High-throughput Screening System Research Articles

Efficient approach to cyclic scheduling of high throughput screening systems for bioengineering

As a standard technology, high throughput screening (HTS) is widely applied for the analysis and detection of new biological substances and drugs in pharmaceutical industries and life science research. Efficient scheduling of HTS systems is a crucial issue for reducing the cost of their operations. As a typical application of high throughput screening, this work studies the scheduling problem of the enzyme-linked immunoassay (ELISA) processes in HTS systems. For this process, it requires a one-microplate cyclic schedule and a microplate visits some processing resources multiple times, making the process deadlock-prone such that its scheduling problem is very challenging. To tackle this problem, the process is modeled by a kind of Petri nets. Based on this model, by analyzing the dynamic properties of the process, it is able to directly deduce an activity sequence to form an optimal cyclic schedule. In this way, it not only efficiently solves the challenging scheduling problem without building a mathematical programming model, but also the obtained schedule is very easy to understand and implement.

The calcium-binding photoprotein clytin II: Expression of the preferred human codon-optimized clytin II gene in Chinese hamster ovary-K1 cells and its use in the G-protein-coupled receptor assays

Clytin II (CLII) is a Ca2+-binding photoprotein and has been identified as an isotype of clytin I (CLI). CLII consists of apoCLII (an apoprotein) and 2-peroxide of coelenterazine (an adduct of molecular oxygen to coelenterazine), which is identical to the widely used Ca2+-binding photoprotein, aequorin (AQ). However, CLII triggered by Ca2+ exhibits a 4.5-fold higher maximum luminescence intensity (Imax) compared to both AQ and CLI, and it is approximately 5 times less sensitive to Ca2+ than AQ. To confirm the suitability of the preferred human codon-optimized CLII (pCLII) gene for cell-based G-protein-coupled receptor (GPCR) assays, a transformant stably expressing apoprotein of pCLII using the pCLII gene in the mitochondria of CHO–K1 cells was established and in situ regenerated pCLII in the cells were applied to the high-throughput screening system. An ATP-stimulated GPCR assay for endogenous P2Y purinergic receptors was confirmed using the established stable transformant.

Discovery of Ten Anti-HIV Hit Compounds and Preliminary Pharmacological Mechanisms Studies.

The research and development of HIV drugs is very important, but at the same time it is a long cycle and expensive system project. High-throughput drug screening systems and molecular libraries of potential hit compounds remain the main ways for the discovery of hit compounds with anti-HIV activity. The aim of this study was to screen out the hit compounds against HIV-1 in the natural product molecule library and the antiviral molecule library, and elucidate the molecular mechanism of their inhibition of HIV-1, so as to provide a new choice for AIDS drug research. In this study, a drug screening system using HIV Rev-dependent indicator cell line (Rev-A3R5-GFP reporter cells) with pseudoviruses (pNL4-3) was used. The natural drug molecule library and antiviral molecule library were screened, and preliminary drug mechanism studies were performed. Ten promising hit compounds were screened. These ten molecules and their drug inhibitory IC50 were as follows: Cephaeline (0.50 μM), Yadanziolide A (8.82 μM), Bruceine D (2.48 μM), Astragaloside IV (4.30 μM), RX-3117 (1.32 μM), Harringtonine (0.63 μM), Tubercidin (0.41 μM), Theaflavine-3, 3'-digallate (0.41 μM), Ginkgetin (10.76 μM), ZK756326 (5.97 μM). The results of the Time of additions showed that except for Astragaloside IV and Theaflavine-3, 3'-digallate had a weak entry inhibition effect, and it was speculated that all ten compounds had an intracellular inhibition effect. Cephaeline, Harringtonine, Astragaloside IV, Bruceine D, and Tubercidin may have pre-reverse transcriptional inhibition. Yadanziolide A, Theaflavine-3, 3'-digallate, Ginkgetin and RX-3117 may be in the post-reverse transcriptional inhibition. The inhibitory effect of ZK 75632 may be in the reverse transcriptional process. A drug screening system using Rev-A3R5-GFP reporter cells with pseudoviruses (pNL4-3) is highly efficient. This study provided potential hit compounds for new HIV drug research.

High-throughput BCRP inhibitors screening system based on styrene maleic acid polymer membrane protein stabilization strategy and surface plasmon resonance biosensor

Multidrug resistance (MDR) is a dominant challenge in cancer chemotherapy failure. The over-expression of breast cancer resistance protein (BCRP) in tumorous cells, along with its extensive substrate profile, is a leading cause of tumor MDR. Herein, on the basis of styrene maleic acid (SMA) polymer membrane protein stabilization strategy and surface plasmon resonance (SPR) biosensor, a novel high-throughput screening (HTS) system for BCRP inhibitors has been established. Firstly, LLC-PK1 and LLC-PK1/BCRP cell membranes were co-incubated with SMA polymers to construct SMA lipid particles (SMALPs). PK1-SMALPs were thus immobilized in channel 1 of the L1 chip as the reference channel, and BCRP-SMALPs were immobilized in channel 2 as the detection channel to establish the BCRP-SMALPs-SPR screening system. The methodological investigation demonstrated that the screening system was highly specific and stable. Three active compounds were screened out from 26 natural products and their affinity constants with BCRP were determined. The KD of xanthotoxin, bergapten, and naringenin were 5.14 μM, 4.57 μM, and 3.72 μM, respectively. The in vitro cell verification experiments demonstrated that xanthotoxin, bergapten, and naringenin all significantly increased the sensitivity of LLC-PK1/BCRP cells to mitoxantrone with possessing reversal BCRP-mediated MDR activity. Collectively, the developed BCRP-SMALPs-SPR screening system in this study has the advantages of rapidity, efficiency, and specificity, providing a novel strategy for the in-depth screening of BCRP inhibitors with less side effects and higher efficacy.

Abstract 2944: A high-throughput screening system for identifying therapeutic targets in immuno-oncology

Abstract INTRODUCTION: Over the last decade, cancer immunotherapy has revolutionized cancer treatment. However, more than half of patients don’t respond, and responders often lack lasting effects. Understanding how genetic variants in cancer cells influence their interaction with immune cells, especially T cells, is crucial to uncover underlying mechanisms. Genetic screens are powerful for uncovering responsible genes for various phenotypes, but challenges in obtaining ample samples to set up a proper immune and cancer co-culture system have limited their use for identifying immune modulators in cancer: primary mouse T cells expressing CD3 against a specific antigen (typically albumin) need to be extracted from transgenic mice and only syngeneic mouse cancer cell lines that match these T cells can be paired, restricting the utilization of human T cells and human cancer cells for this co-culture screening setup. METHODS: Here, we present a highly scalable co-culture system that enables genome-wide CRISPR screens on a pair of human primary T cells and any allogeneic human cancer cell lines. This system utilizes the expression of a membrane-targeted protein in mammalian cells, named as ImBridge, which directly engages and activates CD3+ immune cells. This mechanism bypasses the need for matched MHC on target cancer cells. RESULTS: We validated activation of CD3+ immune cells and selective cancer cell cytotoxicity by ImBridge. A genome-scale CRISPR screen utilizing the co-culture system unveiled known immune-oncology targets including PD-L1 and PTPN2 and revealed MEDiC_01 as a novel therapeutic target. Genetic loss of MEDiC_01 in cancer cells significantly sensitized them to cytotoxic cells, effectively eliminating cancers in the presence of human PBMCs. CONCLUSION: We demonstrated a scalable and efficient approach for conducting high-throughput functional screens to uncover genes that modulate interactions between effector T cells and target cancer cells. This can facilitate the identification of new immuno-oncology therapeutic targets, insights into underlying mechanisms, and genetic factors influencing immune susceptibility and evasion. Citation Format: Dippal Parikh, Soon Youn Choi, Ki Eun Pyo, Hong-Pyo Lee, Kyuho Han. A high-throughput screening system for identifying therapeutic targets in immuno-oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2944.

Fast Algorithm for High-Throughput Screening Scheduling Based on the PERT/CPM Project Management Technique

High-throughput screening systems are robotic cells that automatically scan and analyze thousands of biochemical samples and reagents in real time. The problem under consideration is to find an optimal cyclic schedule of robot moves that ensures maximum cell performance. To address this issue, we proposed a new efficient version of the parametric PERT/CPM project management method that works in conjunction with a combinatorial subalgorithm capable of rejecting unfeasible schedules. The main result obtained is that the new fast PERT/CPM method finds optimal robust schedules for solving large size problems in strongly polynomial time, which cannot be achieved using existing algorithms.

Characterization of two SGNH family cell death-inducing proteins from the horticulturally important fungal pathogen Botrytis cinerea based on the optimized prokaryotic expression system

Botrytis cinerea is one of the most destructive phytopathogenic fungi, causing significant losses to horticultural crops. As a necrotrophic fungus, B. cinerea obtains nutrients by killing host cells. Secreted cell death-inducing proteins (CDIPs) play a crucial role in necrotrophic infection; however, only a limited number have been reported. For high-throughput CDIP screening, we optimized the prokaryotic expression system and compared its efficiency with other commonly used protein expression systems. The optimized prokaryotic expression system showed superior effectiveness and efficiency and was selected for subsequent CDIP screening. The screening system verified fifty-five candidate proteins and identified two novel SGNH family CDIPs: BcRAE and BcFAT. BcRAE and BcFAT exhibited high expression levels throughout the infection process. Site-directed mutagenesis targeting conserved Ser residues abolished the cell death-inducing activity of both BcRAE and BcFAT. Moreover, the transient expression of BcRAE and BcFAT in plants enhanced plant resistance against B. cinerea without inducing cell death, independent of their enzymatic activities. Our results suggest a high-efficiency screening system for high-throughput CDIP screening and provide new targets for further study of B. cinerea-plant interactions.

High-throughput barcoding of nanoparticles identifies cationic, degradable lipid-like materials for mRNA delivery to the lungs in female preclinical models

Lipid nanoparticles for delivering mRNA therapeutics hold immense promise for the treatment of a wide range of lung-associated diseases. However, the lack of effective methodologies capable of identifying the pulmonary delivery profile of chemically distinct lipid libraries poses a significant obstacle to the advancement of mRNA therapeutics. Here we report the implementation of a barcoded high-throughput screening system as a means to identify the lung-targeting efficacy of cationic, degradable lipid-like materials. We combinatorially synthesize 180 cationic, degradable lipids which are initially screened in vitro. We then use barcoding technology to quantify how the selected 96 distinct lipid nanoparticles deliver DNA barcodes in vivo. The top-performing nanoparticle formulation delivering Cas9-based genetic editors exhibits therapeutic potential for antiangiogenic cancer therapy within a lung tumor model in female mice. These data demonstrate that employing high-throughput barcoding technology as a screening tool for identifying nanoparticles with lung tropism holds potential for the development of next-generation extrahepatic delivery platforms.

Recapitulation of the Powassan virus life cycle in cell culture.

Powassan virus (POWV) is a tick-borne flavivirus known for causing fatal neuroinvasive diseases in humans. Recently, there has been a noticeable increase in POWV infections, emphasizing the urgency of understanding viral replication, pathogenesis, and developing interventions. Notably, there are no approved vaccines or therapeutics for POWV, and its classification as a biosafety level-3 (BSL-3) agent hampers research. To overcome these obstacles, we developed a replicon system, a self-replicating RNA lacking structural proteins, making it safe to operate in a BSL-2 environment. We constructed a POWV replicon carrying the Gaussia luciferase (Gluc) reporter gene and blasticidin (BSD) selectable marker. Continuous BSD selection led to obtain a stable POWV replicon-carrying Huh7 cell lines. We identified cell culture adaptive mutations G4079A, G4944T and G6256A, resulting in NS2AR195K, NS3G122G, and NS3V560M, enhancing RNA replication. We demonstrated the utility of the POWV replicon system for high-throughput screening (HTS) assay to identify promising antivirals against POWV replication. We further explored the applications of the POWV replicon system, generating single-round infectious particles (SRIPs) by transfecting Huh7-POWV replicon cells with plasmids encoding viral capsid (C), premembrane (prM), and envelope (E) proteins, and revealed the distinct antigenic profiles of POWV with ZIKV. In summary, the POWV replicon and SRIP systems represent crucial platforms for genetic and functional analysis of the POWV life cycle and facilitating the discovery of antiviral drugs.IMPORTANCEIn light of the recent surge in human infections caused by POWV, a biosafety level-3 (BSL-3) classified virus, there is a pressing need to understand the viral life cycle and the development of effective countermeasures. To address this, we have pioneered the establishment of a POWV RNA replicon system and a replicon-based POWV SRIP system. Importantly, these systems are operable in BSL-2 laboratories, enabling comprehensive investigations into the viral life cycle and facilitating antiviral screening. In summary, these useful tools are poised to advance our understanding of the POWV life cycle and expedite the development of antiviral interventions.

Biological effects of Lippia alba essential oil against Anopheles gambiae and Aedes aegypti

The management of mosquito resistance to chemical insecticides and the biting behaviour of some species are motivating the search for complementary and/or alternative control methods. The use of plants is increasingly considered as a sustainable biological solution for vector control. The aim of this study was to evaluate the biological effects of the essential oil (EO) of Lippia alba harvested in Abidjan (Côte d’Ivoire) against Anopheles gambiae and Aedes aegypti mosquitoes. Phytochemical compounds were identified by GC–MS. Knockdown and mortality were determined according to the WHO test tube protocol. Contact irritancy was assessed by observing the movement of mosquitoes from a treated WHO tube to a second untreated tube. Non-contact repellency was assessed using a standardised high-throughput screening system (HITSS). Blood meal inhibition was assessed using a membrane feeding assay treated with EO. The EO was identified as the citral chemotype. The EO gave 100% KD60 in both species at a concentration of 1%. Mortalities of 100% were recorded with An. gambiae and Ae. aegypti at concentrations of 1% and 5% respectively. The highest proportions of females escaping during the contact irritancy test were 100% for An. gambiae at 1% concentration and 94% for Ae. aegypti at 2.5% concentration. The 1% concentration produced the highest proportions of repelled mosquitoes in the non-contact repellency tests: 76.8% (An. gambiae) and 68.5% (Ae. aegypti). The blood meal inhibition rate at a dose of 10% was 98.4% in Ae. aegypti but only 15.5% in An. gambiae. The citral chemotype of L. alba EO has promising biological effects in both species that make it a potentially good candidate for its use in mosquito control. The results obtained in this study encourage the further evaluation of L. alba EOs from other localities and of different chemotypes, under laboratory and field conditions.

Development of tight junction-strengthening compounds using a high-throughput screening system to evaluate cell surface-localized claudin-1 in keratinocytes

Tight junctions (TJs) are important factors constituting the physical barriers of the skin, and their suppression has been described in various conditions, such as aged skin and atopic dermatitis lesions. However, the methods for improving skin TJ function remain insufficient. Therefore, to obtain compounds that can improve TJ function, we developed a novel high-throughput screening system termed live-cell immunostaining to evaluate cell surface-localized claudin-1 (CLDN1) with high selectivity using normal human epidermal keratinocytes (NHEKs). Heparinoid and phospho-pyridoxal (p-Pyr), a metabolite of pyridoxine, were identified as hit compounds. In addition, heparinoid was strongly suggested to increase CLDN1 expression by inhibiting epidermal growth factor receptor signaling. By contrast, p-Pyr did not enhance CLDN1 expression, but it accelerated the translocation of CLDN1 to the cell surface. Finally, we confirmed that heparinoid and p-Pyr improved barrier function in NHEKs in a transepithelial electrical resistance assay. In conclusion, heparinoid and p-Pyr could potentially ameliorate skin conditions by improving TJ function.

Enhancing Pullulan Production in Aureobasidium pullulans through UV Mutagenesis Breeding and High-Throughput Screening System

This study addresses the production enhancement of pullulan, an extracellular polysaccharide with various applications. Pullulan is primarily produced by Aureobasidium pullulans (A. pullulans), and genetic modification is commonly used to increase its yield. However, there is a need for a more efficient and safer method. To achieve this, we designed a high-throughput screening system utilizing a unique fluorescent protein specific to pullulan. Ultraviolet (UV) mutagenesis was applied to create a pool of mutant strains, and flow cytometry allowed for single-cell screening. Our approach yielded strain M1-B3, which exhibited a substantial increase in pullulan production from 26.5 g/L to 76.88 g/L. Additionally, the molecular weight of the produced pullulan significantly increased, expanding its potential commercial application. This study demonstrates an efficient and safe method to enhance pullulan production in A. pullulans. The UV mutagenesis and flow cytometry based on screening not only increased yield but also improved pullulan’s molecular weight. The adaptability of this method to other polysaccharides and its potential for genomic analysis and broader applications make it a valuable tool in bioproduction.

Unveiling Epigenetic Vulnerabilities in Triple-Negative Breast Cancer through 3D Organoid Drug Screening.

Triple-negative breast cancer (TNBC) poses a therapeutic challenge due to its aggressive nature and lack of targeted therapies. Epigenetic modifications contribute to TNBC tumorigenesis and drug resistance, offering potential therapeutic targets. Recent advancements in three-dimensional (3D) organoid cultures, enabling precise drug screening, hold immense promise for identifying novel compounds targeting TNBC. In this study, we established two patient-derived TNBC organoids and implemented a high-throughput drug screening system using these organoids and two TNBC cell lines. Screening a library of 169 epigenetic compounds, we found that organoid-based systems offer remarkable precision in drug response assessment compared to cell-based models. The top 30 compounds showing the highest drug sensitivity in the initial screening were further assessed in a secondary screen. Four compounds, panobinostat, pacritinib, TAK-901, and JIB-04, targeting histone deacetylase, JAK/STAT, histone demethylases, and aurora kinase pathways, respectively, exhibited potent anti-tumor activity in TNBC organoids, surpassing the effect of paclitaxel. Our study highlights the potential of these novel epigenetic drugs as effective therapeutic agents for TNBC and demonstrates the valuable role of patient-derived organoids in advancing drug discovery.

Identification of a microbial sub-community from the feral chicken gut that reduces Salmonella colonization and improves gut health in a gnotobiotic chicken model.

A complex microbial community in the gut may prevent the colonization of enteric pathogens such as Salmonella. Some individual or a combination of species in the gut may confer colonization resistance against Salmonella. To gain a better understanding of the colonization resistance against Salmonella enterica, we isolated a library of 1,300 bacterial strains from feral chicken gut microbiota which represented a total of 51 species. Using a co-culture assay, we screened the representative species from this library and identified 30 species that inhibited Salmonella enterica subspecies enterica serovar Typhimurium in vitro. To improve the Salmonella inhibition capacity, from a pool of fast-growing species, we formulated 66 bacterial blends, each of which composed of 10 species. Bacterial blends were more efficient in inhibiting Salmonella as compared to individual species. The blend that showed maximum inhibition (Mix10) also inhibited other serotypes of Salmonella frequently found in poultry. The in vivo effect of Mix10 was examined in a gnotobiotic and conventional chicken model. The Mix10 consortium significantly reduced Salmonella load at day 2 post-infection in gnotobiotic chicken model and decreased intestinal tissue damage and inflammation in both models. Cell-free supernatant of Mix10 did not show Salmonella inhibition, indicating that Mix10 inhibits Salmonella through either nutritional competition, competitive exclusion, or through reinforcement of host immunity. Out of 10 species, 3 species in Mix10 did not colonize, while 3 species constituted more than 70% of the community. Two of these species were previously uncultured bacteria. Our approach could be used as a high-throughput screening system to identify additional bacterial sub-communities that confer colonization resistance against enteric pathogens and its effect on the host.IMPORTANCESalmonella colonization in chicken and human infections originating from Salmonella-contaminated poultry is a significant problem. Poultry has been identified as the most common food linked to enteric pathogen outbreaks in the United States. Since multi-drug-resistant Salmonella often colonize chicken and cause human infections, methods to control Salmonella colonization in poultry are needed. The method we describe here could form the basis of developing gut microbiota-derived bacterial blends as a microbial ecosystem therapeutic against Salmonella.

High-throughput screening for a SARS-CoV-2 frameshifting inhibitor using a cell-free protein synthesis system.

Programmed-1 ribosomal frameshifting (-1 PRF) is a translational mechanism adopted by some viruses, including SARS-CoV-2. To find a compound that can inhibit -1 PRF in SARS-CoV-2, we set up a high-throughput screening system using a HeLa cell extract-derived cell-free protein synthesis (CFPS) system. A total of 32,000 compounds were individually incubated with the CFPS system programmed with a -1 PRF-EGFP template. Several compounds were observed to decrease the -1 PRF-driven fluorescence, and one of them had some suppressive effect on -1 PRF of a SARS-CoV-2 genome sequence in transfected cells. Thus the CFPS system can be used as a tool for a high-throughput screening of chemicals.

Fully genotyping and screening of clinically important blood-group antigens by MALDI TOF mass spectrometry.

Several molecular biology methods are available for high-throughput blood typing. In this study, we aimed to build a high-throughput blood-group genetic screening system for high-frequency blood-group antigen-negative rare-blood groups in donors and patients. The amplification primers for all blood-type gene fragments involving the selected alleles were designed for detection. Single-base extend primers were also designed based on specific loci. DNA fragments were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) for the last nucleotide identification of amplification products in the extend step. The accuracy was verified by known samples. Thirty-six random samples were detected by serological tests and sequencing to verify the system stability. After verification, according to the collected known rare-blood-type samples, all the alleles designed to be detected matched with the validated single-nucleotide polymorphisms. The verification tests showed that all genotyping results of the random samples were in accordance with the findings of serotyping and sequencing. Then, 1258 random donor samples were screened by the built typing system after the verification. Three Fy(a-) and four s- were screened out in 1258 random blood samples. The multiple polymerase chain reaction-based MS detection system can be used in rare-blood-type screening with good accuracy and stability.

A nanoluciferase SFTSV for rapid screening antivirals and real-time visualization of virus infection in mice

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen that causes severe hemorrhagic fever in humans, but no FDA-approved specific antivirals or vaccines are available to treat or prevent SFTS. The plasmids construction and transfection were performed to generate the recombinant SFTSV harboring the nanoluciferase gene (SFTSV-Nluc). Immunostaining plaque assay was performed to measure viral titers, and DNA electrophoresis and Sanger sequencing were performed to evaluate the genetic stability. Luciferase assay and quantitative RT-PCR were performed to evaluate the efficacy of antivirals invitro. Bioluminescence imaging, titration of virus from excised organs, hematology, and histopathology and immunohistochemistry were performed to evaluate the efficacy of antivirals invivo. SFTSV-Nluc exhibited high genetic stability and replication kinetics similar to those of wild-type virus (SFTSVwt), then a rapid high-throughput screening system for identifying inhibitors to treat SFTS was developed, and a nucleoside analog, 4-FlU, was identified to effectively inhibit SFTSV invitro. SFTSV-Nluc mimicked the replication characteristics and localization of SFTSVwt in counterpart model mice. Bioluminescence imaging of SFTSV-Nluc allowed real-time visualization and quantification of SFTSV replication in the mice. 4-FlU was demonstrated to inhibit the replication of SFTSV with more efficiency than T-705 and without obvious adverse effect invivo. The high-throughput screening system based on SFTSV-Nluc for use invitro and invivo revealed that a safe and effective antiviral nucleoside analog, 4-FlU, may be a basis for the strategic treatment of SFTSV and other bunyavirus infections, paving the way for the discovery of antivirals. This work was supported by grants from the National Key Research and Development Plan of China (2021YFC2300700 to L. Zhang, 2022YFC2303300 to L. Zhang), Strategic Priority Research Program of Chinese Academy of Sciences (XDB0490000 to L. Zhang), National Natural Science Foundation of China (31970165 to L. Zhang, U22A20379 to G. Xiao), the Science and Technology Commission of Shanghai Municipality (21S11903100 to Y. Xie), Hubei Natural Science Foundation for Distinguished Young Scholars (2022CFA099 to L. Zhang).

Enabling continuous immune cell recirculation on a microfluidic array to study immunotherapeutic interactions in a recapitulated tumour microenvironment.

The effects of immunotherapeutics on interactions between immune and cancer cells are modulated by multiple components in the tumour microenvironment (TME), including endothelium and tumour stroma, which provide both a physical barrier and immunosuppressive stimuli. Herein, we report a recirculating chip to enable continuous immune cell recirculation through a microfluidic cell array to include these crucial players. This system consists of a three-layered cell array (μFCA) spatially emulating the TME, with tailored fluidic circuits establishing T cell recirculation. This platform enables the study of dynamics among the TME, immune cells in a circulatory system and cancer cell responses thereof. Through this system, we found that tumour endothelium hindered T cell infiltration into the reconstructed breast cancer tumour compartment. This negative effect was alleviated when treated with anti-human PD-L1 (programmed cell death ligand 1) antibody. Another key stromal component - cancer associated fibroblasts - attenuated T cell infiltration, compared against normal fibroblasts, and led to reduced apoptotic activity in cancer cells. These results confirm the capability of our tumour-on-a-chip system in identifying some key axes to target in overcoming barriers to immunotherapy by recapitulating immune cell interactions with the reconstructed TME. Our results also attest to the feasibility of scaling up this system for high-throughput cancer immunotherapeutic screening.

Automated Electrolyte Formulation and Coin Cell Assembly for High-Throughput Lithium-Ion Battery Research

Battery research involving performance evaluation by battery cycling is a time-consuming process with sometimes large cell-to-cell variations. To enhance the rate of testing and reproducibility, automation is one accelerator that can be used. In this study, we introduce ODACell1 (Figure 1), an automated system for high-throughput electrolyte screening and Li-ion cell assembly.ODACell enables the assembly of Li-ion battery cells in ambient atmosphere. We use LiFePO4||Li4Ti5O12-based full cells with a dimethyl sulfoxide-based model electrolyte for demonstrating the automated system. We investigate the reproducibility of the automated system on this system as well as the influence of water on the performance of the model electrolyte when exposed to ambient air. Our system is uniquely equipped for combinatorial experimentation, combining electrolyte formulation, batch coin cell assembly, and cycling into a single system. The integration of multiple setups into a single unit is made possible, overcoming the incompatibility issues with integrating multiple, different automated systems together.While there are several other robotic systems besides ODACell for battery material acceleration, ODACell’s unique combination of robots incorporated in the robotic setup allow a broader set of tasks to be automated. For example, Clio by Dave et al. 2 focus on electrolyte formulation and characterization, lacking assembly of battery cells; AutoBASS by Zhang et al. 3 only automates battery assembly; and Poseidon by Svensson et al. 4 can automate electrolyte formulation, characterization, and coin cell assembly, but lack high-throughput batch processing.ODACell ensures high reproducibility, which is needed to establish robust models and determine accurate electrochemical and physical properties. The system assembled 131 coin cells, with a conservative fail rate of only 5%. The relative standard deviation of the discharge capacity after 10 cycles was 2% for the studied system. The investigation into the influence of water on cycling performance show overlapping performance trends between coin cells with 2 vol% and 4 vol% of water in the electrolyte highlighting the nontrivial relationship between water and cycling performance.In conclusion, ODACell is a promising tool for high-throughput electrolyte screening and Li-ion cell assembly. It builds upon previously published robotic setups by automating more of the research workflow. Our study highlights the importance of reproducibility in battery research and the complex relationship between water contaminants in electrolytes and cycling performance. ODACell has the potential to be a useful tool for advancing battery research by enhancing the rate of testing and reducing the variation of results.

Design and Evaluation of a Sticky Attractant Trap for Intra-Domiciliary Surveillance of Aedes aegypti Populations in Mexico.

Surveillance consists of systematic data collection, analysis, and interpretation and is essential for planning and implementing control activities. The lack of success in the control and surveillance of the Ae. aegypti mosquito elsewhere demands the development of new accessible and effective strategies. This work aimed to develop and evaluate an adhesive lure trap for household indoor surveillance of Ae. aegypti. Based on a bibliographic review, four compounds that have significant attraction percentages for Ae. aegypti were considered. Our more effective blend was determined through preliminary bioassays using the high-throughput screening system (HITSS) and 90 × 90 cm mosquito cages. We designed a low-cost, pyramid-shaped, sticky cardboard trap to incorporate the selected blend. Semi-field 2 × 2 m cages and field tests were utilized to evaluate its effectiveness through mosquito capture percentages. In laboratory tests, blend number 2 presented an attraction percentage of 47.5 ± 4.8%; meanwhile, in semi-field cages, a 4-inch, 110 v powered fan was used to disperse the attractants, and then a similar capture percentage of 43.2 ± 4.0% was recorded. Results were recorded during the field evaluation of the at-house indoor environment and were compared with those recorded with the golden-standard BG-Sentinel trap, i.e., our prototype trapped an average of 6.0 ± 1.5 mosquitoes versus 10.0 ± 2.6. In most Latin American countries, there is a lack of formal and accessible strategies for monitoring adult populations of Ae. Aegypti; therefore, we must develop tools that reinforce entomological surveillance methods.