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Introduction: Animals respond to various environmental cues. Animal behavior is complex, and behavior analysis can greatly help to understand brain function. Most of the available behavioral imaging setups are expensive, provide limited options for customization, and allow for behavioral imaging of one animal at a time. Methods: The current study takes advantage of adult zebrafish as a model organism to study behavior in a novel behavioral setup allowing one to concurrently image 8 adult zebrafish. Results: Our results indicate that adult zebrafish show a unique behavioral profile in response to visual stimuli such as moving lines. In the presence of moving lines, the fish spent more time exploring the tank and spent more time toward the edges of the tanks. In addition, the fish moved and oriented themselves against the direction of the moving lines, indicating a negative optomotor response (OMR). With repeated exposure to moving lines, we observed a reduced optomotor response in adult zebrafish. Discussion: Our behavioral setup is relatively inexpensive, provides flexibility in the presentation of various animated visual stimuli, and offers improved throughput for analyzing behavior in adult zebrafish. This behavioral setup shows promising potential to quantify various behavioral measures and opens new avenues to understand complex behaviors. [ABSTRACT FROM AUTHOR]

Growth traits, such as fresh weight, diameter, and leaf area, are pivotal indicators of growth status and the basis for the quality evaluation of lettuce. The time-consuming, laborious and inefficient method of manually measuring the traits of lettuce is still the mainstream. In this study, a threestage multi-branch self-correcting trait estimation network (TMSCNet) for RGB and depth images of lettuce was proposed. The TMSCNet consisted of five models, of which two master models were used to preliminarily estimate the fresh weight (FW), dry weight (DW), height (H), diameter (D), and leaf area (LA) of lettuce, and three auxiliary models realized the automatic correction of the preliminary estimation results. To compare the performance, typical convolutional neural networks (CNNs) widely adopted in botany research were used. The results showed that the estimated values of the TMSCNet fitted the measurements well, with coefficient of determination (R2) values of 0.9514, 0.9696, 0.9129, 0.8481, and 0.9495, normalized root mean square error (NRMSE) values of 15.63, 11.80, 11.40, 10.18, and 14.65% and normalized mean squared error (NMSE) value of 0.0826, which was superior to compared methods. Compared with previous studies on the estimation of lettuce traits, the performance of the TMSCNet was still better. The proposed method not only fully considered the correlation between different traits and designed a novel self-correcting structure based on this but also studied more lettuce traits than previous studies. The results indicated that the TMSCNet is an effective method to estimate the lettuce traits and will be extended to the high-throughput situation. Code is available at https://github.com/lxsfight/TMSCNet.git. [ABSTRACT FROM AUTHOR]

Soil microorganisms can form a stable dynamic system with plant root systems. The composition of the soil microorganism community is related to the growth and stress resistance of plants; in turn, soil microorganisms are also regulated by plant genotypes and root exudates. Therefore, research on how to identify microorganisms that are beneficial or harmful to plants, study the interaction between microorganisms and plants, and form stable microbial communities for better plant growth plays an important role in sustainable agriculture. It is of great significance to identify and analyze rhizosphere microorganisms and plant endophytes through high-throughput methods, especially to analyze which microorganisms are beneficial to plants, which are harmful to plants, and which are opportunistic pathogens. This review provides a theoretical basis and outlook for the utilization of beneficial microbes in sustainable agriculture.

Soil microorganisms can form a stable dynamic system with plant root systems. The composition of the soil microorganism community is related to the growth and stress resistance of plants; in turn, soil microorganisms are also regulated by plant genotypes and root exudates. Therefore, research on how to identify microorganisms that are beneficial or harmful to plants, study the interaction between microorganisms and plants, and form stable microbial communities for better plant growth plays an important role in sustainable agriculture. It is of great significance to identify and analyze rhizosphere microorganisms and plant endophytes through high-throughput methods, especially to analyze which microorganisms are beneficial to plants, which are harmful to plants, and which are opportunistic pathogens. This review provides a theoretical basis and outlook for the utilization of beneficial microbes in sustainable agriculture. [ABSTRACT FROM AUTHOR]

Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H2 and CO2 that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen Methanococcus maripaludis strain S2 and the rumen methanogen Methanobrevibacter species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against M. maripaludis as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against M. maripaludis and M. AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions. [ABSTRACT FROM AUTHOR]

Microfluidic systems offer precisely controlled experimental conditions for efficient study of mammalian cells, ranged from a single cell to matrixbased three-dimensional (3D) in vitro culture models. droplet-based microfluidic system is a robust and highly reproducible device enabling encapsulation of a cell or cells within well-confined microenvironment. Recently, such droplet-based cell culture models have drawn much attention due to their unique properties such that conventional culture systems couldn't provide. The encapsulation of cells in specifically designed aqueous phase of a microfluidic system can provide profound understand of cell to cell and cell to extracellular matrix interactions, also can be used to regulate various cell behaviors. A droplet-based cell culture system allows better control over confinement for culturing, maintaining, and analyzing cells, such as high-throughput screening. In this review, we discuss recent researches on microdroplet-based 3D cell culture models, and advanced applications of microfluidic systems. [ABSTRACT FROM AUTHOR]

Background: Volatile fatty acid intoxication (acidosis), a common process failure recorded in anaerobic reactors, leads to drastic losses in methane production. Unfortunately, little is known about the microbial mechanisms underlining acidosis and the potential to recover the process. In this study, triplicate mesophilic anaerobic reactors of 100 L were exposed to acidosis resulting from an excessive feeding with sugar beet pulp and were compared to a steadystate reactor. Results: Stable operational conditions at the beginning of the experiment initially led to similar microbial populations in the four reactors, as revealed by 16S rRNA gene T-RFLP and high-throughput amplicon sequencing. Bacteroidetes and Firmicutes were the two dominant phyla, and although they were represented by a high number of operational taxonomic units, only a few were dominant. Once the environment became deterministic (selective pressure from an increased substrate feeding), microbial populations started to diverge between the overfed reactors. Interestingly, most of bacteria and archaea showed redundant functional adaptation to the changing environmental conditions. However, the dominant Bacteroidales were resistant to high volatile fatty acids content and low pH. The severe acidosis did not eradicate archaea and a clear shift in archaeal populations from acetotrophic to hydrogenotrophic methanogenesis occurred in the overfed reactors. After 11 days of severe acidosis (pH 5.2 ± 0.4), the process was quickly recovered (restoration of the biogas production with methane content above 50 %) in the overfed reactors, by adjusting the pH to around 7 using NaOH and NaHCO3. Conclusions: In this study we show that once the replicate reactors are confronted with sub-optimal conditions, their microbial populations start to evolve differentially. Furthermore the alterations of commonly used microbial parameters to monitor the process, such as richness, evenness and diversity indices were unsuccessful to predict the process failure. At the same time, we tentatively propose the replacement of the dominant Methanosaeta sp. in this case by Methanoculleus sp., to be a potential warning indicator of acidosis. [ABSTRACT FROM AUTHOR]

ABSTRACT Grooved substrates are commonly used to guide cell alignment and produce in vitro tissues that mimic certain aspects of in vivo cellular organization. These more sophisticated tissues provide valuable in vitro models for testing drugs and for dissecting out molecular mechanisms that direct tissue organization. To increase the accessibility of these tissue models we describe a simple and yet reproducible strategy to produce 1 µm-spaced grooved well plates suitable for conducting automated analysis of cellular responses. We characterize the alignment of four human cell types: retinal epithelial cells, umbilical vein endothelial cells, foreskin fibroblasts, and human pluripotent stem-cell-derived cardiac cells on grooves. We find all cells align along the grooves to differing extents at both sparse and confluent densities. To increase the sophistication of in vitro tissue organization possible, we also created hybrid substrates with controlled patterns of microgrooved and flat regions that can be identified in real-time using optical microscopy. Using our hybrid patterned surfaces we explore: (i) the ability of neighboring cells to provide a template to organize surrounding cells that are not directly exposed to grooved topographic cues, and (ii) the distance over which this template effect can operate in confluent cell sheets. We find that in fibroblast sheets, but not epithelial sheets, cells aligned on grooves can direct alignment of neighboring cells in flat regions over a limited distance of approximately 200 μm. Our hybrid surface plate provides a novel tool for studying the collective response of groups of cells exposed to differential topographical cues. Biotechnol. Bioeng. 2014;111: 2537-2548. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Highlights: [•] A new micro-neutralization high-throughput centrifugation-free method is described. [•] Host cell monolayers were fixed to poly-d-Lys coated solid-phases. [•] Saponin-containing buffers were used for immunostaining. [•] Cells were detached by trypsin digestion for batch flow cytometry. [•] Viral-neutralizing antibodies were successfully assayed in plasma from VHSV-immunized zebrafish (Danio rerio). [ABSTRACT FROM AUTHOR]

Human macrophages are a natural host of many mycobacterium species, including Mycobacterium abscessus (M. abscessus), an emerging pathogen affecting immunocompromised and cystic fibrosis patients with few available treatments. The search for an effective treatment is hindered by the lack of a tractable in vitro intracellular infection model. Here, we established a reliable model for M. abscessus infection using human pluripotent stem cell-derived macrophages (hPSC-macrophages). hPSC differentiation permitted reproducible generation of functional macrophages that were highly susceptible to M. abscessus infection. Electron microscopy demonstrated that M. abscessus was present in the hPSC-macrophage vacuoles. RNA sequencing analysis revealed a time-dependent host cell response, with differing gene and protein expression patterns post-infection. Engineered tdTOMATO-expressing hPSC-macrophages with GFP-expressing mycobacteria enabled rapid image-based high-throughput analysis of intracellular infection and quantitative assessment of antibiotic efficacy. Our study describes the first to our knowledge hPSC-based model for M. abscessus infection, representing a novel and accessible system for studying pathogen-host interaction and drug discovery., Competing Interests: Conflicts of interest E.G.S. is member of the editorial board of Stem Cell Reports., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Helicobacter pylori infection is associated with severe gastrointestinal disease including cancer. It induces complex antibody responses that might vary depending on disease state but currently cannot be assessed adequately. The objective of this work was the development of a sensitive and specific H. pylori multiplex serology assay with high-throughput capability that allows simultaneous detection of antibodies to a protein array. Methods: Seventeen proteins of up to three H. pylori strains (26695, G27, 151), including CagA, VacA, UreA, Catalase, Omp, and GroEL, were recombinantly expressed as glutathione- S-transferase fusion proteins, affinity-purified, and used as antigens in a fluorescent bead-based antibody-binding assay. Reference sera (n = 317) characterized by commercial assays (screening ELISA with Western blot confirmation) were used for validation. Results: H. pylori seropositivity by multiplex serology defined as reactivity with at least four proteins showed good agreement (kappa: 0.70) with commercial serologic assay classification, and a sensitivity of 89% and specificity of 82%. For individual antigens, agreement with Western blot was good for CagA (kappa: 0.77), moderate for UreA (kappa: 0.53), and weak for VacA (kappa: 0.12). Of the 13 proteins expressed from two strains, only VacA showed serologic strain differences. High antibody reactivity to CagA (Type I infection) was negatively associated with antibodies to GroEL, Cad, CagM, catalase, HcpC, NapA, and UreA, suggesting type-specific differences in protein expression patterns and/or immune response. Conclusion: With its high-throughput and simultaneous detection abilities, H. pylori multiplex serology appears suited as tool for large seroepidemiologic studies assessing H. pylori prevalence, antibody patterns, and associations with specific diseases. [ABSTRACT FROM AUTHOR]

Abstract: The potential genotoxicity of drug candidates is a serious concern during drug development. Therefore, it is important to assess the potential genotoxicity and mutagenicity of a compound early in the discovery phase of drug development. AMES Salmonella assay is the most widely used assay for the assessment of mutagenicity and genotoxicity. However, the AMES assay is not readily adaptable to highthroughput screening and several strains of Salmonella must be employed to ensure that different types of DNA damage can be studied. Therefore, an additional robust highthroughput genotoxicity screen would be of significant value in the early detection and elimination of genotoxicity. The complexity of DNA damage requires numerous cellular pathways, thus using single model organism to predict genotoxicity in early stage is challenging. Another critical component of such screens is that they incorporate the capability of metabolic activation to ensure that no genotoxic metabolites are generated. We have developed a novel highthroughput reporter assay for DNA repair that detects genotoxicity, and which incorporates metabolic activation. The assay has a low compound requirement as compared to Ames, and relies upon two different reporter genes cotransfected into a yeast strain. The gene encoding Renilla luciferase is fused to the constitutive 3-phosphoglycerate kinase (PGK1) promoter and integrated into the yeast genome to provide a control for cell numbers. The firefly luciferase gene is fused to the RAD51 (bacterial RecA homolog) promoter and used to report an increase in DNA repair activity. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. The result is expressed as the ratio of the two luciferase activities; changes from the base level (control) are interpreted as induction of the RAD51 promoter and evidence of DNA repair activity in eukaryote cells due to DNA damage. The yeast dual luciferase reporter has been characterized with and without S-9 activation using positive and negative control agents. This assay is efficient, requires little time and low amounts of compound. The assay is compatible with metabolic activation, adaptable to a highthroughput platform, and yields data that accurately and reproducibly detects DNA damage. Whereas the normal yeast cell wall, plasma membrane composition and the presence of active transporters can prevent the entry or persistence of some compounds internally in yeast cells, our assay did show concordance with regulatory mutagenicity assays, many of which require metabolic activation and are poorly detected by bacterial mutagenicity assays. Although there were false negative results, in our hands this assay performs as well as or better than other commercially available genetox assays. Furthermore, the RAD51 gene is strongly inducible by homologous intrachromosomal recombination; thus this assay may provide a means to detect clastogens. The RAD51 promoter fused dual luciferase assay represents a valuable addition to the armamentarium for the early detection of genotoxic compounds. [Copyright &y& Elsevier]

We develop Well-Deposition Particle Templating (W-DePT), a fast approach for mass-producing monodisperse shape-designed colloids, or LithoParticles. W-DePT is based on a single solid template that has been permanently etched in negative relief to provide one or more wells of desired shapes of particles. This "well-template" is coated with a sacrificial release layer, and the desired particle material, whether organic, inorganic, or metal, or a combination thereof, is deposited into the wells. By simply dissolving the sacrificial layer in a fluid, particles having the desired shapes can be released and the well-template can be re-used. Parallel production of many particles using multiple well templates is an inexpensive alternative to optical-exposure based approaches. The particles are released from the wells with greater than 99% efficiency, and they have less than 5% polydispersity. Using W-DePT, LithoParticles are made without either repeated optical exposure or imprinting, which involves mechanically contacting a patterned template with a second flat plate. [ABSTRACT FROM AUTHOR]