High-throughput Format Research Articles

Mechanical characterization of freestanding lipid bilayers with temperature-controlled phase.

Coexistence of lipid domains in cell membranes is associated with vital biological processes. Here, we investigate two such membranes: a multi-component membrane composed of DOPC and DPPC lipids with gel and fluid separated domains, and a single component membrane composed of PMPC lipids forming ripples. We characterize their mechanical properties below their melting point, where ordered and disordered regions coexist, and above their melting point, where they are in fluid phase. To conduct these inquiries, we create lipid bilayers in a microfluidic chip interfaced with a heating system and optical tweezers. The chip features a bubble trap and enables high-throughput formation of planar bilayers. Optical tweezers experiments reveal interfacial hydrodynamics (fluid-slip) and elastic properties (membrane tension and bending rigidity) at various temperatures. For PMPC bilayers, we demonstrate a higher fluid slip at the interface in the fluid-phase compared to the ripple phase, while for the DOPC:DPPC mixture, similar fluid slip is measured below and above the transition point. Membrane tension for both compositions increases after thermal fluidization. Bending rigidity is also measured using the forces required to extend a lipid nanotube pushed out of the freestanding membranes. This novel temperature-controlled microfluidic platform opens numerous possibilities for thermomechanical studies on freestanding planar membranes.

A Hybrid 2D-to-3D in vitro Differentiation Platform Improves Outcomes of Cerebral Cortical Organoid Generation in hiPSCs.

Three-dimensional (3D) cerebral cortical organoids are popular in vitro cellular model systems widely used to study human brain development and disease, compared to traditional stem cell-derived methods that use two-dimensional (2D) monolayer cultures. Despite the advancements made in protocol development for cerebral cortical organoid derivation over the past decade, limitations due to biological, mechanistic, and technical variables remain in generating these complex 3D cellular systems. Building from our previously established differentiation system, we have made modifications to our existing 3D cerebral cortical organoid protocol that resolve several of these technical and biological challenges when working with diverse groups of human induced pluripotent stem cell (hiPSC) lines. This improved protocol blends a 2D monolayer culture format for the specification of neural stem cells and expansion of neuroepithelial progenitor cells with a 3D system for improved self-aggregation and subsequent organoid development. Furthermore, this "hybrid" approach is amenable to both an accelerated cerebral cortical organoid protocol as well as an alternative long-term differentiation protocol. In addition to establishing a hybrid technical format, this protocol also offers phenotypic and morphological characterization of stage-specific cellular profiles using antibodies and fluorescent-based dyes for live cell imaging. © 2024 Wiley Periodicals LLC. Basic Protocol 1: hiPSC-based 2D monolayer specification into neural stem cells (NSCs) Basic Protocol 2: Serial passaging and 2D monolayer expansion of neuroepithelial progenitor cells (NPCs) Support Protocol 1: Direct cryopreservation and rapid thawing of NSCs and NPCs Basic Protocol 3: Bulk aggregation of 3D neurospheres and accelerated cerebral cortical organoid differentiation Alternate Protocol 1: Bulk aggregation of 3D neurospheres and long-term cerebral cortical organoid differentiation Support Protocol 2: High-throughput 3D neurosphere formation and 2D neurosphere migration assay Support Protocol 3: LIVE/DEAD stain cell imaging assay of 3D neurospheres Support Protocol 4: NeuroFluor NeuO live cell dye for 3D cerebral cortical organoids.

A Novel Acoustic Modulation of Oscillating Thin Elastic Membrane for Enhanced Streaming in Microfluidics and Nanoscale Liposome Production.

Liposomes are widely utilized in therapeutic nanosystems as promising drug carriers for cancer treatment, which requires a meticulous synthesis approach to control the nanoprecipitation process. Acoustofluidic platforms offer a favorable synthesis environment by providing robust agitation and rapid mixing. Here, a novel high-throughput acoustofluidic micromixer is presented for a solvent and solvent-free synthesis of ultra-small and size-tunable liposomes. The size-tunability is achieved by incorporating glycerol as a new technique into the synthesis reagents, serving as a size regulator. The proposed device utilizes the synergistic effects of vibrating trapped microbubbles and an oscillating thin elastic membrane to generate vigorous acoustic microstreaming. The working principle and mixing mechanism of the device are explored numerically and experimentally. The platform exhibits remarkable mixing efficacy for aqueous and viscous solutions at flow rates up to 8000 µL/h, which makes it unique for high-throughput liposome formation and preventing aggregation. As a proof of concept, this study investigates the impact of phospholipid type and concentration, flow rate, and glycerol on the size and size distribution of liposomes. The results reveal a significant size reduction, from ≈900 nm to 40 nm, achieved by merely introducing 75% glycerol into the synthesis reagents, highlighting an innovative approach toward size-tunable liposomes.

New Highly Selective Antivirals for Chikungunya Virus identified from the Screening of a Drug-Like Compound Library.

Chikungunya fever is a mosquito-borne disease caused by Chikungunya virus (CHIKV). Treatment of CHIKV infections is currently supportive and does not limit viral replication or symptoms of persistent chronic arthritis. Although there are multiple compounds reported as antivirals active against CHIKV in vitro, there are still no effective and safe antivirals. Thus, active research aims at the identification of new chemical structures with antiviral activity. Here, we report the screen of the Pandemic Response Box library of small molecules against a fully infectious CHIKV reporter virus. Our screening approach successfully identified previously reported CHIKV antiviral compounds within this library and further expanded potentially active hits, supporting the use of reporter-virus-based assays in high-throughput screening format as a reliable tool for antiviral drug discovery. Four molecules were identified as potential drug candidates against CHIKV: MMV1634402 (Brilacidin) and MMV102270 (Diphyllin), which were previously shown to present broad-spectrum antiviral activities, in addition to MMV1578574 (Eravacycline), and the antifungal MMV689401 (Fluopicolide), for which their antiviral potential is uncovered here.

Fluorescence-Based HTS Assays for Ion Channel Modulation in Drug Discovery Pipelines.

Ion channels represent a druggable family of transmembrane pore-forming proteins with important (patho)physiological functions. While electrophysiological measurement (manual patch clamp) remains the only direct method for detection of ion currents, it is a labor-intensive technique. Although automated patch clamp instruments have become available to date, their high costs limit their use to large pharma companies or commercial screening facilities. Therefore, fluorescence-based assays are particularly important for initial screening of compound libraries. Despite their numerous disadvantages, they are highly amenable to high-throughput screening and in many cases, no sophisticated instrumentation or materials are required. These features predispose them for implementation in early phases of drug discovery pipelines (hit identification), even in an academic environment. This review summarizes the advantages and pitfalls of individual methodological approaches for identification of ion channel modulators employing fluorescent probes (i.e., membrane potential and ion flux assays) with emphasis on practical aspects of their adaptation to high-throughput format.

A homo-FRET assay for patatin-specific proteolytic activity

The potato protein patatin embeds bioactive peptides that require targeted hydrolysis to be released as promising food additives. This study presents a patatin-specific protease assay for assessing a wide range of protease activities in high-throughput format. Conjugating patatin to the amine reactive fluorogenic BODIPY FL dye provided a stable protease substrate with efficient homo-FRET quenching at a low degree (7–8) of labeling. Compared to commercial BODIPY-casein, BODIPY-patatin provided higher fluorescence enhancement (by de-quenching) at high protease concentrations, while the sensitivity was generally comparable for both highly specific (e.g. Trypsin) and industrial relevant proteases (e.g. Alcalase and Neutrase) at low doses. For Chymotrypsin, BODIPY-patatin provided a 39 % response improvement at 5 ng dose. A peptide-centric analysis of mass spectrometry-based bottom-up proteomics data identified several BODIPY-labeling sites with varying occupancies in patatin, indicating heterogenous labeling under the applied conjugation conditions. BODIPY-labeled patatin complements commercial BODIPY-labeled casein as a globular, plant-based alternative for screening of proteolytic activity.

High-Throughput Preosteoblastic Spheroids Elevate Fibroblast Growth Factor 23 via Parathyroid Hormone Signaling Pathway.

Fibroblast growth factor 23 (FGF23) plays a crucial role in managing renal phosphate and the synthesis of 1,25(OH)2-vitamin D3, which is essential for bone homeostasis. Developing robust in vitro systems to study FGF23-regulating mechanisms is crucial for advancing our knowledge and identifying potential therapeutic targets. The traditional in vitro 2D culture system results in relatively low expression of FGF23, complicating further exploration of its regulatory mechanisms and potential therapeutic targets. Herein, we reported a high-throughput approach to generate preosteoblastic cell spheroids with enhanced FGF23 production. For this purpose, murine preosteoblast cell line (MC3T3-E1) was cultured in our previously reported nonadherent microwells (200 µm in diameter, 148 µm in depth, and 100 µm space in between) and self-assembled into spheroids with a diameter of 92.3 ± 15.0 µm after 24 h. Compared with monolayer culture, the MC3T3-E1 spheroids showed a significant upregulation of FGF23 in both gene and protein levels after 24 h of serum-free induction. RNA sequencing and western blotting analysis further suggested that the enhanced FGF23 production in MC3T3-E1 spheroids was attributed to the activation of the parathyroid hormone (PTH)/PTH1R signaling pathway. Impressively, inhibition of PTH signaling through small molecular inhibitors or short hairpin RNA targeting PTH1R effectively reduced FGF23 production. In summary, the current study revealed the efficacy of the high-throughput formation of preosteoblast cell spheroid in stimulating FGF23 expression for mechanistic studies. Importantly, our findings highlight the potential of the current 3D spheroid system for target identification and drug discovery.

An Experimental Protocol for the Boyden Chamber Invasion Assay With Absorbance Readout.

The phenomenon of cell invasion is an essential step in angiogenesis, embryonic development, immune responses, and cancer metastasis. In the course of cancer progression, the ability of neoplastic cells to degrade the basement membrane and penetrate neighboring tissue (or blood vessels and lymph nodes) is an early event of the metastatic cascade. The Boyden chamber assay is one of the most prevalent methods implemented to measure the pro- or anti-invasive effects of drugs, investigate signaling pathways that modulate cell invasion, and characterize the role of extracellular matrix proteins in metastasis. However, the traditional protocol of the Boyden chamber assay has some technical challenges and limitations. One such challenge is that the endpoint of the assay involves photographing and counting stained cells (in multiple fields) on porous filters. This process is very arduous, requires multiple observers, and is very time-consuming. Our improved protocol for the Boyden chamber assay involves lysis of the dye-stained cells and reading the absorbance using an ELISA reader to mitigate this challenge. We believe that our improved Boyden chamber methodology offers a standardized, high-throughput format to evaluate the efficacy of various drugs and test compounds in influencing cellular invasion in normal and diseased states. We believe that our protocol will be useful for researchers working in the fields of immunology, vascular biology, drug discovery, cancer biology, and developmental biology. Key features • Measurement of tumor invasion using human cancer cells. • Ability to measure the pro-invasive/anti-invasive activity of small molecules and biological modifiers. • Measurement of chemotaxis, chemokines, trafficking of immune cells, and proteolytic activity of matrix metalloproteinases, lysosomal hydrolysates, collagenases, and plasminogen activators in physiological and pathological conditions. • Investigation of the role of extracellular matrix proteins in the crosstalk between endothelial, epithelial, muscle, or neuronal cells and their adjacent stroma.

Evaluation of Fluorescence-Based Screening Assays for the Detection and Quantification of Silyl Hydrolase Activity.

This study reports the development of fluorometric assays for the detection and quantification of silyl hydrolase activity using silicatein as a model enzyme. These assays employed a series of organosilane substrates containing either mycophenolate or umbelliferone moieties, which become fluorescent upon hydrolysis of a scissile Si-O bond. Among these substrates, the mycophenolate-derived molecule MycoF, emerged as the most promising candidate due to its relative stability in aqueous media, which resulted in good differentiation between the enzyme-catalyzed and uncatalyzed background hydrolysis. The utility of MycoF was also demonstrated in the detection of enzyme activity in cell lysates and was found to be capable of qualitative identification of positive "hit" candidates in a high-throughput format. These fluorogenic substrates were also suitable for use in quantitative kinetic assays, as demonstrated by the acquisition of their Michaelis-Menten parameters.

Predicting the formation enthalpy and phase stability of (Ti,Al,TM)N (TM = III-VIB group transition metals) by high-throughput ab initio calculations and machine learning

The development of transition-metal-alloyed (Ti,Al)N thin films has become a common strategy to achieve optimized mechanical and thermal properties. Selection of a suitable alloying element, however, should consider the effect on Al solubility, directly influencing phase stability during the deposition. Here we use high-throughput ab initio formation enthalpy calculations to assess stability of the cubic (c) vs. hexagonal wurtzite-type (w-) phase of TM-alloyed (Ti,Al,TM)N. This compositionally-limited ab initio dataset serves to fit several machine-learning (ML) models enabling phase stability predictions over the entire compositional range. Of all the models, the linear regression using Magpie feature descriptor pre-processed by a genetic algorithm has the highest accuracy. For Ta, Nb, Mo, and W addition below ∼10 at.%, our ML model predicts enhanced stability of c-(Ti,Al,TM)N due to increased solubility of Al. Other alloying elements, especially Sc and Y from IIIB group and Hf and Zr from IVB group, decrease the cubic metastable solubility limit. In agreement with available experimental data, all transition metals except for Cr and V increase the volume of c-(Ti,Al,TM)N and w-(Ti,Al,TM)N.

High-content analysis of proteostasis capacity in cellular models of amyotrophic lateral sclerosis (ALS)

Disrupted proteome homeostasis (proteostasis) in amyotrophic lateral sclerosis (ALS) has been a major focus of research in the past two decades. However, the proteostasis processes that become disturbed in ALS are not fully understood. Obtaining more detailed knowledge of proteostasis disruption in association with different ALS-causing mutations will improve our understanding of ALS pathophysiology and may identify novel therapeutic targets and strategies for ALS patients. Here we describe the development and use of a novel high-content analysis (HCA) assay to investigate proteostasis disturbances caused by the expression of several ALS-causing gene variants. This assay involves the use of conformationally-destabilised mutants of firefly luciferase (Fluc) to examine protein folding/re-folding capacity in NSC-34 cells expressing ALS-associated mutations in the genes encoding superoxide dismutase-1 (SOD1A4V) and cyclin F (CCNFS621G). We demonstrate that these Fluc isoforms can be used in high-throughput format to report on reductions in the activity of the chaperone network that result from the expression of SOD1A4V, providing multiplexed information at single-cell resolution. In addition to SOD1A4V and CCNFS621G, NSC-34 models of ALS-associated TDP-43, FUS, UBQLN2, OPTN, VCP and VAPB mutants were generated that could be screened using this assay in future work. For ALS-associated mutant proteins that do cause reductions in protein quality control capacity, such as SOD1A4V, this assay has potential to be applied in drug screening studies to identify candidate compounds that can ameliorate this deficiency.

A real-time biochemical assay for quantitative analyses of APOBEC-catalyzed DNA deamination

Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a need for biochemical tools that can be used to identify and characterize potential inhibitors of this enzyme family. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination (RADD) assay. This assay offers a single-step set-up and real-time fluorescent read-out, and it is capable of providing insights into enzyme kinetics and also offering a high-sensitivity and easily scalable method for identifying APOBEC3 inhibitors. This assay serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit and possesses the versatility to be readily adapted into a high-throughput format for inhibitor discovery.

Allele-level visualization of transcription and chromatin by high-throughput imaging

The spatial arrangement of the genome within the nucleus is a pivotal aspect of cellular organization and function with implications for gene expression and regulation. While all genome organization features, such as loops, domains, and radial positioning, are nonrandom, they are characterized by a high degree of single-cell variability. Imaging approaches are ideally suited to visualize, measure, and study single-cell heterogeneity in genome organization. Here, we describe two methods for the detection of DNA and RNA of individual gene alleles by fluorescence in situ hybridization (FISH) in a high-throughput format. We have optimized combined DNA/RNA FISH approaches either using simultaneous or sequential detection of DNA and nascent RNA. These optimized DNA and RNA FISH protocols were implemented in a 384-well plate format alongside automated image and data analysis and enable accurate detection of individual gene alleles and their gene expression status across a large cell population. We successfully visualized MYC and EGFR DNA and nascent RNA with allele-level resolution in multiple cell types, and we determined the radial position of active and inactive MYC and EGFR alleles. These optimized DNA/RNA detection approaches are versatile and sensitive tools for mapping of chromatin features and gene activity at the single-allele level and at high throughput.

Abstract 221: Novel organotypic patient derived primary tumor tissues for oncology drug safety and efficacy studies

Abstract Organotypic, three-dimensional (3D) cancer tissue models (OCTM) have enabled investigators to develop predictive markers that can be used in preclinical cancer drug development. However, a drawback of the existing models is that they are mostly based on cell lines based are not physiological. In vitro development of 3D tumoroids using patient derived primary tumors (PDPT) in tumor microenvironment (TME) will have clinical significant and can predict human responses to cancer therapeutics. Here, we describe the generation of OCTM from patient-derived metastatic colorectal cancer, the second most deadly cancer. PDPT were grown in Matrigel for 5 passages and for biobanking purposes tumoroids were further expanded in faCellitate 384 well plates or Sun Bioscience plates to harvest large amounts of well-defined tumoroids cultured for over 21 days. Expanded tumoroids were collected and cryopreserved or gently mixed several times using a pipet tip to break up tumoroids into smaller fragments. These tumor fragments were mixed with endothelial cells, dendritic cells, fibroblasts and embedded in a collagen matrix to simulate TME. The collagen-cell mixture was then added to Transwells and induced to gel to form a collagen gel matrix that simulates TME. The tumoroids were cultured with a specialized medium that supports the growth of complex cell types. The tumor development was monitored daily using bright field microscopy and image analysis was used to monitor tumor size. To evaluate the utility of the OCTM, treatment was initiated at day 10 of the culture period by exposing tissues with the chemotherapeutic drug Cisplatin on days 0, 2, 4, 7, 9, and 11. After each treatment, OCTM tissues (N=2) were fixed for H&E and live/dead staining. Overall, the results of the study showed that: 1) PDPT can be expanded for 5 passages (this low passage number is not expected to change the phenotype of the tumor) and further expanded in high throughput format, 384 well plates, 2) the PDPT could be cultured with multiple cell types in a collagen-gel matrix to recreate the TME, 3) histological evaluation of the OCTM showed a glandular-like tumoroid, 4) immunohistochemical staining revealed that the PDPT in the in vivo-like microenvironment were positive for CK20, and 5) the PDPT shrank following 3 treatment cycles with Cisplatin (day 4). Conclusion: The data shows metastatic colorectal PDPT can be expanded in vitro and cultured in a complex tumor microenvironment on tissue culture inserts. These OCTMs can be used to screen drug safety and efficacy. This study opens new avenues for high-throughput cancer drug screening format using PDPT embedded in a in vivo like TME. This model can be used as a tool for personalized oncology drug testing and for translational assays tailored to other cancer types from our patient derived primary tumor pool. Citation Format: Seyoum Ayehunie, Megan Groves, Bryda Bryda, Alex Armento, Anthony Tolcher. Novel organotypic patient derived primary tumor tissues for oncology drug safety and efficacy studies [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 221.

Abstract 4698: Measuring cellular PARylation to gain insight in PARP/PARG-targeted drug discovery

Abstract Inhibitors of poly (ADP-ribose) polymerases (PARPs) have revolutionized cancer therapy by highlighting the potential of synthetic lethal drugs to target the DNA damage response (DDR) network. More recently, the development of PARG inhibitors was spurred by the discovery that inhibiting PARG (poly (ADP-ribose) glycohydrolase) results in the accumulation of poly ADP-ribose on DDR proteins and induces cell death. Adding PARG or PARP inhibitors to a cell of interest and quantifying the resulting levels of PARylation can provide valuable insight on compound membrane permeability and target engagement in a cellular context. Nevertheless, assessing PARylation (poly-ADP-ribosylation in which multiple ribose units are added in linear or branched forms) in cells is difficult compared to biochemical assays, impeding the progress of candidate drug development. A sandwich ELISA-based assay was developed to analyze the total protein PARylation present in cellular extracts. Experimental protocols were optimized to discern differences in cellular PARylation levels resulting from activating the DDR and from exposure to PARP inhibitors and/or PARG inhibitors. It is worth noting that our assay specifically detects PARylation, measuring the effects associated with PARP family members 1 to 5, as other family members primarily catalyze MARylation (mono-ADP ribosylation in which a single ADP-ribose unit is added). Titration of known PARP inhibitors and PARG inhibitors was performed to validate the assay. 1) PARG inhibitor PDD00017273, used in combination with DNA damaging agent H2O2, increased the level of cellular PARylation, exhibiting a similar IC50 value across the four cell lines tested. This is consistent with the notion that PARG is the major de-PARylating enzyme in most cells. The potency of other known PARG inhibitors PDD00017272 and PDD00017238 ranked the compounds as expected from published results (PDD00017272< PDD00017273< PDD00017238). 2) Titration of the three PARP1/2 inhibitors Talazoparib, Olaparib and AZD5305, in combination with a DNA damage agent and PDD00017273 inhibitor, shows a dose-dependent reduction in cellular PARylation in HEK293 cells, as expected since PARP1 is activated in response to DNA damage and this activation is blocked by the inhibitors (IC50 values, Olaparib = 0.013 µM, AZD5305 = 0.0024 µM, Talazoparib = 0.0016 µM). These IC50 are consistent with published values and underscore the usefulness of the assay in measuring dose response effects, allowing accurate determination of compound IC50 in living cells. In summary, our optimized experimental cell treatment protocols and ELISA-based assay allow quantification of total cellular PARylation and accurately rank candidate drugs targeting PAR erasers or PAR writers in a high-throughput format, using living cells. Citation Format: Caila Pilo, Mohammad Awad, Veronique T. Baron, Fernando Martins, Jonathan Mikolosko, Steven Plouffe, Zhihua Tao, Katarzyna Zientara-Rytter, Henry Zhu, Pavel Shashkin. Measuring cellular PARylation to gain insight in PARP/PARG-targeted drug discovery [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 4698.

Abstract 3096: CETSA Navigate HT: A new paradigm for high-throughput screening

Abstract The CEllular Thermal Shift Assay (CETSA) technology is a powerful way to assess target engagement across various matrixes. By the introduction of the high-throughput format (CETSA Navigate HT), this technology offers a reliable high-throughput screening method in natural cellular environments that can successfully be applied in compound screening for hit identification and confirmation already during lead generation. Due to its broad applicability to native, full length protein targets in the live cellular environment, without the need for expensive and time-consuming protein and/or compound labelling efforts, CETSA Navigate HT may be regarded as a new paradigm for high-throughput screening and actives generation. As a case study to demonstrate the applicability of CETSA Navigate HT in hit identification, we have applied the technology for primary screening of a 12000 compound library for CDK4, a protein targeted in several cancers. Hits identified from this screen are privileged from the onset by binding to the target in a physiologically relevant cellular matrix and in a binding-mode agnostic way. This is in contrast to traditional screening methods targeting activity or in vitro interactions where cellular activity is addressed in later stages. Data from follow up studies using functional assays will be presented as well as mass spectrometry based CETSA selectivity profiling and comparison to commercially available de novo CDK4/6 inhibitors. Citation Format: Helena Almqvist, Victoria Brehmer, Laurence Arnold, Tomas Friman, Stina Lundgren, Daniel Martinez Molina. CETSA Navigate HT: A new paradigm for high-throughput screening [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 3096.

Establishment, optimization and validation of a fluorescence polarization-based high-throughput screening assay targeting cathepsin L inhibitors

Cathepsin L (CTSL), a lysosomal cysteine proteinase, is primarily dedicated to the metabolic turnover of intracellular proteins. It is involved in various physiological processes and contributes to pathological conditions such as viral infection, tumor invasion and metastasis, inflammatory status, atherosclerosis, renal disease, diabetes, bone diseases, and other ailments. The coronavirus disease 2019 (COVID-19), with its rapid global spread and significant mortality, has been a worldwide epidemic since the late 2019s. Notably, CTSL plays a role in the processing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, providing a potential avenue to block coronavirus host cell entry and thereby inhibit SARS-CoV-2 infection in humans. In this study, we have developed a novel method using fluorescence polarization (FP) for screening CTSL inhibitors in a high-throughput format. The optimized assay demonstrated its appropriateness for high-throughput screening (HTS) with a Z-factor of 0.9 in a 96-well format. Additionally, the IC50 of the known inhibitor, Z-Phe-Tyr-CHO, was determined to be 188.50 ± 46.88 nM. Upon screening over 2000 small molecules, we identified, for the first time, the anti-CTSL properties of a benzothiazoles derivative named IMB 8015. This work presents a novel high-throughput approach and its application in discovering and evaluating CTSL inhibitors.

Harnessing HetHydrogel: A Universal Platform to Dropletize Single-Cell Multiomics.

A universal platform is developed for dropletizing single cell plate-based multiomic assays, consisting of three main pillars: a miniaturized open Heterogeneous Hydrogel reactor (abbreviated HetHydrogel) for multi-step biochemistry, its tunable permeability that allows Tn5 tagmentation, and single cell droplet barcoding. Through optimizing the HetHydrogel manufacturing procedure, the chemical composition, and cell permeation conditions, simultaneous high-throughput mitochondrial DNA genotyping and chromatin profiling at the single-cell level are demonstrated using a mixed-species experiment. This platform offers a powerful way to investigate the genotype-phenotype relationships of various mtDNA mutations in biological processes. The HetHydrogel platform is believed to have the potential to democratize droplet technologies, upgrading a whole range of plate-based single cell assays to high throughput format.

TTLL12 has a potential oncogenic activity, suppression of ligation of nitrotyrosine to the C-terminus of detyrosinated α-tubulin, that can be overcome by molecules identified by screening a compound library.

Tubulin tyrosine ligase 12 (TTLL12) is a promising target for therapeutic intervention since it has been implicated in tumour progression, the innate immune response to viral infection, ciliogenesis and abnormal cell division. It is the most mysterious of a fourteen-member TTL/TTLL family, since, although it is the topmost conserved in evolution, it does not have predicted enzymatic activities. TTLL12 seems to act as a pseudo-enzyme that modulates various processes indirectly. Given the need to target its functions, we initially set out to identify a property of TTLL12 that could be used to develop a reliable high-throughput screening assay. We discovered that TTLL12 suppresses the cell toxicity of nitrotyrosine (3-nitrotyrosine) and its ligation to the C-terminus of detyrosinated α-tubulin (abbreviated to ligated-nitrotyrosine). Nitrotyrosine is produced by oxidative stress and is associated with cancer progression. Ligation of nitrotyrosine has been postulated to be a check-point induced by excessive cell stress. We found that the cytotoxicities of nitrotyrosine and tubulin poisons are independent of one another, suggesting that drugs that increase nitrotyrosination could be complementary to current tubulin-directed therapeutics. TTLL12 suppression of nitrotyrosination of α-tubulin was used to develop a robust cell-based ELISA assay that detects increased nitrotyrosination in cells that overexpress TTLL12 We adapted it to a high throughput format and used it to screen a 10,000 molecule World Biological Diversity SETTM collection of low-molecular weight molecules. Two molecules were identified that robustly activate nitrotyrosine ligation at 1 μM concentration. This is the pioneer screen for molecules that modulate nitrotyrosination of α-tubulin. The molecules from the screen will be useful for the study of TTLL12, as well as leads for the development of drugs to treat cancer and other pathologies that involve nitrotyrosination.

FlgI Is a Sec-Dependent Effector of Candidatus Liberibacter asiaticus That Can Be Blocked by Small Molecules Identified Using a Yeast Screen.

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The phloem-restricted bacterium Candidatus Liberibacter asiaticus (CLas) is considered to be the main pathogen responsible for HLB. There is currently no effective practical strategy for the control of HLB. Our understanding of how pathogens cause HLB is limited because CLas has not been artificially cultured. In this study, 15 potential virulence factors were predicted from the proteome of CLas through DeepVF and PHI-base searches. One among them, FlgI, was found to inhibit yeast growth when expressed in Saccharomyces cerevisiae. The expression of the signal peptide of FlgI fused with PhoA in Escherichia coli resulted in the discovery that FlgI was a novel Sec-dependent secretory protein. We further found that the carboxyl-terminal HA-tagged FlgI was secreted via outer membrane vesicles in Sinorhizobium meliloti. Fluoresence localization of transient expression FlgI-GFP in Nicotiana benthamiana revealed that FlgI is mainly localized in the cytoplasm, cell periphery, and nuclear periphery of tobacco cells. In addition, our experimental results suggest that FlgI has a strong ability to induce callose deposition and cell necrosis in N. benthamiana. Finally, by screening a large library of compounds in a high-throughput format, we found that cyclosporin A restored the growth of FlgI-expressing yeast. These results confirm that FlgI is a novel Sec-dependent effector, enriching our understanding of CLas pathogenicity and helping to develop new and more effective strategies to manage HLB.