High-throughput Testing Research Articles

Recent advances and application of liquid chromatography in pharmaceutical industry

Liquid chromatography (LC) and its associated techniques are essential in the pharmaceutical industry for optimizing drug formulations, ensuring product quality, and meeting regulatory standards. Thin Layer Chromatography (TLC) is widely used for rapid screening and chiral separations in early-stage development due to its simplicity and cost-effectiveness. High-Performance Liquid Chromatography (HPLC) remains the gold standard for routine analysis, offering high resolution and reproducibility for complex sample analysis and quantification of active pharmaceutical ingredients (APIs). Ultra-Performance Liquid Chromatography (UPLC) builds on HPLC, providing faster analysis, higher sensitivity, and greater efficiency, making it ideal for high-throughput testing. Supercritical Fluid Chromatography (SFC) is increasingly used for separating thermally labile and polar compounds, offering advantages in solvent usage and analysis time. This review will discuss the evolution of these chromatographic techniques and their critical role in optimizing pharmaceutical processes, ensuring product quality, and facilitating regulatory compliance throughout the drug development lifecycle.

Development and validation of a high-throughput qPCR platform for the detection of soil-transmitted helminth infections.

Historically, soil-transmitted helminth (STH) control and prevention strategies have relied on mass drug administration efforts targeting preschool and school-aged children. While these efforts have succeeded in reducing morbidity associated with STH infection, recent modeling efforts have suggested that expanding intervention to treatment of the entire community could achieve transmission interruption in some settings. Testing the feasibility of such an approach requires large-scale clinical trials, such as the DeWorm3 cluster randomized trial. In addition, accurate interpretation of trial outcomes requires diagnostic platforms capable of accurately determining infection prevalence (particularly as infection intensity is reduced) at large population scale and with significant throughput. Here, we describe the development and validation of such a high-throughput molecular testing platform. Through the development, selection, and validation of appropriate controls, we have successfully created and evaluated the performance of a testing platform capable of the semi-automated, high-throughput detection of four species of STH in human stool samples. Comparison of this platform with singleplex reference assays for the detection of these same pathogens has demonstrated comparable performance metrics, with index assay accuracy measuring at or above 99.5% and 98.1% for each target species at the level of the technical replicate and individual extraction respectively. Through the implementation of a rigorous validation program, we have developed a diagnostic platform capable of providing the necessary throughput and performance required to meet the needs of the DeWorm3 cluster randomized trial and other large-scale operational research efforts for STH. Resulting from the rigorous developmental approach taken, the platform we describe here provides the needed confidence in testing outcomes when utilized in conjunction with large-scale efforts such as the DeWorm3 trial. Additionally, the successful development of an evaluation and validation strategy provides a template for the creation of similar diagnostic platforms for other neglected tropical diseases.

Accelerating the Discovery of Abiotic Vesicles with AI-Guided Automated Experimentation.

The first protocells are speculated to have arisen from the self-assembly of simple abiotic carboxylic acids, alcohols, and other amphiphiles into vesicles. To study the complex process of vesicle formation, we combined laboratory automation with AI-guided experimentation to accelerate the discovery of specific compositions and underlying principles governing vesicle formation. Using a low-cost commercial liquid handling robot, we automated experimental procedures, enabling high-throughput testing of various reaction conditions for mixtures of seven (7) amphiphiles. Multitemplate multiscale template matching (MMTM) was used to automate confocal microscopy image analysis, enabling us to quantify vesicle formation without tedious manual counting. The results were used to create a Gaussian process surrogate model, and then active learning was used to iteratively direct the laboratory experiments to reduce model uncertainty. Mixtures containing primarily trimethyl decylammonium and decylsulfate in equal amounts formed vesicles at submillimolar critical vesicle concentrations, and more than 20% glycerol monodecanoate prevented vesicles from forming even at high total amphiphile concentrations.

Bioluminescent Pseudomonas aeruginosa and Escherichia coli for whole-cell screening of antibacterial and adjuvant compounds

Continued efforts to discover new antibacterial molecules are critical to achieve a robust pre-clinical pipeline for new antibiotics. Screening of compound or natural product extract libraries remains a widespread approach and can benefit from the development of whole cell assays that are robust, simple and versatile, and allow for high throughput testing of antibacterial activity. In this study, we created and validated two bioluminescent reporter strains for high-throughput screening, one in Pseudomonas aeruginosa, and another in a hyperporinated and efflux-deficient Escherichia coli. We show that the bioluminescent strains have a large dynamic range that closely correlates with cell viability and is superior to conventional optical density (OD600) measurements, can detect dose-dependent antibacterial activity and be used for different drug discovery applications. We evaluated the assays’ performance characteristics (signal to background ratio, signal window, Z’ robust) and demonstrated their potential utility for antibiotic drug discovery in two examples. The P. aeruginosa bioluminescent reporter was used in a pilot screen of 960 repurposed compound libraries to identify adjuvants that potentiate the fluoroquinolone antibiotic ofloxacin. The E. coli bioluminescent reporter was used to test the antibacterial activity of bioactive bacterial supernatants and assist with bioassay-guided fractionation of the crude extracts.

Prevalence Estimation of Dysfibrinogenemia Using the Clauss-CWA Approach.

The actual prevalence of the qualitative fibrinogen abnormalities dysfibrinogenemia and hypodysfibrinogenemia is unknown. The major reasons are that patients with dysfibrinogenemia are frequently asymptomatic, and a recommended screening test, the Clauss fibrinogen assay, cannot completely distinguish qualitative from quantitative abnormalities. We previously established a high-throughput screening test (Clauss-CWA) to identify dysfibrinogenemia with high specificity and sensitivity by the Clauss fibrinogen assay alone. This was a single-center, observational study to estimate the prevalence of dysfibrinogenemia using Clauss-CWA technology. A total of 25 471 patients in Nagoya University Hospital were screened to identify patients with suspected dysfibrinogenemia. The suspected patients were investigated by further confirmatory analyses, such as antigenic fibrinogen determination, reptilase time, fibrin polymerization analysis, and scanning electron microscopy. Of the 25 471 enrolled patients, five with suspected dysfibrinogenemia were identified. Unfortunately, one patient was not confirmed due to a lack of plasma samples. The ratio of functional to antigenic fibrinogen was decreased, and the reptilase time was prolonged in the four patients. Interestingly, two of them showed normal functional fibrinogen levels due to acute inflammatory responses. Fibrin polymerization was impaired, and structural abnormalities were found in the fibrinogen from the patients. In some cases, functional fibrinogen levels may not be effective for identifying functional fibrinogen abnormalities. Further nationwide studies are needed to more precisely understand the epidemiology of dysfibrinogenemia.

NR1D1exon6::MAML2exon2 Fusion-Positive Sarcomas: Two Additional Cases of an Emerging Soft Tissue Tumor Entity.

NR1D1::MAML1/L2 fusion-positive sarcomas constitute an emerging subtype of undifferentiated sarcomas, histopathologically composed of epithelioid and spindle cells. We describe two NR1D1exon6::MAML2exon2 fusion-positive sarcomas occurring in the occipital region of a 53-year-old female patient and the left shoulder of a 25-year-old male patient. Histopathologically, the former tumor comprised spindle and polygonal-shaped/epithelioid cells, while the latter comprised epithelioid cells. Immunohistochemically, the cells of the first tumor were positive for keratin cocktail (AE1/AE3), EMA, and p40. The other tumor lacked any epithelial marker immunoexpression and showed SATB2 positivity. Both the tumors were SMARCB1 proficient. While the former tumor was initially diagnosed as a sarcomatoid carcinoma, the latter was diagnosed as an undifferentiated sarcoma.Both patients underwent surgical resection as the primary treatment. One patient received adjuvant radiotherapy, whereas the other received ifosfamide and doxorubicin-based therapy. However, both the patients developed progressive disease, with one developing lung metastasis during relapse. There was a suboptimal response to chemotherapy in both patients.This study highlights the clinicopathological features of two rare and emerging sarcomas. It further emphasizes the value of high-throughput molecular testing, such as next-generation sequencing in the undifferentiated epithelioid, and spindle-cell sarcomas to identify the prognostic subtypes and differentiate these neoplasms from their histopathological mimics.

High-throughput formulation of reproducible 3D cancer microenvironments for drug testing in myeloid leukemia

Leukemic microenvironment has been recognized as a factor that strongly supports the mechanisms of resistance. Therefore, targeting the microenvironment is currently one of the major directions in drug development and preclinical studies in leukemia. Despite the variety of available leukemia 3D culture models, the reproducible generation of miniaturized leukemic microenvironments, suitable for high-throughput drug testing, has remained a challenge. Here, we use droplet microfluidics to generate tens of thousands of highly monodisperse leukemic-bone marrow microenvironments within minutes. We employ gelatin methacryloyl (GelMA) as a model extracellular matrix (ECM) and tune the concentration of the biopolymer, check the impact of other components of the ECM (hyaluronic acid), cell concentration and the ratio of leukemic cells to bone marrow cells within the microbeads to establish the optimal conditions for microtissue formation. We administer model kinase inhibitor, imatinib, at various concentrations to the encapsulated leukemic microtissues, and, via comparing mono- and co-culture conditions (cancer alone vs cancer-stroma), we find that the stroma-leukemia crosstalk systematically protects the encapsulated cells against the drug-induced cytotoxicity. With that we demonstrate that our system mimics the physiological stroma-dependent protection. We discuss applicability of our model to (i) studying the role of direct- or close-contact interactions between the leukemia and bone marrow cells embedded in microscale 3D ECM on the stroma-mediated protection, and (ii) high-throughput screening of anti-cancer therapeutics in personalized leukemia therapies.

Symplectic contact analysis of a horizontally laminated magneto-electro-elastic finite specimen

This work formulates a symplectic framework for contact analysis of a horizontally laminated, magneto-electro-elastic, finite specimen. By incorporating dual variables and deriving the Hamiltonian operator matrix within the symplectic space, the constraints on the state variables are fulfilled through the property of symplectic orthogonality. The state variables between different layers are connected via the transfer matrices, and the Hamiltonian mixed energy variational principle is generalized for the laminated medium. Distinct from traditional methods, the symplectic framework offers a more realistic and efficient analysis approach for a finite-sized specimen, applicable to indenters with arbitrary profiles but predetermined contact region. The interfacial and boundary effects are explored, with remarkably accurate results as compared to the finite element simulations. This research not only enhances our understanding of the underlying phenomena but also lays a foundation for materials characterization using high-throughput testing techniques.

Human genetic variation reveals FCRL3 is a lymphocyte receptor for Yersinia pestis.

Yersinia pestis is the gram-negative bacterium responsible for plague, one of the deadliest and most feared diseases in human history. This bacterium is known to infect phagocytic cells, such as dendritic cells and macrophages, but interactions with non-phagocytic cells of the adaptive immune system are frequently overlooked despite the importance they likely hold for human infection. To discover human genetic determinants of Y. pestis infection, we utilized nearly a thousand genetically diverse lymphoblastoid cell lines in a cellular genome-wide association study method called Hi-HOST (High-throughput Human in-vitrO Susceptibility Testing). We identified a nonsynonymous SNP, rs2282284, in Fc receptor like 3 (FCRL3) associated with bacterial invasion of host cells (p=9×10-8). FCRL3 belongs to the immunoglobulin superfamily and is primarily expressed in lymphocytes. rs2282284 is within a tyrosine-based signaling motif, causing an asparagine-to-serine mutation (N721S) in the most common FCRL3 isoform. Overexpression of FCRL3 facilitated attachment and invasion of non-opsonized Y. pestis. Additionally, FCRL3 colocalized with Y. pestis at sites of cellular attachment, suggesting FCRL3 is a receptor for Y. pestis. These properties were variably conserved across the FCRL family, revealing molecular requirements of attachment and invasion, including an Ig-like C2 domain and a SYK interaction motif. Direct binding was confirmed with purified FCRL5 extracellular domain. Following attachment, invasion of Y. pestis was dependent on SYK and decreased with the N721S mutation. Unexpectedly, this same variant is associated with risk of chronic hepatitis C virus infection in BioBank Japan. Thus, Y. pestis hijacks FCRL proteins, possibly taking advantage of an immune receptor to create a lymphocyte niche during infection.

3D-printed suction clamps for tensile testing of brain tissue.

The conventional mounting of ultra-soft biological tissues often involves gluing it between two plates or manually tightening grips. Both methods demand delicate handling skills and are time-consuming. This study outlines the design and practical application of 3D-printed suction clamps for uniaxial tension tests on brain samples. Successful testing was defined by the absence of relevant slippage or the sample being drawn into the clamp. A total of 112 deer brain samples underwent testing using a universal testing machine after one freeze-thaw cycle. These samples were obtained from eight different brain regions. During sample preparation, 7 out of all samples failed. Among the 105 tests, 89 (85%) were successful. Of the 16 unsuccessful tests, 15 samples (14%) slipped, while only one sample (1%) was drawn into the clamp to an extent that testing became impossible. Medulla oblongata samples exhibited exceptionally high slippage at 38%, whereas samples from the temporal cortex, external capsule, and putamen had the lowest slippage in only one single case. In conclusion, suction clamps facilitate high-throughput testing through user-friendly and rapid sample mounting. Testing success is contingent on the specific brain site, with sample slippage being the primary reason for testing failures, while sample inspiration into the clamp is negligible.

Colony Fingerprinting: Image-Based Pathogen Identification for High-Throughput Food Microbiological Testing

Introduction: The contamination of foodborne pathogens in food products poses a substantial risk to public health. Precise detection and identification of pathogens is important for food safety. Conventional identification techniques rely on biochemical metabolic profiling and 16S ribosomal RNA sequencing. Recently, mass spectrometry-based approaches, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been employed to distinguish pathogens. These methodologies require expensive reagents and equipment, expertise, and prolonged incubation. To overcome these issues, we introduce colony fingerprinting (CFP) that identify bacterial species based on image analysis of bacterial micro-colony without additional works [1,2]. CFP utilizes machine learning technology to distinguish bacterial species based on image characteristics, such as the shape or optical patterns. In this study, we newly developed a line image sensor-based CFP system [3]. The imaging unit offers practical throughput by covering the entire surface of the Petri dish used in the actual field. To demonstrate the efficacy of the system, we collected datasets of bacterial colony images using the system and trained a bacterial species identification model. The model enables the detection of Staphylococcus aureus inoculated in food samples with high accuracy in less than half the time required by conventional methods. This result indicates the usefulness of the CFP system for easy and rapid pathogen testing. Materials and Methods: The CFP system consisted of an imaging unit and an incubation unit (124 cm in width, 44 cm in height, 56 cm in depth). It captured colony images using a line image sensor consisting of 16,384 pixels (with a pixel size of 3.52 μm) under a collimated blue light-emitting diode emitting light (470 nm). The imaging area of this system for a single scan was 57.5 mm × 95 mm with a resolution of 44.2 μm. The incubation unit was equipped with an electric motor-driven Petri dish (92 mm) holder designed to be moved into the imaging unit during the culture process. To maintain the incubation temperature, the incubator unit employed an air heater based on a proportional-integral-differential controller. Bacteria were cultured on solid agar medium at the appropriate temperatures for durations ranging from 24 to 72 h. Less than 100 bacterial cells were inoculated onto a Petri dish for each species. In this study, fifteen bacterial species were analyzed, including four foodborne pathogens: S. aureus, Bacillus cereus, Escherichia coli, and Salmonella enterica. Results & Discussion: We monitored the growth of 1335 colonies (15 species × 89 colonies) at 5-min intervals to collect datasets for a machine learning. To extract image characteristics from colony images, colony regions were segmented using a trained fully convolutional network. Fifty-nine discrimination parameters were calculated to characterize the colony images in terms of luminance, texture, geometric details, and growth rate at 13 distinct growth stages (0.025 to 1.0 mm2) in an individual colony. The dataset was split into two subsets, with 80% of the data used to train the XGBoost classification model. The remaining 20% was allocated for validation to assess the accuracy of the model. The validation results show that an average accuracy of 92% was achieved when the colony size exceeded 0.3 mm2, and the accuracy increased to 96% at 0.6 mm2. This result suggests that the model constructed in this study is capable of identifying microbial species with high accuracy even in early stage of culturing.To verify the generalization performance of this model, we conducted validations for the detection of S. aureus, which is a significant foodborne pathogen, in spiked milk samples. We monitored three Petri dishes inoculated with diluted milk samples and analysed image parameters from 239 colonies. As a result, 96% S. aureus were successfully detected when the colony size exceeded 0.3 mm2, requiring 10 hours of incubation. Our model enables the rapid detection of pathogenic bacteria compared with MALDI-TOF-MS. On the other hand, our system could not perfectly detect S. aureus, despite the distinct colony colors of the misidentified species. This limitation arises from the property of imaging unit that can only acquire grayscale data. To improve the discrimination accuracy, the line image sensor was replaced with one equipped with RGB elements. Consequently, misidentified species in grayscale were clearly discriminated. These results highlighted the potential of color image analysis for enhanced discrimination.

A novel ionic liquid-based approach for DNA and RNA extraction simplifies sample preparation for bacterial diagnostics

DNA- and RNA-based diagnostics play a pivotal role in accurately detecting and characterizing health-relevant bacteria, offering insights into bacterial presence, viability and treatment efficacy. Herein, we present the development of a novel extraction protocol for both DNA and RNA, designed to enable simple and rapid molecular diagnostics. The extraction method is based on the hydrophilic ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate and silica-coated magnetic beads. First, we developed an IL-based cell lysis protocol for bacteria that operates at room temperature. Subsequently, we established a magnetic bead purification procedure to efficiently and reproducibly extract DNA and RNA from the IL-lysates. The IL not only lyses the cells, but also facilitates the adsorption of nucleic acids (NAs) onto the surface of the magnetic beads, eliminating the need for a chaotropic binding buffer and allowing for purification of NAs without significant effort and materials required. Lastly, we combined the cell lysis step and the purification step and evaluated the novel IL-based extraction method on periopathogenic bacterial cultures, comparing it to commercial DNA and RNA extraction kits via (RT)-qPCR. In comparison to the reference methods, the IL-based extraction protocol yielded similar or superior results. Furthermore, costs are lower, required materials and equipment are minimal and the process is fast (30 min), simple and automatable. These characteristics favour the developed method for use in routine and high-throughput testing as well as in point-of-care, on-site and low-resource settings, thereby advancing the field of molecular diagnostics.Graphical

Molecular and functional profiling of primary normal ovarian cells defines insights into cancer development and drug responses

Patients with ovarian cancer, especially the high-grade serous (HGSOC) subtype, face poor prognosis due to late diagnosis and treatment resistance. Owing to the high heterogeneity of HGSOC, identifying the origin of the disease and optimal treatments is difficult. Here, we characterized two primary immortalized human ovarian cell lines, HOSE1C and HOSE2C, comparing their molecular profiling with representative HGSOC cells. We identified molecular features associated with normal and malignant phenotype of ovarian cells by applying single-cell transcriptomics, high-content imaged-based Cell Painting, and high-throughput drug testing. Our findings reveal distinct transcriptomic and morphological profiles for the two HOSEs, with a stromal phenotype. Moreover, their responses to the tumor microenvironment differ, exemplified by STAT1 and GREM1 upregulation in HOSE1C and HOSE2C, respectively. We identified selective activation of ERK/MEK targeted inhibitors in cancer cells compared to HOSEs. This study offers insights into the normal and malignant ovarian cells, shedding light on cancer development and drug responses.

Microdissected tumor cuboids: a microscale cancer model for large-scale testing that retains a complex tumor microenvironment.

To bridge the gap between bench and bedside, there is a need for more faithful models of human cancers that can recapitulate key features of the human tumor microenvironment (TME) and simultaneously facilitate large-scale drug tests. Our recently developed microdissection method optimizes the yield of large numbers of cuboidal microtissues (″cuboids″, ~(400 µm) 3 ) from a tumor biopsy. Here we demonstrate that cuboids from syngeneic mouse tumor models and human tumors retain a complex TME, making them amenable for drug and immunotherapy evaluation. We characterize relevant TME parameters, such as cellular architecture, cytokine secretion, proteomics profiles, and response to drug panels in multi-well arrays. Despite the cutting procedure and the time spent in culture (up to 7 days), the cuboids display strong cytokine expression and drug responses, including to immunotherapy. Overall, our results suggest that cuboids could provide essential therapeutic information for personalized oncology applications and could help the development of TME-dependent therapeutics and cancer disease models, including for clinical trials.

CRISPR/Cas9-Mediated Genome Editing of T4 Bacteriophage for High-Throughput Antimicrobial Susceptibility Testing.

The accurate and effective determination of antimicrobial resistance is essential to limiting the spread of infectious diseases and ensuring human health. Herein, a simple, accurate, and high-throughput phage-based colorimetric sensing strategy was developed for antimicrobial susceptibility testing (AST). Taking advantage of the CRISPR/Cas9 system, the genome of the T4 phage was modularly engineered to carry lacZ-α (lacZa), a marker gene encoding the α-fragment of β-galactosidase (β-gal). T4lacZa phages were identified by blue-white selection and then used for a biosensing application. In this strategy, the bacterial solution is exposed to the T4lacZa phage, causing target bacteria to overexpress β-gal. Upon the addition of a colorimetric substrate, the β-gal initiates an enzymatic reaction, resulting in a solution color change from yellow to red. This sensing strategy offers a visual way to monitor bacterial growth in the presence of antibiotics, enabling the determination of bacterial antimicrobial susceptibility. As a proof of concept, our developed sensing strategy was successfully applied to identify 9 different multidrug-resistant Escherichia coli (E. coli) in urine samples with 100% specificity. Compared with conventional disk diffusion susceptibility tests, the engineered phage-based sensing strategy can shorten the detection time by at least half without losing detection sensitivity, providing an alternative high-throughput method for AST in clinical diagnosis.

High-throughput phenotype-to-genotype testing of meningococcal carriage and disease isolates detects genetic determinants of disease-relevant phenotypic traits.

Genome-wide association studies (GWAS) with binary or single phenotype data have successfully identified disease-associated genotypes and determinants of antimicrobial resistance. We describe a novel phenotype-to-genotype approach for a major bacterial pathogen that involves simultaneously testing for associations among multiple disease-related phenotypes and linkages between phenotypic variation and genetic determinants. High-throughput assays quantified variation among 163 Neisseria meningitidis serogroup W ST-11 clonal complex isolates for 11 phenotypic traits. A comparison of carriage and two disease subgroups detected significant differences between groups for eight phenotypic traits. Candidate genotypic testing indicated that indels in csw, a capsular biosynthesis gene, were associated with reduced survival in antibody-depleted heat-inactivated serum. GWAS testing detected 341 significant genetic variants (3 single-nucleotide polymorphisms and 338 unitigs) across all traits except serum bactericidal antibody-depleted assays. Growth traits were associated with variants of capsular biosynthesis genes, carbonic anhydrase, and an iron-uptake system while adhesion-linked variation was in pilC2, marR, and mutS. Multiple phase variation states or combinatorial phasotypes were associated with significant differences in multiple phenotypes. Controlling for group effects through regression and recursive random forest approaches detected group-independent effects for nalP with biofilm formation and fetA with a growth trait. Through random forest testing, nine phenotypes were weakly predictive of MenW:cc11 sub-lineage, original or 2013, for disease isolates while three characteristics separated carriage and disease isolates with >80% accuracy. This study demonstrates the power of combining high-throughput phenotypic testing of pathogenically relevant isolate collections with genomics for identifying genetic determinants of specific disease-relevant phenotypes and the pathobiology of microbial pathogens.IMPORTANCENext-generation sequencing technologies have led to the creation of extensive microbial genome sequence databases for several bacterial pathogens. Mining of these databases is now imperative for unlocking the maximum benefits of these resources. We describe a high-throughput methodology for detecting associations between phenotypic variation in multiple disease-relevant traits and a range of genetic determinants for Neisseria meningitidis, a major causative agent of meningitis and septicemia. Phenotypic variation in 11 disease-related traits was determined for 163 isolates of the hypervirulent ST-11 lineage and linked to specific single-nucleotide polymorphisms, short sequence variants, and phase variation states. Application of machine learning algorithms to our data outputs identified combinatorial phenotypic traits and genetic variants predictive of a disease association. This approach overcomes the limitations of generic meta-data, such as disease versus carriage, and provides an avenue to explore the multi-faceted nature of bacterial disease, carriage, and transmissibility traits.

On-Chip NADH Detection in Multicellular Models Using an AlGaN/GaN Photodetector Array with Enhanced Sensitivity.

Nicotinamide adenine dinucleotide (NAD) is a pivotal coenzyme, existing in its oxidized form (NAD+) and reduced form (NADH). Both are essential in cellular redox reactions and are implicated in energy production and cancer. Current NADH detection methods often involve complex optical measurements. We propose a miniaturized, on-chip photoelectric sensor array using AlGaN/GaN two-dimensional electron gas (2DEG) photodetectors for NADH quantification. The device exhibits an ultralow dark current and ultrahigh UV light responsivity, enabling sensitive NADH detection. By exploiting the absorbance disparity between NADH and NAD+, our sensor achieves rapid, sensitive detection, surpassing commercial assays. It effectively detects NADH levels in 3D multicellular models, promising cancer screening and monitoring. This sensor platform offers a significant advancement in NADH quantification, with the potential for high-throughput testing and point-of-care diagnostics. Our study presents an efficient approach for NADH sensing, addressing the need for rapid and sensitive detection methods in biomedical research and clinical practice.

Zebrafish arterial valve development occurs through direct differentiation of second heart field progenitors.

Bicuspid Aortic Valve (BAV) is the most common congenital heart defect, affecting at least 2% of the population. The embryonic origins of BAV remain poorly understood, with few assays for validating patient variants, limiting the identification of causative genes for BAV. In both human and mouse, the left and right leaflets of the arterial valves arise from the outflow tract cushions, with interstitial cells originating from neural crest cells and the overlying endocardium through endothelial-to-mesenchymal transition (EndoMT). In contrast, an EndoMT-independent mechanism of direct differentiation of cardiac progenitors from the second heart field (SHF) is responsible for the formation of the anterior and posterior leaflets. Defects in either of these developmental mechanisms can result in BAV. Although zebrafish have been suggested as a model for human variant testing, their naturally bicuspid arterial valve has not been considered suitable for understanding human arterial valve development. Here, we have set out to investigate to what extent the processes involved in arterial valve development are conserved in zebrafish and ultimately, whether functional testing of BAV variants could be carried out. Using a combination of live imaging, immunohistochemistry and Cre-mediated lineage tracing, we show that the zebrafish arterial valve primordia develop directly from SHF progenitors with no contribution from EndoMT or neural crest, in keeping with the human and mouse anterior and posterior leaflets. Moreover, once formed, these primordia share common subsequent developmental events with all three aortic valve leaflets. Our work highlights a conserved ancestral mechanism of arterial valve leaflet formation from the SHF and identifies that development of the arterial valve is distinct from that of the atrioventricular valve in zebrafish. Crucially, this confirms the utility of zebrafish for understanding the development of specific BAV subtypes and arterial valve dysplasia, offering potential for high-throughput variant testing.

Full-Thickness Perfused Skin-on-a-Chip with In Vivo-Like Drug Response for Drug and Cosmetics Testing.

In this study, we present a novel 3D perfused skin-on-a-chip model fabricated using micro-precision 3D printing, which offers a streamlined and reproducible approach for incorporating perfusion. Perfused skin models are well-regarded for their advantages, such as improved nutrient supply, enhanced barrier function, and prolonged tissue viability. However, current models often require complex setups, such as self-assembled endothelial cells or sacrificial rods, which are prone to variability and time-consuming. Our model uses projection micro-stereolithography 3D printing to create precise microcapillary-like channels using a biocompatible resin, overcoming the drug-absorbing properties of PDMS. A customized chip holder allows for the simultaneous culture of six perfused chips, enabling high-throughput testing. The engineered skin-on-a-chip features distinct dermis and epidermis layers, confirmed via H&E staining and immunostaining. To evaluate drug screening capabilities, inflammation was induced using TNF-α and treated with dexamethasone, with cytokine levels compared to 2D cultures and human skin biopsies. Our 3D model exhibited drug response trends similar to human skin, while showing reduced cytotoxicity over time compared to biopsies. This perfused skin-on-a-chip provides a reliable, physiologically relevant alternative for drug and cosmetics screening, simplifying perfusion setup while preserving key benefits.

Application of high-throughput drug sensitivity testing in children with relapsed and refractory acute leukemia

To explore the current application of high-throughput drug sensitivity (HDS) testing in children with relapsed and refractory acute leukemia (RR-AL) and analyze the feasibility of salvage treatment plans. A retrospective collection of clinical data from children with RR-AL who underwent HDS testing at the Department of Children's Hematology and Oncology of the First Affiliated Hospital of Zhengzhou University from November 2021 to October 2023 was conducted, followed by an analysis of drug sensitivity results and treatment outcomes. A total of 17 children with RR-AL underwent HDS testing, including 7 cases of relapsed refractory acute myeloid leukemia and 10 cases of relapsed refractory acute lymphoblastic leukemia. The detection rate of highly sensitive chemotherapy drugs/regimens was 53% (9/17), while the detection rate of moderately sensitive chemotherapy drugs/regimens was 100% (17/17). Among the 17 RR-AL patients with highly and moderately sensitive chemotherapy drugs and regimens, the MOACD regimen (mitoxantrone + vincristine + cytarabine + cyclophosphamide + dexamethasone) accounted for 100%, with the highest inhibition rate for single-agent mitoxantrone (94%, 16/17), and the highest inhibition rate for targeted therapy being bortezomib (94%, 16/17). Nine patients adjusted their chemotherapy based on HDS testing results, with 4 undergoing hematopoietic stem cell transplantation. Four patients achieved disease-free survival, while 5 died. Eight patients received empirical chemotherapy, with 2 undergoing hematopoietic stem cell transplantation; 4 achieved disease-free survival, while 4 died. HDS testing can identify highly sensitive drugs/regimens for children with RR-AL, improving the rate of re-remission and creating conditions for subsequent hematopoietic stem cell transplantation.