Biology subjects

Gao, Z.

Publications and source records attributed to Gao, Z..

At least 145 records · Page 8Linked to original sources

bioRxiv2023

Structural determinants of pure antiestrogenicity

Inhibitory activities of antiestrogens on estrogen receptor alpha (ER) range from mixed antagonism/agonism (selective ER modulators; SERMs) to complete antiestrogenicity associated with accelerated ER turnover (selective ER degraders; SERDs). Here, we show using a panel of SERMs, SERDs and a PROTAC that efficient induction of ER SUMOylation better accounts for complete transcriptional repression than increased suppression of coactivator recruitment or accelerated ER degradation. Antiestrogen-induced ER SUMOylation depends on the hydrophobicity of N-terminal residues of ligand binding domain (LBD) helix 12 (H12). L536 mutations, including those occurring in endocrine therapy-resistant breast cancer, abolished SUMOylation with all antiestrogens. Structures of the L536S human ER LBD bound to fulvestrant analogs and molecular dynamics simulations predict dynamic side chain interactions with wild-type ER H12 in the coactivator-binding groove and model the impact of L536 mutations. Together, our results show that induced SUMOylation contributes to the complete suppression of ER activity and provide insights into its structural determinants.

molecular biology↗

bioRxiv2023

Proteomic Stratification of Prognosis and Treatment Options for Small Cell Lung Cancer

Small cell lung cancer (SCLC) is a highly malignant and heterogeneous cancer with limited therapeutic options and prognosis prediction models. Here, we analyzed formalin-fixed, paraffin-embedded (FFPE) samples of surgical resections by proteomic profiling, and stratified SCLC into three proteomic subtypes (S-I, S-II, and S-III) with distinct clinical outcomes and chemotherapy responses. The proteomic subtyping was an independent prognostic factor and performed better than current TNM or Veterans Administration Lung Study Group (VALG) staging methods. The subtyping results could be further validated using FFPE biopsy samples from an independent center, extending the analysis to both surgical and biopsy samples. The signatures of the S-II subtype in particular suggest potential benefits from immunotherapy. Differentially overexpressed proteins in S-III, the worst prognostic subtype, allowed us to nominate potential therapeutic targets, indicating that patient selection may bring new hope for previously failed clinical trials. Finally, analysis of an independent cohort of SCLC patients who had received immunotherapy validated the prediction that the S-II patients had better Progression Free Survival (PFS) and Overall Survival (OS) after first-line immunotherapy. Collectively, our study provides the rationale for future clinical investigations to validate the current findings for more accurate prognosis prediction and precise treatments.

cancer biology↗

bioRxiv2023

Membrane Mimetic-Dependence of GPCR Energy Landscapes

Protein function strongly depends on temperature, which is related to temperature-dependent changes in the equilibria of protein conformational states. We leveraged variable-temperature 19F-NMR spectroscopy to interrogate the temperature dependence of the conformational landscape of the human A2A adenosine receptor (A2AAR), a class A GPCR. Temperature-induced changes in the conformational equilibria of A2AAR in lipid nanodiscs were markedly dependent on the efficacy of bound drugs. While antagonist complexes displayed only modest changes as the temperature rose, both full and partial agonist complexes exhibited substantial increases in the active state population. Importantly, the temperature-dependent response of complexes with both full and partial agonists exhibited a pronounced sensitivity to the specific membrane mimetic employed. In striking contrast to observations within lipid nanodiscs, in detergent micelles the active state population exhibited different behavior for A2AAR complexes with both full and partial agonists. This underscores the importance of the protein environment in understanding the thermodynamics of GPCR activation. HighlightsO_LIActive A2AAR population increases with increasing temperature in lipid nanodiscs C_LIO_LIActive A2AAR population exhibits different temperature dependence in detergents C_LIO_LIPartial agonist complexes present a unique conformational state in nanodiscs C_LIO_LITemperature dependence of partial agonist equilibria depends on membrane mimetic C_LI

biophysics↗

bioRxiv2023

Mesenchymal-specific Alms1 knockout in mice recapitulates key metabolic features of Alström Syndrome

BackgroundAlstrom Syndrome (AS), a multi-system disease caused by mutations in the ALMS1 gene, includes obesity with disproportionately severe insulin resistant diabetes, dyslipidemia, and hepatosteatosis. How loss of ALMS1 causes this phenotype is poorly understood, but prior studies have circumstancially implicated impaired adipose tissue expandability. We set out to test this by comparing the metabolic effects of selective Alms1 knockout in mesenchymal cells including preadipocytes to those of global Alms1 knockout. MethodsGlobal Alms1 knockout (KO) mice were generated by crossing floxed Alms1 and CAG-Cre mice. A Pdgfr-Cre driver was used to abrogate Alms1 function selectively in mesenchymal stem cells (MSCs) and their descendants, including preadipocytes. We combined metabolic phenotyping of global and Pdgfr+ Alms1-KO mice on a 45% fat diet with measurements of body composition and food intake, and histological analysis of metabolic tissues. ResultsGlobal Alms1 KO caused hyperphagia, obesity, insulin resistance, dyslipidaemia, and fatty liver. Pdgfr-cre driven KO of Alms1 (MSC KO) recapitulated insulin resistance, fatty liver, and dyslipidaemia in both sexes. Other phenotypes were sexually dimorphic: increased fat mass was only present in female Alms1 MSC KO mice. Hyperphagia was not evident in male Alms1 MSC KO mice, but was found in MSC KO females, despite no neuronal Pdgfr expression. ConclusionsMesenchymal deletion of Alms1 recapitulates the metabolic features of AS, including severe fatty liver. This confirms a key role for Alms1 in the adipose lineage, where its loss is sufficient to cause systemic metabolic effects and damage to remote organs. AS should be regarded as a forme fruste of lipodystrophy. Therapies should prioritise targeting positive energy balance.

physiology↗

bioRxiv2023

Collaborative Augmented Reconstruction for Scaled Production of 3D Neuron Morphology in Mouse and Human Brains

Digital reconstruction of the intricate 3D morphology of individual neurons from microscopic images is widely recognized as a crucial challenge in both individual research laboratories and large-scale scientific projects focusing on cell types and brain anatomy. This task often fails both conventional manual reconstruction and state-of-the-art automatic reconstruction algorithms, even many of which are based on artificial intelligence (AI). It is also critical but challenging to organize multiple neuroanatomists to produce and cross-validate biologically relevant and agreeable reconstructions in scaled data production. Here we propose an approach based on collaborative human intelligence augmented by AI. Specifically, we have developed a Collaborative Augmented Reconstruction (CAR) platform for neuron reconstruction at scale. This platform allows for immersive interaction and efficient collaborative-editing of neuron anatomy using a variety of client devices, such as desktop workstations, virtual reality headsets, and mobile phones, enabling users to contribute anytime and anywhere and take advantage of several AI-based automation tools. We have tested CARs applicability for challenging mouse and human neurons towards a scaled and faithful data production. Our data indicate that the CAR platform is suitable for generating tens of thousands of neuronal reconstructions used in our companion studies.

neuroscience↗

bioRxiv2023

Comprehensive Metabolomic Analysis of Human Heart Tissue Enabled by Parallel Metabolite Extraction and High-Resolution Mass Spectrometry

The heart contracts incessantly and requires a constant supply of energy, utilizing numerous metabolic substrates such as fatty acids, carbohydrates, lipids, and amino acids to supply its high energy demands. Therefore, a comprehensive analysis of various metabolites is urgently needed for understanding cardiac metabolism; however, complete metabolome analyses remain challenging due to the broad range of metabolite polarities which makes extraction and detection difficult. Herein, we implemented parallel metabolite extractions and high-resolution mass spectrometry (MS)-based methods to obtain a comprehensive analysis of the human heart metabolome. To capture the diverse range of metabolite polarities, we first performed six parallel liquid-liquid extractions (three monophasic, two biphasic, and one triphasic extractions) of healthy human donor heart tissue. Next, we utilized two complementary MS platforms for metabolite detection - direct-infusion ultrahigh-resolution Fourier-transform ion cyclotron resonance (DI-FTICR) and high-resolution liquid chromatography quadrupole time-of-flight tandem MS (LC-Q-TOF MS/MS). Using DI-FTICR MS, 9,521 metabolic features were detected where 7,699 were assigned a chemical formula and 1,756 were assigned an annotated by accurate mass assignment. Using LC-Q-TOF MS/MS, 21,428 metabolic features were detected where 626 metabolites were identified based on fragmentation matching against publicly available libraries. Collectively, 2276 heart metabolites were identified in this study which span a wide range of polarities including polar (benzenoids, alkaloids and derivatives and nucleosides) as well as non-polar (phosphatidylcholines, acylcarnitines, and fatty acids) compounds. The results of this study will provide critical knowledge regarding the selection of appropriate extraction and MS detection methods for the analysis of the diverse classes of human heart metabolites. Table of Contents Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/558013v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1db91c8org.highwire.dtl.DTLVardef@400d81org.highwire.dtl.DTLVardef@136b221org.highwire.dtl.DTLVardef@1e8d3b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

A minimum threshold for myelination of pyramidal cells in human and mouse neocortex

Neocortical pyramidal neurons are frequently myelinated. Diversity in the topography of axonal myelination in the cerebral cortex has been attributed to a combination of electrophysiological activity, axonal morphology, and neuronal-glial interactions. Previously, we showed that axonal segment length and calibre are critical determinants of fast-spiking interneuron myelination (Stedehouder, J. et al (2019). However, the factors that determine the myelination of individual axonal segments along neocortical pyramidal neurons remain largely unexplored. Here, we used structured illumination microscopy and cell type-specific manipulations to examine the extent to which axonal morphology determines the topography of axonal myelination in mouse neocortical pyramidal neurons. We found that, unlike what was determined for fast-spiking interneurons, the joint combination of axonal calibre and interbranch distance does not predict axonal myelination in pyramidal neurons, rather it provides a minimum threshold for myelination; pyramidal neurons with an axon calibre and interbranch distance lower than 0.24 {micro}m and 19 {micro}m, respectively, are almost never myelinated. Moreover, we further confirmed that these findings in mice also extend to human neocortical pyramidal cell myelination, suggesting that this mechanism is evolutionarily conserved. Taken together, our findings suggest that axonal morphology is highly deterministic of the topography and cell-type specificity of neocortical myelination.

neuroscience↗

bioRxiv2023

Accurate Prediction of Antibody Function and Structure Using Bio-Inspired Antibody Language Model

In recent decades, antibodies have emerged as indispensable therapeutics for combating diseases, particularly viral infections. However, their development has been hindered by limited structural information and labor-intensive engineering processes. Fortunately, significant advancements in deep learning methods have facilitated the precise prediction of protein structure and function by leveraging co-evolution information from homologous proteins. Despite these advances, predicting the conformation of antibodies remains challenging due to their unique evolution and the high flexibility of their antigen-binding regions. Here, to address this challenge, we present the Bio-inspired Anti-body Language Model (BALM). This model is trained on a vast dataset comprising 336 million 40% non-redundant unlabeled antibody sequences, capturing both unique and conserved properties specific to antibodies. Notably, BALM showcases exceptional performance across four antigen-binding prediction tasks. Moreover, we introduce BALMFold, an end-to-end method derived from BALM, capable of swiftly predicting full atomic antibody structures from individual sequences. Remarkably, BALMFold outperforms those well-established methods like AlphaFold2, IgFold, ESMFold, and OmegaFold in the antibody benchmark, demonstrating significant potential to advance innovative engineering and streamline therapeutic antibody development by reducing the need for unnecessary trials.

bioinformatics↗

bioRxiv2023

Dissecting the neurotropism and neurovirulence of MPXV using human stem cell-based models

Mpox is a zoonotic illness of international concern that can lead to severe disease including neurological sequelae. However, the neurotropism of monkeypox virus (MPXV) and the mechanisms regulating cell-intrinsic antiviral immunity within the central nervous system (CNS) remain poorly understood. Here, we investigated the neurotropism of MPXV using astrocytes, cortical neurons, and microglia derived from human pluripotent stem cells (hPSCs) and ex vivo human brain tissue to demonstrate that MPXV infects and replicates more efficiently in astrocytes and microglia compared to cortical neurons. Upon MPXV exposure, glial cells, in contrast to cortical neurons, inhibit type I IFN antiviral programs potentially conferring differential susceptibility to MPXV. Furthermore, we demonstrate that treatment using either IFN-beta or tecovirimat inhibits MPXV infection. Together, our results suggest that MPXV has a broad tropism within the CNS and that differential type I IFN signaling underpins cell type-specific susceptibility to MPXV infection.

microbiology↗

bioRxiv2023

Preclinical evaluation of the efficacy and safety of AAV1-hOTOF in mice and non-human primates

Pathogenic mutations in the OTOF gene cause autosomal recessive hearing loss 9 (DFNB9), one of the most common forms of auditory neuropathy. There is no biological treatment for DFNB9. Here, we designed an OTOF gene therapy agent by dual AAV1 carrying human OTOF coding sequences with the expression driven by the hair cell-specific promoter Myo15, AAV1-hOTOF. To develop a clinical application of AAV1-hOTOF gene therapy, we evaluated its efficacy and safety in animal models by pharmacodynamics, behavior, and histopathology. AAV1-hOTOF inner ear delivery significantly improved hearing in Otof -/- mice without affecting normal hearing in wild-type mice. AAV1 was predominately distributed to the cochlea although it was detected in other organs such as the central nervous system and the liver, and no obvious toxic effects of AAV1-hOTOF were observed in mice. To further evaluate the safety of Myo15 promoter-driven AAV1-transgene, AAV1-GFP was delivered into the inner ear of Macaca fascicularis via the round window membrane. AAV1-GFP transduced 60-94% of the inner hair cells along the cochlear turns. AAV1-GFP was detected in isolated organs and no significant adverse effects were detected. These results suggest that AAV1-hOTOF is well tolerated and effective in animals, providing critical support for its clinical translation.

genetics↗

bioRxiv2023

YY1 Binding to Regulatory Elements that Lack Enhancer Activity Promotes Locus Folding and Gene Activation

Enhancers activate their cognate promoters over huge distances but how enhancer/promoter interactions become established is not completely understood. There is strong evidence that cohesin-mediated loop extrusion is involved but this does not appear to be a universal mechanism. Here, we identify an element within the mouse immunoglobulin lambda (Ig{lambda}) light chain locus, HSC{lambda}1, that has characteristics of active regulatory elements but lacks intrinsic enhancer or promoter activity. Remarkably, knock-out of the YY1 binding site from HSC{lambda}1 reduces Ig{lambda} transcription significantly and disrupts enhancer/promoter interactions, even though these elements are >10 kb from HSC{lambda}1. Genome-wide analyses of mouse embryonic stem cells identified 3503 similar YY1-bound, putative genome organizing elements that lie within CTCF/cohesin loop boundaries but that lack intrinsic enhancer activity. We suggest that such elements play a fundamental role in locus folding and in facilitating enhancer/promoter interactions. HighlightsHow long-range enhancer-promoter interactions are established is not fully understood An element in the lambda light chain locus, HSC{lambda}1, lacks intrinsic enhancer activity Removal of YY1 binding from HSC{lambda}1 disrupts neighbouring enhancer/promoter contacts Genome-wide analyses detect similar elements that lack enhancer or promoter activity We propose these elements aid locus folding and nearby enhancer-promoter interactions O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/554459v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@562875org.highwire.dtl.DTLVardef@7a1a85org.highwire.dtl.DTLVardef@634652org.highwire.dtl.DTLVardef@19ddfda_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

A vast repertoire of secondary metabolites influences community dynamics and biogeochemical processes in cold seeps

In deep sea cold seeps, diverse microbial communities thrive on the geological seepage of hydrocarbons and inorganic compounds. These chemosynthetically-driven communities are unique in composition, ecology, and biogeochemical activities compared to photosynthetically-driven ecosystems. However, their biosynthetic capabilities remain largely unexplored. Here, we analyzed 81 metagenomes, 33 metatranscriptomes, and seven metabolomes derived from nine globally distributed areas of cold seeps to investigate the secondary metabolites produced by cold seep microbiomes. Cold seep microbiomes encode diverse, abundant, and novel biosynthetic gene clusters (BGCs). Most BGCs are affiliated with understudied bacteria and archaea, including key mediators of methane and sulfur cycling, and multiple candidate phyla. The BGCs encode diverse antimicrobial compounds (e.g. NRPS, PKSs, RiPPs) that potentially shape community dynamics, as well as compounds predicted to influence biogeochemical cycling, such as phosphonates, iron-acquiring siderophores, nitrogenase-protecting glycolipids, and methyl-CoM reductase-modifying proteins. BGCs from key players in cold seeps are widely distributed and highly expressed, with their abundance and expression levels varying with different sediment depths. Numerous unique natural products were detected through untargeted sediment metabolomics, demonstrating a vast, unexplored chemical space and validating in situ expression of the BGCs in cold seep sediments. Overall, these results demonstrate cold seep sediments potentially serve as a reservoir of hidden natural products and provide insights into microbial adaptation in chemosynthetically-driven ecosystems.

microbiology↗

bioRxiv2023

An adult clock component links circadian rhythms to pancreatic β-cell maturation

Circadian clocks attune metabolism to daily energy cycles, but how they regulate functional maturation of metabolic tissues is poorly understood. Here we show that DEC1, a clock transcription factor induced in adult islet {beta} cells, coordinates their glucose responsiveness by synchronizing energetic and secretory rhythms. DEC1 binds and regulates maturity-linked genes to integrate insulin exocytosis with {beta}-cell energy metabolism, and {beta}-cell-specific Dec1 ablation disrupts their transcription synchrony. Dec1-disrupted mice develop lifelong glucose intolerance and insulin deficiency, despite normal islet formation. Metabolic dysfunction upon {beta}-cell Dec1 loss stems from poor coupling of islet insulin secretion to glucose metabolism, reminiscent of fetal/neonatal islet immaturity. We link stunted maturation to a deficit in circadian bioenergetics, prompted by compromised glucose utilization, mitochondrial dynamics, and respiratory metabolism, which is rescued by increased metabolic flux. Thus, DEC1 links circadian clockwork to {beta}-cell metabolic maturation, revealing a hierarchy for how the clock programs metabolic tissue specialization.

cell biology↗

bioRxiv2023

The Extent of Edgetic Perturbations in the Human Interactome Caused by Population-Specific Mutations

Until recently, efforts in population genetics have been focused primarily on people of European ancestry. To attenuate the bias, global population studies, such as the 1,000 Genomes Project, have revealed differences in genetic variation across ethnic groups. How much of these differences would attribute to the population-specific traits? To answer this question, the mutation data must be linked with the functional outcomes. A new "edgotype" concept has been proposed that emphasizes the interaction-specific, "edgetic", perturbations caused by mutations in the interacting proteins. In this work, we performed a systematic in-silico edgetic profiling of [~]50,000 non-synonymous SNVs (nsSNVs) from 1,000 Genomes Project by leveraging our semi-supervised learning approach SNP-IN tool on a comprehensive set of over 10,000 protein interaction complexes. We interrogated functional roles of the variants and their impact on the human interactome and compared the results with the pathogenic variants disrupting PPIs in the same interactome. Our results demonstrated that a considerable number of nsSNVs from healthy populations could rewire the interactome. We also showed that the proteins enriched with the interaction-disrupting mutations were associated with diverse functions and had implications in a broad spectrum of diseases. Further analysis indicated that distinct gene edgetic profiles among major populations could shed light on the molecular mechanisms behind the population phenotypic variances. Finally, the network analysis revealed that the disease-associated modules surprisingly harbored a higher density of interaction-disrupting mutations from the healthy populations. The variation in the cumulative network damage within these modules could potentially account for the observed disparities in disease susceptibility, which are distinctly specific to certain populations. Our work demonstrates the feasibility of a large-scale in-silico edgetic study and reveals insights into the orchestrated play of the population-specific mutations in the human interactome.

bioinformatics↗

bioRxiv2023

T2T-YAO: a Telomere-to-telomere Assembled Diploid Reference Genome for Han Chinese

Since its initial release in 2001, the human reference genome has been continuously improved in both continuity and accuracy, and the recently-released telomere-to-telomere version--T2T-CHM13--reaches its top quality after 20 years of effort. However, T2T-CHM13 does not represent an authentic diploid human genome, but rather one derived from a simplified, nearly homozygous genome of a hydatidiform mole cell line. To address this limitation and provide an alternative pertinent to the Chinese population, the largest ethnic group in the world, we have assembled a complete diploid human genome of a male Han Chinese, T2T-YAO, which includes telomere-to-telomere assemblies for all the 22+X+M and 22+Y chromosomes in his two haploids inherited separately from his parents. Both haplotypes contain no artificial sequences or model nucleotides and possess a high quality comparable to CHM13, with fewer than one error per [~]14 Mb. Derived from the individual who lives in the aboriginal region of Han Chinese, T2T-YAO shows clear ancestry and potential genetic continuity from the ancient ancestors of the Han population. Each haplotype of T2T-YAO possesses [~]340 Mb exclusive sequences and [~]3100 unique genes as compared to CHM13, and their genome sequences show greater genetic distance to CHM13 than to each other in terms of nucleotide polymorphism and structural variations. The construction of T2T-YAO would serve as a high-quality diploid reference that enables precise delineation of genomic variations in a haplotype-sensitive manner, which could advance our understandings in human evolution, hereditability of diseases and phenotypes, especially within the context of the unique variations of the Chinese population.

genetics↗

bioRxiv2023

EpiGePT: a Pretrained Transformer model for epigenomics

The inherent similarities between natural language and biological sequences have given rise to great interest in adapting the transformer-based large language models (LLMs) underlying recent breakthroughs in natural language processing (references), for applications in genomics. However, current LLMs for genomics suffer from several limitations such as the inability to include chromatin interactions in the training data, and the inability to make prediction in new cellular contexts not represented in the training data. To mitigate these problems, we propose EpiGePT, a transformer-based pretrained language model for predicting context-specific epigenomic signals and chromatin contacts. By taking the context-specific activities of transcription factors (TFs) and 3D genome interactions into consideration, EpiGePT offers wider applicability and deeper biological insights than models trained on DNA sequence only. In a series of experiments, EpiGePT demonstrates superior performance in a diverse set of epigenomic signals prediction tasks when compared to existing methods. In particular, our model enables cross-cell-type prediction of long-range interactions and offers insight on the functional impact of genetic variants under different cellular contexts. These new capabilities will enhance the usefulness of LLM in the study of gene regulatory mechanisms. We provide free online prediction service of EpiGePT through http://health.tsinghua.edu.cn/epigept/.

genomics↗

bioRxiv2023

Age-related spatial memory differences are correlated with neural remapping in CA1 of the human hippocampus

Older adults show declines in spatial memory, although the extent of these alterations is not uniform across the healthy older population. Here, we investigate the stability of neural representations for the same and different spatial environments in a sample of younger and older adults using high-resolution functional magnetic resonance imaging (fMRI) of the medial temporal lobe. Older adults showed, on average, lower neural pattern similarity for retrieving the same environment and more variable neural patterns compared to young adults. We also found a positive association between spatial distance discrimination and the distinctiveness of neural patterns between environments. Our analyses suggested that one source for this association was the extent of informational connectivity to CA1 from other subfields, which was dependent on age, while another source was the fidelity of signals within CA1 itself, which was independent of age. Together, our findings suggest both age-dependent and independent neural contributions to spatial memory performance. Significance StatementSpatial memory declines with age, although some older adults show little cognitive decline, even into their 80s. One important lead for age-related changes comes from electrophysiological studies of older rats, who have less stable neural representations for a spatial environment, termed "place cells." Using high-resolution fMRI targeting the human hippocampus, we demonstrate that older adults also have less stable neural representations within the same environment. Our results reveal, however, that there are both age and performance dependent differences driving these effects. While older adults, on average, showed alterations in information flow within the hippocampal circuit, better performing individuals showed more differentiated neural representations regardless of age. Together, these findings provide novel insight into the impact of age on spatial memory.

neuroscience↗

bioRxiv2023

Development and Optimisation of in vitro Sonodynamic Therapy for Glioblastoma

BackgroundSonodynamic therapy (SDT) is currently on critical path for glioblastoma therapeutics. However, the molecular mechanisms underlying the therapeutic functions of SDT remain enigmatic due to the lack of intricately optimised instrumentation capable of modulating SDT delivery in vitro. We established and validated an automated in vitro SDT system and assessed its SDT efficacy. MethodsAn automated in vitro SDT system was established and validated to allow the application and mapping of focused ultrasound fields under varied acoustic exposure conditions and setup configurations. Ultrasound field simulations were performed to assess the acoustic energy exposure. Systematic in vitro investigations were performed to optimise ultrasound frequency, intensity, plate base material, thermal effect, and the integration of live cells. ResultsIn the presence of 5-ALA, focused ultrasound induces apoptotic cell death in primary patient-derived glioma cells with concurrent upregulation of intracellular reactive oxygen species. Intriguingly, primary glioma stem neurospheres also exhibit remarkably reduced 3D growth upon SDT exposure. Further, ultrasound field simulations performed established acoustic field distribution and the impact of acoustic standing waves within the well-plates. ConclusionsThe optimised in vitro SDT system and associated ultrasound field simulations establish the basis for further understanding of SDT and its therapeutic potentials in cancer.

bioengineering↗