High-throughput Platform Research Articles

Multicenter Clinical Performance Evaluation of the NeuMoDx™ CT/NG Assay 2.0.

Given the continued increases in rates of both Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infection, additional diagnostic assays may be useful in increasing access to testing for these sexually transmitted infections. We evaluated the performance of the NeuMoDx™ CT/NG Assay 2.0 on the NeuMoDx-96 and NeuMoDx-288 Molecular Systems. The clinical sensitivity and specificity of the assay was assessed when used with: 1) endocervical swabs; 2) self-, and clinician-collected vaginal swabs; and 3) first-catch urine specimens (female and male). Results were compared to a patient infection status based on US Food and Drug Administration (FDA)-cleared assays. The NeuMoDx CT/NG Assay 2.0 demonstrated high sensitivity and specificity in both symptomatic and asymptomatic participants. All specimen types other than endocervical swabs had >95% sensitivity and > 99% specificity for both pathogens. For endocervical samples, sensitivity was 93.2% and 93.3% for CT and NG, respectively. There was no difference in performance based on platform. The frequency of invalid results was low (<1%). The NeuMoDx CT/NG Assay 2.0 demonstrated performance similar to currently FDA-cleared assays, with the added choice of a moderate- (96-sample) or a high-throughput (288-sample) platform. The system therefore offers solutions to laboratories running lower volumes of testing that may obviate the need for outsourcing to larger reference laboratories.

Quantitative Top-down Proteomics Revealed Kinase Inhibitor-Induced Proteoform-Level Changes in Cancer Cells.

Quantitative analysis of proteins and their post-translational modifications (PTMs) in complex biological samples is critical to understanding cellular biology as well as disease detection and treatment. Top-down proteomics methods provide a "bird's eye" view of the proteome by directly detecting and quantifying intact proteoforms. Here, we developed a high-throughput quantitative top-down proteomics platform to probe intact proteoform and phosphoproteoform abundance changes in HeLa cells as a result of treatment with staurosporine (STS), a broad-spectrum kinase inhibitor. In total, we identified and quantified 1187 proteoforms from 215 proteoform families. Among them, 55 proteoforms from 37 proteoform families were significantly changed upon STS treatment. These proteoforms were primarily related to catabolic, metabolic, and apoptotic pathways that are expected to be impacted as a result of kinase inhibition. In addition, we manually evaluated 25 proteoform families that expressed one or more phosphorylated proteoforms. We observed that phosphorylated proteoforms in the same proteoform family, such as eukaryotic initiation factor 4E binding protein 1 (4EBP1), were differentially regulated relative to the unphosphorylated proteoforms. Combining relative profiling of proteoforms within these proteoform families with individual proteoform profiling results in a more comprehensive picture of STS treatment-induced proteoform abundance changes that cannot be achieved using bottom-up methods.

Identification of VISTA regulators in macrophages mediating cancer cell survival.

Numerous human cancers have exhibited the ability to elude immune checkpoint blockade (ICB) therapies. This type of resistance can be mediated by immune-suppressive macrophages that limit antitumor immunity in the tumor microenvironment (TME). Here, we elucidate a strategy to shift macrophages into a proinflammatory state that down-regulates V domain immunoglobulin suppressor of T cell activation (VISTA) via inhibiting AhR and IRAK1. We used a high-throughput microfluidic platform combined with a genome-wide CRISPR knockout screen to identify regulators of VISTA levels. Functional characterization showed that the knockdown of these hits diminished VISTA surface levels on macrophages and sustained an antitumor phenotype. Furthermore, targeting of both AhR and IRAK1 in mouse models overcame resistance to ICB treatment. Tumor immunophenotyping indicated that infiltration of cytotoxic CD8+ cells, natural killer cells, and antitumor macrophages was substantially increased in treated mice. Collectively, AhR and IRAK1 are implicated as regulators of VISTA that coordinate a multifaceted barrier to antitumor immune responses.

High-throughput expansion microscopy enables scalable super-resolution imaging.

Expansion microscopy (ExM) enables nanoscale imaging using a standard confocal microscope through the physical, isotropic expansion of fixed immunolabeled specimens. ExM is widely employed to image proteins, nucleic acids, and lipid membranes in single cells; however, current methods limit the number of samples that can be processed simultaneously. We developed High-throughput Expansion Microscopy (HiExM), a robust platform that enables expansion microscopy of cells cultured in a standard 96-well plate. Our method enables ~4.2 x expansion of cells within individual wells, across multiple wells, and between plates. We also demonstrate that HiExM can be combined with high-throughput confocal imaging platforms to greatly improve the ease and scalability of image acquisition. As an example, we analyzed the effects of doxorubicin, a known cardiotoxic agent, on human cardiomyocytes (CMs) as measured by the Hoechst signal across the nucleus. We show a dose-dependent effect on nuclear DNA that is not observed in unexpanded CMs, suggesting that HiExM improves the detection of cellular phenotypes in response to drug treatment. Our method broadens the application of ExM as a tool for scalable super-resolution imaging in biological research applications.

High-throughput expansion microscopy enables scalable super-resolution imaging

Expansion microscopy (ExM) enables nanoscale imaging using a standard confocal microscope through the physical, isotropic expansion of fixed immunolabeled specimens. ExM is widely employed to image proteins, nucleic acids, and lipid membranes in single cells; however, current methods limit the number of samples that can be processed simultaneously. We developed High-throughput Expansion Microscopy (HiExM), a robust platform that enables expansion microscopy of cells cultured in a standard 96-well plate. Our method enables ~4.2 x expansion of cells within individual wells, across multiple wells, and between plates. We also demonstrate that HiExM can be combined with high-throughput confocal imaging platforms to greatly improve the ease and scalability of image acquisition. As an example, we analyzed the effects of doxorubicin, a known cardiotoxic agent, on human cardiomyocytes (CMs) as measured by the Hoechst signal across the nucleus. We show a dose-dependent effect on nuclear DNA that is not observed in unexpanded CMs, suggesting that HiExM improves the detection of cellular phenotypes in response to drug treatment. Our method broadens the application of ExM as a tool for scalable super-resolution imaging in biological research applications.

High-throughput capture of transcription factor-driven epigenome dynamics using PHILO ChIP-seq.

Assessing the dynamics of chromatin features and transcription factor (TF) binding at scale remains a significant challenge in plants. Here, we present PHILO (Plant HIgh-throughput LOw input) ChIP-seq, a high-throughput ChIP-seq platform that enables the cost-effective and extensive capture of TF binding and genome-wide distributions of histone modifications. The PHILO ChIP-seq pipeline is adaptable to many plant species, requires very little starting material (1mg), and provides the option to use MNase (micrococcal nuclease) for chromatin fragmentation. By employing H3K9ac PHILO ChIP-seq on eight Arabidopsis thaliana jasmonic acid (JA) pathway mutants, with the simultaneous processing of over 100 samples, we not only recapitulated but also expanded the current understanding of the intricate interplay between the master TFs MYC2/3/4 and various chromatin regulators. Additionally, our analyses brought to light previously unknown histone acetylation patterns within the regulatory regions of MYC2 target genes in Arabidopsis, which is also conserved in tomato (Solanum lycopersicum). In summary, our PHILO ChIP-seq platform demonstrates its high effectiveness in investigating TF binding and chromatin dynamics on a large scale in plants, paving the way for the cost-efficient realization of complex experimental setups.

HTBPPS: A high-throughput behavioral phenotyping platform for shrimp

In shrimp breeding, phenotypic measurement is crucial for identifying key traits. Meanwhile, behavioral traits typically exhibiting moderate to high heritability are gaining recognition as novel traits in aquatic species breeding. However, traditional shrimp behavioral phenotype measurement is unsuitable for large-scale evaluations as it relies primarily on time-consuming and error-prone manual observations. With advances in computer vision, various behavior recognition and tracking algorithms have effectively overcome these limitations. Yet, such algorithms often prove inadequate for long-duration behavioral video data. Inspired by high-throughput phenotyping platforms in plant research, this study designed specialized temporary housing and observation apparatus to track shrimp behavior and the shrimp.tracker software based on a boundary-constrained Kalman filter algorithm. Additionally, this study established an analysis system for shrimp behavioral phenotypic data and a framework for long-term high-throughput behavioral data acquisition. Ultimately, these hardware and software systems form a high-throughput behavioral phenotyping platform for shrimp. The platform's algorithm scored 92.4 in sMOTSA, 99.5 in MOTA, and 95.5 in MOTSP, with a detection accuracy of 99.79 %, surpassing the deep learning-based Track-RCNN algorithm. Case studies illustrated the platform's capability to track various shrimp species, multiple individuals over extended periods, and shrimp under high- temperature stress. The experimental results revealed significant behavioral phenotype differences among different types and sizes of shrimp. During long-term behavioral observations, shrimp behaviors exhibited 24-h cyclical rhythm changes. Different types of shrimp exhibited the same M-shaped trend in mobility during linear temperature increases, with the last peak of the M shape moderately correlating with shrimp heat tolerance. These findings emphasize the importance of behavioral phenotypes in shrimp breeding, serving as potential indicators for assessing heat tolerance in shrimp breeding. Thus, the proposed platform has significant potential for future applications in shrimp breeding.

Heteroatom-regulated iron single-atom nanozymes for universal colorimetric discrimination of mycotoxins and metal ions

Single-atom nanozymes (SAzymes) have garnered considerable attentions in biosensing due to their superior catalytic properties. Although significant progress has been made in this field, designing SAzymes with satisfactory catalytic performance and constructing high-throughput sensor platforms remains a major challenge. Herein, we design and fabricate an iron single-atom catalyst with the optimal Fe-N3PS active moiety supported on N, P, S co-doped hollow carbon nanocage (Fe-N3PS/HC). As expected, the prepared Fe-N3PS/HC SAzyme shows outstanding peroxidase-like activity and kinetics. Moreover, the density-functional theory (DFT) calculations reveal that the doped P and S atoms endow Fe moiety with unique electronic structure due to the regulation of atomic configuration, and thus improve the peroxide-like activity of Fe-N3PS/HC SAzyme kinetically and thermodynamically. A three-channel nanozyme sensor array combing Fe-N3PS/HC SAzyme and three colorimetric reactions is established for colorimetric discrimination of mycotoxins and metal ions. Different analytes have varying interactions with Fe-N3PS/HC and diverse degrees of catalytic oxidation abilities, thus resulting in fingerprint-like colorimetric response for the analytes. The statistical analysis results show that the sensor array displays excellent discrimination ability for five mycotoxins and ten metal ions, and perform well in quantitative determination at the level of ng/mL and μM. Additionally, the sensor array allows precise identification even in multicomponent mixtures and real samples (e.g., corn juice and actual water sample), demonstrating its potential in the applications of food safety and environment monitoring.

Analysis of the virulence of a lethal, carbapenem-resistant hypervirulent KPC-33-producing Klebsiella pneumoniae: Emergence of ST11-KL64 hv-CRKP in ICU

ObjectiveHypervirulent and carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) poses a serious threat to public health. Here, we analyse a case of systemic infection caused by a hv-CRKP, which ultimately led to the patient's death from sepsis. And a total of 30 CRKPs were analyzed to elucidate the molecular epidemiological features of CRKPs in the hospital, and to provide a basis for clinical anti-infective therapy. MethodsIn this case, a total of 7 K. pneumoniae strains were isolated from the blood, sputum, urine, and feces of the patient. The Vitek-2 compact system was used to identify the strains and perform antimicrobial susceptibility testing. Biofilm formation, siderophore production assays and Galleria mellonella infection model were used to verify the virulence phenotypes of the strains in the case. Whole-genome sequencing was conducted on the four hv-CRKP isolated from different samples in the case and 26 other CRKP collected in our hospital from September to November in 2022, using the Illumina Hiseq 6000 high-throughput sequencing platform to analyse the resistance and virulence genes. ResultsIn the case, after 7 days of treatment with ceftazidime-avibactam (CZA), the resistance profile of the strains changed. The strain that was initially sensitive to CZA developed to resistant, resistant to imipenem (IPM) developed to sensitive, and resistant to meropenem (MEM) developed to intermediate. Whole-genome sequencing revealed that the four strains in the case were all ST11-KL64 K. pneumoniae, and the change in resistance phenotype was due to the mutation from blaKPC-2 to blaKPC-33. KPN7 had a total of six plasmids, with siderophore-related genes iucABCD and iutA, and mucoid phenotype-related gene rmpA2 located on plasmid p4-KPN7; resistance genes blaKPC-33, blaTEM-1B, and blaCTX-M-65 located on plasmid p5-KPN7; and virulence genes fim, irp, iutA, and ybt located on the chromosome. Biofilm formation and siderophore production assays confirmed that the seven K. pneumoniae strains isolated in this case had strong biofilm formation and siderophore production capabilities. Galleria mellonella Infection Model showed that KPN4 and KPN7 was phenotypically highly virulent and KPN7 performed lower virulence compared to KPN4. Apart from the 4 hv-CRKP strains, other 26 CRKP strains all carried blaKPC-2, and 69.2% (18/26) were ST-11 and 30.8%(8/26) were ST-15. And 83.3% (15/18) were ST11-KL64 strains, followed by ST11-KL25 strains 11.1%(2/18) and ST11-KL47 strain 5.6%(1/18). All the eight ST-15 strains were KL-19. ConclusionThe ST11-KL64 hv-CRKP clone spread widely in ICU carried numerous resistance and virulence genes, and under antibiotic pressure, they easily underwent mutations resulting in changes in resistance phenotypes, especially in mutations of blaKPC-2 gene in acquiring resistance to CZA. Therefore, clinical attention should be paid to such strains, and the use of antibiotics should be adjusted promptly based on the susceptibility of the strains to antimicrobial agents.

Resistomes from oxytetracycline-treated pigs are readily transferred to untreated pen mates

Pork is currently a major part of Danish food export and is also a key dietary source of protein across the world. Industrial pork production, however, comes with high antibiotic usage in many countries, including Denmark. This has created consumer demand for meat Raised Without Antibiotics (RWA). Previous work has demonstrated that levels of antibiotic resistance genes (ARGs) are indeed increased in antibiotically treated animals, but also suggest that these ARGs are transferred to untreated pen-mates. In a Danish commercial farm, we studied four groups of physically separated pigs: one group of only antibiotic treated pigs (n = 20), one group of only untreated pigs (n = 30 total, n = 15 analysed), and one group combining treated (n = 15) and untreated pigs (n = 15). These groups were followed for 16 weeks during which all pigs were profiled for both their faecal microbiome (through 16 S rRNA gene sequencing) and resistome (by use of a high-throughput qPCR platform targeting 82 ARGs and their variants). We found that the resistome of treated pigs was substantially enriched in resistance genes compared to untreated pigs but, importantly, observed that untreated pigs co-reared with treated pigs had levels of resistance genes approaching their treated pen mates, suggesting that the treated enterotype is readily transferred to the untreated animal. From this, we conclude that mixing of treated and untreated pigs causes spill-over of antibiotic resistant bacteria and/or resistance genes from treated pigs when these are co-reared. To optimize RWA production, treated and untreated pigs should be physically separated to limit the proliferation of ARGs.

Pooled CRISPR screens with joint single-nucleus chromatin accessibility and transcriptome profiling.

Pooled single-cell CRISPR screens have profiled either gene expression or chromatin accessibility but not both modalities. Here we develop MultiPerturb-seq, a high-throughput CRISPR screening platform with joint single-nucleus chromatin accessibility, transcriptome and guide RNA capture using combinatorial indexing combined with droplet microfluidics to scale throughput and integrate all three modalities. We identify key differentiation genes in a rare pediatric cancer and establish ZNHIT1 as a potential target for cancer reprogramming therapy.

A high-throughput method for monitoring growth of lettuce seedlings in greenhouses based on enhanced Mask2Former

Monitoring plant growth is crucial for cultivation management. Agronomists can assess the health status of lettuce seedlings based on monitoring results to implement relevant management measures for improving the quality and yield of lettuce seedlings. This study developed a non-destructive, high-throughput growth monitoring method suitable for large-scale assessment of lettuce seedling quality in nurseries. The method utilizes a plant high-throughput phenotyping platform to acquire 10-day time-series imagery data. An Mask2Former network model enhanced by multidimensional collaborative attention mechanism, combined with sliding window and morphological operations, achieves precise recognition and localization of seedling trays, varieties, and individual seedling plants in a progressive manner. Based on individual seedling localization and segmentation results, the method estimates emergence numbers and rates for each variety, and further achieves instance segmentation and counting of individual seedling leaves, innovatively constructing leaf segmentation results of different varieties across the entire seedling tray. Applied to time-series images, the method automatically monitored seedling emergence changes and growth trends for 1,086 lettuce varieties. In monitoring these varieties, the method achieved a coefficient of determination (R2) of 0.96 for emergence number estimation. The extraction of all six key phenotypic parameters demonstrated exceptionally high correlations: projected area, projected perimeter, convex hull area, and convex hull perimeter all showed R2 above 0.99, while leaf compactness R2 was 0.9698, and leaf count R2 was 0.91. Results demonstrate that this high-throughput, reliable method can effectively monitor the growth status of large-scale lettuce seedlings and provide technical support for lettuce nursery quality assessment.

Advancements in Single-Cell Proteomics and Mass Spectrometry-Based Techniques for Unmasking Cellular Diversity in Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive and complex subtype of breast cancer characterized by a lack of targeted treatment options. Intratumoral heterogeneity significantly drives disease progression and complicates therapeutic responses, necessitating advanced analytical approaches to understand its underlying biology. This review aims to explore the advancements in single-cell proteomics and their application in uncovering cellular diversity in TNBC. It highlights innovations in sample preparation, mass spectrometry-based techniques, and the potential for integrating proteomics into multi-omics platforms. The review discusses the combination of improved sample preparation methods and cutting-edge mass spectrometry techniques in single-cell proteomics. It emphasizes the challenges associated with protein analysis, such as the inability to amplify proteins akin to transcripts, and examines strategies to overcome these limitations. Single-cell proteomics provides a direct link to phenotype and cell behavior, complementing transcriptomic approaches and offering new insights into the mechanisms driving TNBC. The integration of advanced techniques has enabled deeper exploration of cellular heterogeneity and disease mechanisms. Despite the challenges, single-cell proteomics holds immense potential to evolve into a high-throughput and scalable multi-omics platform. Addressing existing hurdles will enable deeper biological insights, ultimately enhancing the diagnosis and treatment of TNBC.

Barcoding of small extracellular vesicles with CRISPR-gRNA enables comprehensive, subpopulation-specific analysis of their biogenesis and release regulators

Small extracellular vesicles (sEVs) are important intercellular information transmitters in various biological contexts, but their release processes remain poorly understood. Herein, we describe a high-throughput assay platform, CRISPR-assisted individually barcoded sEV-based release regulator (CIBER) screening, for identifying key players in sEV release. CIBER screening employs sEVs barcoded with CRISPR-gRNA through the interaction of gRNA and dead Cas9 fused with an sEV marker. Barcode quantification enables the estimation of the sEV amount released from each cell in a massively parallel manner. Barcoding sEVs with different sEV markers in a CRISPR pooled-screening format allows genome-wide exploration of sEV release regulators in a subpopulation-specific manner, successfully identifying previously unknown sEV release regulators and uncovering the exosomal/ectosomal nature of CD63+/CD9+ sEVs, respectively, as well as the synchronization of CD9+ sEV release with the cell cycle. CIBER should be a valuable tool for detailed studies on the biogenesis, release, and heterogeneity of sEVs.

Highly multiplexed design of an allosteric transcription factor to sense new ligands

Allosteric transcription factors (aTF) regulate gene expression through conformational changes induced by small molecule binding. Although widely used as biosensors, aTFs have proven challenging to design for detecting new molecules because mutation of ligand-binding residues often disrupts allostery. Here, we develop Sensor-seq, a high-throughput platform to design and identify aTF biosensors that bind to non-native ligands. We screen a library of 17,737 variants of the aTF TtgR, a regulator of a multidrug exporter, against six non-native ligands of diverse chemical structures – four derivatives of the cancer therapeutic tamoxifen, the antimalarial drug quinine, and the opiate analog naltrexone – as well as two native flavonoid ligands, naringenin and phloretin. Sensor-seq identifies biosensors for each of these ligands with high dynamic range and diverse specificity profiles. The structure of a naltrexone-bound design shows shape-complementary methionine-aromatic interactions driving ligand specificity. To demonstrate practical utility, we develop cell-free detection systems for naltrexone and quinine. Sensor-seq enables rapid and scalable design of new biosensors, overcoming constraints of natural biosensors.

Decoding the Double Stress Puzzle: Investigating Nutrient Uptake Efficiency and Root Architecture in Soybean Under Heat- and Water-Stresses.

In the context of climate change, associated with increasingly frequent water deficits and heat waves, there is an urgent need to maintain the performance of soybean, a leading legume crop worldwide, before its yield declines. The objective of this study was to explore which plant traits improve soybean tolerance to heat and/or water stress, with a focus on traits involved in plant architecture and nutrient uptake. For this purpose, two soybean genotypes were grown under controlled conditions in a high-throughput phenotyping platform where either optimal conditions, heat waves, water stress or both heat waves and water stresses were applied during the vegetative stage. By correlating architectural to functional traits, related to water, carbon allocation and nutrient absorption, we were able to explain the stress susceptibility level of the two genotypes. We have shown that water flow in the plant is central to the uptake and allocation of mineral elements in the plant, despite its modulation by stress and in a genotype-dependent manner. This cross-analysis of plant ecophysiology and plant nutrition under different stresses provides new information, especially on the importance of mineral elements in the different plant organs, and can inform future crop design, particularly under changing climatic conditions.

Integrative Multi-PTM Proteomics Reveals Dynamic Global, Redox, Phosphorylation, and Acetylation Regulation in Cytokine-Treated Pancreatic Beta Cells

Studying regulation of protein function at a systems level necessitates an understanding of the interplay among diverse post-translational modifications (PTMs). A variety of proteomics sample processing workflows are currently used to study specific PTMs but rarely characterize multiple types of PTMs from the same sample inputs. Method incompatibilities and laborious sample preparation steps complicate large-scale physiological investigations and can lead to variations in results. The single-pot, solid-phase-enhanced sample preparation (SP3) method for sample cleanup is compatible with different lysis buffers and amenable to automation, making it attractive for high-throughput multi-PTM profiling. Herein, we describe an integrative SP3 workflow for multiplexed quantification of protein abundance, cysteine thiol oxidation, phosphorylation, and acetylation. The broad applicability of this approach is demonstrated using cell and tissue samples, and its utility for studying interacting regulatory networks is highlighted in a time-course experiment of cytokine-treated β-cells. We observed a swift response in global regulation of protein abundances consistent with rapid activation of JAK-STAT and NF-κB signaling pathways. Regulators of these pathways as well as proteins involved in their target processes displayed multi-PTM dynamics indicative of a complex cellular response stages: acute, adaptation, and chronic (prolonged stress). PARP14, a negative regulator of JAK-STAT, had multiple co-localized PTMs that may be involved in intraprotein regulatory crosstalk. Our workflow provides a high-throughput platform that can profile multi-PTMomes from the same sample set, which is valuable in unraveling the functional roles of PTMs and their co-regulation.

Rare copy number variant analysis in case–control studies using snp array data: a scalable and automated data analysis pipeline

BackgroundRare copy number variants (CNVs) significantly influence the human genome and may contribute to disease susceptibility. High-throughput SNP genotyping platforms provide data that can be used for CNV detection, but it requires the complex pipelining of bioinformatic tools. Here, we propose a flexible bioinformatic pipeline for rare CNV analysis from human SNP array data.ResultsThe pipeline consists of two major sub-pipelines: (1) Calling and quality control (QC) analysis, and (2) Rare CNV analysis. It is implemented in Snakemake following a rule-based structure that enables automation and scalability while maintaining flexibility.ConclusionsOur pipeline automates the detection and analysis of rare CNVs. It implements a rigorous CNV quality control, assesses the frequencies of these rare CNVs in patients versus controls, and evaluates the impact of CNVs on specific genes or pathways. We hence aim to provide an efficient yet flexible bioinformatic framework to investigate rare CNVs in biomedical research.

CRISPR-Cas9 mediated knockout of NDUFS4 in human iPSCs: A model for mitochondrial complex I deficiency

Mitochondrial diseases, often caused by defects in complex I (CI) of the oxidative phosphorylation system, currently lack curative treatments. Human-relevant, high-throughput drug screening platforms are crucial for the discovery of effective therapeutics, with induced pluripotent stem cells (iPSCs) emerging as a valuable technology for this purpose. Here, we present a novel iPSC model of NDUFS4-related CI deficiency that displays a strong metabolic phenotype in the pluripotent state. Human iPSCs were edited using CRISPR-Cas9 to target the NDUFS4 gene, generating isogenic NDUFS4 knockout (KO) cell lines. Sanger sequencing detected heterozygous biallelic deletions, whereas no indel mutations were found in isogenic control cells. Western blotting confirmed the absence of NDUFS4 protein in KO iPSCs and CI enzyme kinetics showed a ~56 % reduction in activity compared to isogenic controls. Comprehensive metabolomic profiling revealed a distinct metabolic phenotype in NDUFS4 KO iPSCs, predominantly associated with an elevated NADH/NAD+ ratio, consistent with alterations observed in other models of mitochondrial dysfunction. Additionally, β-lapachone, a recognized NAD+ modulator, alleviated reductive stress in KO iPSCs by modifying the redox state in both the cytosol and mitochondria. Although undifferentiated iPSCs cannot fully replicate the complex cellular dynamics of the disease seen in vivo, these findings highlight the utility of iPSCs in providing a relevant metabolic milieu that can facilitate early-stage, high-throughput exploration of therapeutic strategies for mitochondrial dysfunction.

An iridium(III) complex-based luminogenic probe for high-throughput screening of hydrogen sulfide donors in living cells

The scarcity of suitable high-throughput screening technology for hydrogen sulfide (H2S) donors has hampered the discovery of H2S donors. In this study, a long-lived cyclometalated iridium complex was rationally designed as a mitochondria-targeted H2S probe to monitor the real-time dynamic change of H2S. By using the time-resolved emission spectroscopy (TRES) technique, an anti-interference high-throughput screening system was developed to monitor H2S in living cells with decreased false negative results. As a proof-of-concept, three natural products were identified as potential H2S donors from a natural product library using the developed TRES probe. Notably, the discovery of allicin and diallyl trisulfide demonstrated the feasibility of this screening platform, while garlic-derived allyl methyl sulfide was explored as a H2S donor candidate. The results were further validated by a commercial assay. We anticipate this high-throughput platform could facilitate the discovery of H2S donors by discriminating the endogenous interfering fluorescence from biological systems.