to ensure environmental and human health. Multiple instrument-based methods exist for sensitive and selective detection of PFAS in a variety of matrices, but these methods suffer from expensive costs and the need for a laboratory and highly trained personnel. There is a big need for fast, inexpensive, robust, and portable methods to detect PFAS in the field. This would allow environmental laboratories and other agencies to perform more frequent testing to comply with regulations. In addition, the general public would benefit from a fast method to evaluate the drinking water in their homes for PFAS contamination. A PFAS sensor would provide almost real-time data on PFAS concentrations that can also provide actionable information for water quality managers and consumers around the planet. In this review, we discuss the sensors that have been developed up to this point for PFAS detection by their molecular detection mechanism as well as the goals that should be considered during sensor development. Future research needs and commercialization challenges are also highlighted., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Sensitive, reliable and cost-effective detection of pathogens has wide ranging applications in clinical diagnostics and therapeutics, water and food safety, environmental monitoring, biosafety and epidemiology. Nucleic acid amplification tests (NAATs) such as PCR and isothermal amplification methods provide excellent analytical performance and significantly faster turnaround times than conventional culture-based methods. However, the inherent cost and complexity of NAATs limit their application in resource-limited settings and the developing world. To help address this urgent need, we have developed a sensitive method for nucleic acid analysis based on padlock probe rolling circle amplification (PLRCA), nuclease protection (NP) and lateral flow detection (LFA), referred to as PLAN-LFA, that can be used in resource-limited settings. The assay involves solution-phase hybridization of a padlock probe to target, sequence-specific ligation of the probe to form a circular template that undergoes isothermal rolling circle amplification in the presence of a polymerase and a labeled probe DNA. The RCA product is a long, linear concatenated single-stranded DNA that contains binding sites for the labeled probe. The sample is then exposed to a nuclease which selectively cleaves single-stranded DNA, the double-stranded labeled probe is protected from nuclease digestion and detected in a lateral flow immunoassay format to provide a visual, colorimetric readout of results. We have developed specific assays targeting beta-lactamase resistance gene for monitoring of antimicrobial resistance and Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2, the novel coronavirus discovered in 2019) using the PLAN-LFA platform. The assay provides a limit of detection of 1.1 pM target DNA (or 1.3 × 106 copies per reaction). We also demonstrate the versatility and robustness of the method by performing analysis on DNA and RNA targets, and perform analysis in complex sample matrices like saliva, plant tissue extract and bacterial culture without any sample pretreatment steps.

Heavy metals are the main pollutants present in aquatic environments and their presence in human organisms can lead to many different diseases. While many methods exist for analysis, colorimetric and electrochemistry are particularly attractive for on-site analysis and their integration on a single platform can improve multiplexed metals analysis. This report describes for the first time a "plug-and-play" (PnP) assembly for coupling a microfluidic paper-based device (μPAD) and a screen-printed electrochemical paper-based device (ePAD) using a vertical and reversible foldable mechanism for multiplexed detection of Fe, Ni, Cu, Zn, Cd and Pb in river water samples. The integration strategy was based on a reversible assembly, allowing the insertion of a pretreatment zone to minimize potential chemical interfering agents and providing a better control of the aspirated sample volume as well as to a lower sample evaporation rate. In comparison with lateral flow and electrochemical assays performed using independent devices, the integrated prototype proved that the reversible coupling mechanism does not interfere on the analytical performance (95% confidence interval). The limit of detection (LOD) values calculated for metals determined varied from 0.1 to 0.3 mg L-1 (colorimetric) and from 0.9 to 10.5 μg L-1 (electrochemical). When compared to other integrated devices based on horizontal designs, the use of a foldable coupling mechanism offered linear response in a lower concentration range and better LOD values for Fe, Ni and Cu. The proposed method successfully measured heavy metals in river water samples with concentrations ranging from 16 to 786 μg L-1, with recovery studies ranging from 76 to 121%. The new method also showed good correlation with conventional atomic absorption spectroscopic methods (95% significance level). Thus, the integration of μPADs and ePADs by a vertical folding mechanism was efficient for multiplexed heavy metal analysis and could be exploited for environmental monitoring.

Environmental factors, mucosal permeability and defective immunoregulation drive overactive immunity to a subset of resident intestinal bacteria that mediate multiple inflammatory conditions. GUT-103 and GUT-108, live biotherapeutic products rationally designed to complement missing or underrepresented functions in the dysbiotic microbiome of IBD patients, address upstream targets, rather than targeting a single cytokine to block downstream inflammation responses. GUT-103, composed of 17 strains that synergistically provide protective and sustained engraftment in the IBD inflammatory environment, prevented and treated chronic immune-mediated colitis. Therapeutic application of GUT-108 reversed established colitis in a humanized chronic T cell-mediated mouse model. It decreased pathobionts while expanding resident protective bacteria; produced metabolites promoting mucosal healing and immunoregulatory responses; decreased inflammatory cytokines and Th-1 and Th-17 cells; and induced interleukin-10-producing colonic regulatory cells, and IL-10-independent homeostatic pathways. We propose GUT-108 for treating and preventing relapse for IBD and other inflammatory conditions characterized by unbalanced microbiota and mucosal permeability.

A new paper-based analytical device design was fabricated by a wax printing method for simultaneous determination of Cu(ii), Co(ii), Ni(ii), Hg(ii), and Mn(ii). Colorimetry was used to quantify these heavy metal ions using bathocuproine (Bc), dimethylglyoxime (DMG), dithizone (DTZ), and 4-(2-pyridylazo) resorcinol (PAR) as complexing agents. The affinity of complexing agents to heavy metal ions is dependent on the formation constant (K f ). To enhance the selectivity for heavy metal ion determination, the new device was designed with two pretreatment zones, where masking agents remove the interfering ions. It was found that two pretreatment zones worked better than a single pretreatment zone at removing interferences. The reaction time, sample and complexing agent volumes, and complexing agent concentrations were optimized. The analytical results were achieved with the lowest detectable concentrations of 0.32, 0.59, 5.87, 0.20, and 0.11 mg L -1 for Cu(ii), Co(ii), Ni(ii), Hg(ii), and Mn(ii), respectively. The linear ranges were found to be 0.32-63.55 mg L -1 (Cu(ii)), 0.59-4.71 mg L -1 (Co(ii)), 5.87-352.16 mg L -1 (Ni(ii)), 0.20-12.04 mg L -1 (Hg(ii)), and 0.11-0.55 mg L -1 (Mn(ii)). The lowest detectable concentration and linearity for the five metal ions allow the application of this device for the determination of heavy metal ions in various water samples. The sensor showed high selectivity and efficiency for simultaneous determination of Cu(ii), Co(ii), Ni(ii), Hg(ii), and Mn(ii) in drinking, tap, and pond water samples on a single device and detection with the naked eye. The results illustrated that the proposed sensor showed good accuracy and precision agreement with the standard ICP-OES method.

The reconstruction of bacterial and archaeal genomes from shotgun metagenomes has enabled insights into the ecology and evolution of environmental and host-associated microbiomes. Here we applied this approach to >10,000 metagenomes collected from diverse habitats covering all of Earth's continents and oceans, including metagenomes from human and animal hosts, engineered environments, and natural and agricultural soils, to capture extant microbial, metabolic and functional potential. This comprehensive catalog includes 52,515 metagenome-assembled genomes representing 12,556 novel candidate species-level operational taxonomic units spanning 135 phyla. The catalog expands the known phylogenetic diversity of bacteria and archaea by 44% and is broadly available for streamlined comparative analyses, interactive exploration, metabolic modeling and bulk download. We demonstrate the utility of this collection for understanding secondary-metabolite biosynthetic potential and for resolving thousands of new host linkages to uncultivated viruses. This resource underscores the value of genome-centric approaches for revealing genomic properties of uncultivated microorganisms that affect ecosystem processes.

Metabolic engineering uses enzymes as parts to build biosystems for specified tasks. Although a part's working life and failure modes are key engineering performance indicators, this is not yet so in metabolic engineering because it is not known how long enzymes remain functional in vivo or whether cumulative deterioration (wear-out), sudden random failure, or other causes drive replacement. Consequently, enzymes cannot be engineered to extend life and cut the high energy costs of replacement. Guided by catalyst engineering, we adopted catalytic cycles until replacement (CCR) as a metric for enzyme functional life span in vivo. CCR is the number of catalytic cycles that an enzyme mediates in vivo before failure or replacement, i.e., metabolic flux rate/protein turnover rate. We used estimated fluxes and measured protein turnover rates to calculate CCRs for ∼100-200 enzymes each from Lactococcus lactis , yeast, and Arabidopsis CCRs in these organisms had similar ranges (<10 3 to >10 7 ) but different median values (3-4 × 10 4 in L. lactis and yeast versus 4 × 10 5 in Arabidopsis ). In all organisms, enzymes whose substrates, products, or mechanisms can attack reactive amino acid residues had significantly lower median CCR values than other enzymes. Taken with literature on mechanism-based inactivation, the latter finding supports the proposal that 1) random active-site damage by reaction chemistry is an important cause of enzyme failure, and 2) reactive noncatalytic residues in the active-site region are likely contributors to damage susceptibility. Enzyme engineering to raise CCRs and lower replacement costs may thus be both beneficial and feasible., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Thermodynamics plays a crucial role in regulating the metabolic processes in all living organisms. Accurate determination of biochemical and biophysical properties is important to understand, analyze, and synthetically design such metabolic processes for engineered systems. In this work, we extensively performed first-principles quantum mechanical calculations to assess its accuracy in estimating free energy of biochemical reactions and developed automated quantum-chemistry (QC) pipeline (https://appdev.kbase.us/narrative/45710) for the prediction of thermodynamics parameters of biochemical reactions. We benchmark the QC methods based on density functional theory (DFT) against different basis sets, solvation models, pH, and exchange-correlation functionals using the known thermodynamic properties from the NIST database. Our results show that QC calculations when combined with simple calibration yield a mean absolute error in the range of 1.60-2.27 kcal/mol for different exchange-correlation functionals, which is comparable to the error in the experimental measurements. This accuracy over a diverse set of metabolic reactions is unprecedented and near the benchmark chemical accuracy of 1 kcal/mol that is usually desired from DFT calculations., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Capillary-driven microfluidic devices are of significant interest for on-site analysis because they do not require external pumps and can be made from inexpensive materials. Among capillary-driven devices, those made from paper and polyester film are among the most common and have been used in a wide array of applications. However, since capillary forces are the only driving force, flow is difficult to control, and passive flow control methods such as changing the geometry must be used to accomplish various analytical applications. This study presents several new flow control methods that can be utilized in a laminate capillary-driven microfluidic device to increase available functionality. First, we introduce push and burst valve systems that can stop and start flow. These valves can stop flow for >30 min and be opened by either pressing the channel or inflowing other fluids to the valve region. Next, we propose flow control methods for Y-shaped channels that enable more functions. In one example, we demonstrate the ability to accurately control concentration to create laminar, gradient, and fully mixed flows. In a second example, flow velocity in the main channel is controlled by adjusting the length of the inlet channel. In addition, the flow velocity is constant as the inlet length increases. Finally, the flow velocity in the Y-shaped device as a function of channel height and fluid properties such as viscosity and surface tension was examined. As in previous studies on capillary-driven channels, the flow rate was affected by each parameter. The fluidic control tools presented here will enable new designs and functions for low cost point of need assays across a variety of fields.

This study reports a new electrochemical method for tryptamine determination using a paper-based microfluidic device and a thermoplastic electrode (TPE) as an amperometric detector. Tryptamine (Tryp) is a biogenic amine present in drinks and foods. Even though this compound has some beneficial effects on human health, the ingestion of foods with high concentrations of Tryp may be detrimental, which justifies the need for monitoring the Tryp levels. The TPEs were made from 50% carbon black and 50% polycaprolactone and characterized by cyclic voltammetry, demonstrating enhancement in the analytical response compared to other carbon composites. TPEs also showed a better antifouling effect for Tryp compared to conventional glassy carbon electrodes. Once characterized, the electrodes were incorporated into the microfluidic device to determine Tryp in water and cheese samples using amperometry. A linear range was achieved from 10 to 75 μmol L -1 with limits of detection and quantification of 3.2 and 10.5 μmol L -1 , respectively. Therefore, this work shows promising findings of the electrochemical determination of Tryp, bringing valuable results regarding the electrochemical properties of thermoplastic composites., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Thermoplastic electrodes (TPEs) are carbon composite electrodes consisting of graphite and thermoplastic polymer binder. TPE production is a solvent-based method, which makes it easy to fabricate and pattern into complex geometries, contrary to classical carbon composite electrodes. Depending on the composition (carbon type, binder, and composition ratio), TPEs can give excellent electrochemical performance and high conductivity. However, these TPEs are relatively new electrode materials, and thorough electrochemical characterization is still missing to understand and predict why large differences between TPEs exist. We used scanning electrochemical microscopy (SECM) as a screening tool to characterize TPEs. SECM data treatment based on scanning probe microscopy imaging allows a fast and easy comparison of the numerous images, as well as the optimization of the preparation. Experiments suggest that TPEs behave as a network of interacting microelectrodes made by electrochemically active islands isolated between less active areas. Higher carbon content in TPEs is not always indicative of more uniform electrodes with better electrochemical performances. Using various SECM redox probes, it is possible to select a specific graphite or polymer type for the analyte of interest. For example, TPEs made with COC:3569 are the best compromise for general detection, whereas PMMA:11 μm is better suited for catechol-like polyphenol analysis.

Stencil-printing conductive carbon inks has revolutionized the development of inexpensive, disposable and portable electrochemical sensors. However, stencil-printed carbon electrodes (SPCEs) typically suffer from poor electrochemical properties. While many surface pretreatments and modifications have been tested to improve the electrochemical activity of SPCEs, the bulk composition of the inks used for printing has been largely ignored. Recent studies of other carbon composite electrode materials show significant evidence that the conductive carbon particle component is strongly related to electrochemical performance. However, such a study has not been carried out with SPCEs. In this work, we perform a systematic characterization of SPCEs made with different carbon particle types including graphite particles, glassy carbon microparticles and carbon black. The relationship between carbon particle characteristics including particle size, particle purity, and particle morphology as well as particle mass loading on the fabrication and electrochemical properties of SPCEs is studied. SPCEs were plasma treated for surface activation and the electrochemical properties of both untreated and plasma treated SPCEs are also compared. SPCEs displayed distinct analytical utilities characterized through solvent window and double layer capacitance. Cyclic voltammetry (CV) of several standard redox probes, FcTMA + , ferri/ferrocyanide, and pAP was used to establish the effects of carbon particle type and plasma treatment on electron transfer kinetics of SPCEs. CV of the biologically relevant molecules uric acid, NADH and dopamine was employed to further illustrate the differences in sensing and fouling characteristics of SPCEs fabricated with different carbon particle types. SEM imaging revealed significant differences in the SPCE surface microstructures. This systematic study demonstrates that the electrochemical properties of SPCEs can be tuned and significantly improved through careful selection of carbon particle type and plasma cleaning with a goal toward the development of better performing electrochemical point-of-need sensors., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A paper-based method for heating preconcentration (PAD-HP) has been developed for the determination of Pb 2+ , Cd 2+ , Fe 3+ , and Ni 2+ . The design of our heating system was evaluated for dual quantification of ions using electrochemical and colorimetric methods simultaneously. The PAD-HP was used to detect Pb 2+ and Cd 2+ by anodic stripping voltammetry and to detect Fe 3+ and Ni 2+ by colorimetric reactions. Assay conditions were optimized by evaluating performance when changing the concentration of the colorimetric reagent, eluent volume, electrolyte concentration, and electrochemical parameters. Limits of detection (LOD) were determined to be 0.97 and 2.33 μg L -1 for Pb 2+ and Cd 2+ (via voltammetry) and 0.03 and 0.04 mg L -1 for Fe 3+ and Ni 2+ (via colorimetric assay), respectively. The relative standard deviations for assays were in the range of 5.76 to 10.12%. We observed that the PAD-HP method significantly enhanced the signal of all metals ions (14-100-fold, depending on the metal) in comparison to paper-based devices that did not use a heating preconcentration system. This PAD-HP method was successfully applied to the determination of metals ions in samples of drinking water, tap water, pond water, and wastewater. These results suggest that our approach can provide a convenient strategy to monitor aqueous samples for heavy metals with high sensitivity and selectivity.

Crop disease management depends on efficient and adequate pesticide distribution to reduce pest population. Instrument-based methods to evaluate the spatial distribution of pesticides are available, but they are not field-compatible because of instrument size, cost, and extensive sample preparation. The current gold standard of water-sensitive papers is field-compatible; however, these papers often produce false positives due to reaction with water from sources other than the pesticide mixture. Thus, we developed a novel method in which a fluorescent dye is sprayed over a crop with circles of filter paper (samplers) attached to the leaves. After collection, a lightbox is used to take pictures of the samplers, and an algorithm analyzes each image for percent coverage to visualize the pesticide distribution within the crop. Our method produces results quickly and inexpensively compared to current methods and can be applied to any crop to inform best pesticide application strategies.

Microfluidic magnetophoresis is a powerful technique that is used to separate and/or isolate cells of interest from complex matrices for analysis. However, mechanical pumps are required to drive flow, limiting portability and making translation to point-of-care (POC) settings difficult. Microfluidic paper-based analytical devices (μPADs) offer an alternative to traditional microfluidic devices that do not require external pumps to generate flow. However, μPADs are not typically used for particle analysis because most particles become trapped in the porous fiber network. Here we report the ability of newly developed fast-flow microfluidic paper-based analytical devices (ffPADs) to perform magnetophoresis. ffPADs use capillary action in a gap between stacked layers of paper and transparency sheets to drive flow at higher velocities than traditional μPADs. The multi-layer ffPADs allow particles and cells to move through the gap without being trapped in the paper layers. We first demonstrate that ffPADs enable magnetic particle separations in a μPAD with a neodymium permanent magnet and study key factors that affect performance. To demonstrate utility, E. coli was used as a model analyte and was isolated from human urine before detection with a fluorescently labeled antibody. A capture efficiency of 61.5% was then obtained of E. coli labeled magnetic beads in human urine. Future studies will look at the improvement of the capture efficiency and to make this assay completely off-chip without the need of a fluorescent label. The assay and device described here demonstrate the first example of magnetophoresis in a paper based, pump free microfluidic device.

Human exposure to air pollution is associated with increased risk of morbidity and mortality. However, personal air pollution exposures can vary substantially depending on an individual's daily activity patterns and air quality within their residence and workplace. This work developed and validated an adaptive buffer size (ABS) algorithm capable of dynamically classifying an individual's time spent in predefined microenvironments using data from global positioning systems (GPS), motion sensors, temperature sensors, and light sensors. Twenty-two participants in Fort Collins, CO were recruited to carry a personal air sampler for a 48-h period. The personal sampler was retrofitted with a GPS and a pushbutton to complement the existing sensor measurements (temperature, motion, light). The pushbutton was used in conjunction with a traditional time-activity diary to note when the participant was located at "home", "work", or within an "other" microenvironment. The ABS algorithm predicted the amount of time spent in each microenvironment with a median accuracy of 99.1%, 98.9%, and 97.5% for the "home", "work", and "other" microenvironments. The ability to classify microenvironments dynamically in real time can enable the development of new sampling and measurement technologies that classify personal exposure by microenvironment.

We present here the draft genome sequence of a pyridine-degrading bacterium, Micrococcus luteus ATCC 49442, which was reclassified as Pseudarthrobacter sp. strain ATCC 49442 based on its draft genome sequence. Its genome length is 4.98 Mbp, with 64.81% GC content., (Copyright © 2020 Gupta et al.)

It has recently been determined that Pseudomallada prasinus (Burmeister, 1839) is part of a complex of prasinoid species (Duelli Obrist, 2019). Apart from P. prasinus s.s. and P. abdominalis s.s., each at the opposite end of the prasinoid spectrum, some populations in Switzerland and surrounding countries show consistently differing morphological and biological traits, while nevertheless living in the same habitats. To test whether they constitute reproductively isolated species, seven strains of three morphologically distinctive prasinoid morphs (Pp1, Pp2, Pp3) were reared and the virgin offspring (F1) paired with single partners of each of the six other strains. The four strains of morph Pp3 were unable to produce any viable offspring when paired with the two other morphs, suggesting that Pp3 is a distinct species. The Mediterranean morph Pp1 produced some offspring with the two strains of morph Pp2, but the crossed offspring (F2) of the hybrids yielded less than 2% viable larvae. Furthermore, in a cage experiment with free choice of partners between Pp1 and Pp2, all females produced offspring of their own morph; there were no hybrids. We conclude that north and south of the Alps there are at least three reproductively isolated prasinoid species in addition to P. prasinus and P. abdominalis. Their distinctiveness is principally based on live color traits, but also on the presence or absence of the "furwing" phenotype in males (double number of setae on wing veins). Therefore, many old museum specimens, especially females, cannot be identified with confidence. Because most of the dozens of synonymized type specimens of prasinoid species and varieties in museums are females, and all have lost their color, it is presently not possible to assign valid names to the three "new" species. We call them Pseudomallada prasinoid 1 (Pp1), P. prasinoid 2 (Pp2), and P. prasinoid 3 (Pp3). Using the distinctive traits given here, most specimens that have retained their original colors (live, recently pinned, or deep frozen) can be identified.

Here, we report the draft genome sequence of Arthrobacter sp. strain ATCC 49987, consisting of three contigs with a total length of 4.4 Mbp. Based on the genome sequence, we suggest reclassification of Arthrobacter sp. strain ATCC 49987 as Pseudarthrobacter sp. strain ATCC 49987., (Copyright © 2020 Gupta et al.)

Major changes in the microbiome are associated with health and disease. Some microbiome states persist despite seemingly unfavorable conditions, such as the proliferation of aerobe-anaerobe communities in oxygen-exposed environments in wound infections or small intestinal bacterial overgrowth. Mechanisms underlying transitions into and persistence of these states remain unclear. Using two microbial taxa relevant to the human microbiome, we combine genome-scale mathematical modeling, bioreactor experiments, transcriptomics, and dynamical systems theory to show that multistability and hysteresis (MSH) is a mechanism describing the shift from an aerobe-dominated state to a resilient, paradoxically persistent aerobe-anaerobe state. We examine the impact of changing oxygen and nutrient regimes and identify changes in metabolism and gene expression that lead to MSH and associated multi-stable states. In such systems, conceptual causation-correlation connections break and MSH must be used for analysis. Using MSH to analyze microbiome dynamics will improve our conceptual understanding of stability of microbiome states and transitions between states., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Microfluidics has revolutionized the fields of bioanalytical chemistry, cellular biology, and molecular biology. Advancements in microfluidic technologies, however, are often limited by labor, time, and resource-intensive fabrication methods, most commonly a form of photolithography. The advent of 3D printing has helped researchers fabricate proof-of-concept microfluidics more rapidly and at lower costs but suffers from poor resolution and tedious post-processing to remove uncured resin from enclosed channels. Additionally, custom resins and printers are often needed to create entirely enclosed channels, which increases cost and complexity of fabrication. In this work we demonstrate the ability to create microfluidic devices by covalently sealing 3D-printed parts with open-faced channels to polydimethylsiloxane (PDMS). Open-faced channels are easier to print than fully enclosed channels and can be printed using an inexpensive and commercially available stereolithography 3D printer and resin. The 3D-printed parts are sealed to PDMS, a common substrate used in traditional microfluidic fabrication, using two different techniques. The first involves coating the part with a commercially available silicone spray before sealing to PDMS via plasma treatment. In the second technique, the cured methacrylate resin is silanized with (3-Aminopropyl)triethoxysilane (APTES) before binding to PDMS with plasma treatment. Both methods create a strong seal between the two substrates, which is demonstrated with several types of microfluidic devices including droplet and gradient generators., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Micromolded carbon paste electrodes are easily fabricated, disposable, and can be integrated into microfluidic devices to fabricate inexpensive sensors and biosensors. In this work, carbon paste microelectrodes were fabricated in poly(dimethylsiloxane) using micromolding techniques and were coupled to a microfluidic channel to fabricate electrogenerated chemiluminescent (ECL) sensors. ECL was generated using both the tris(2,2'-bipyridyl)ruthenium(II)-tripropylamine system and the hydrogen peroxide and luminol system. For each of these ECL systems, the sensor fabrication method was optimized, along with key experimental parameters (applied voltage, solution flow rate, buffer species and luminol concentration). The limit of detection (S/N = 3) for TPrA was ~2.4 μM with a linear range of 10-100μM. For hydrogen peroxide the LOD was ~11 μM and the electrodes gave a linear response between 30 μM and 200 μM hydrogen peroxide. Electrodes containing glucose oxidase were fabricated using this new method, demonstrating that glucose could be indirectly detected via generation of hydrogen peroxide by the enzymatic reaction at the micromolded biosensor.