Biology subjects

Hu, J.

Publications and source records attributed to Hu, J..

At least 235 records · Page 13Linked to original sources

bioRxiv2024

Stearoyl coenzyme A desaturase 1 (SCD1) regulates foot-and-mouth disease virus replication by modulating host cell lipid metabolism and 2C-mediated replication complex formation

The life cycle of foot-and-mouth disease virus (FMDV) is tightly regulated by host cell lipid metabolism. In a previous study, we successfully established a BHK-21 cell model (BHK-Op) for persistent FMDV infection by single-cell clone selection and isolated a virus-negative cell line (BHK-VEC cells) from high-passage BHK-Op cells. BHK-VEC cells could be not infected with FMDV. By conforming the transcriptome data of BHK-VEC cells and BHK-21 cells, we identified that the stearoyl coenzyme A desaturase 1 (SCD1), a key enzyme for the fatty acid metabolism, regulates FMDV replication. SCD1 overexpression or exogenous addition of oleic acid (OA), a product of SCD1 enzyme activity, promoted or upregulated FMDV replication in BHK-21 cells or SCD1 knockdown cells, respectively. Interestingly, overexpression of SCD1 or exogenous addition of OA restored the FMDV infection and replication in BHK-VEC cells. Exogenous addition of OA also promoted FMDV replication in BHK-Op. SCD1 recruited the nonstructural FMDV protein 2C to the detergent-resistant membrane located in the perinuclear nucleus to form replication complex. Inhibition of SCD1 enzyme activity resulted in significantly decreased number of FMDV replication complexes with also abnormal morphology. Importantly, inhibition of SCD1 enzyme activity effectively suppressed replication of other positive-sense RNA viruses, such as REO176, PV1 and EV71. Our results demonstrated that, as a key host regulator of RNA virus replication, SCD1 is a potential target for developing novel drugs against positive-sense RNA virus infection.

microbiology↗

bioRxiv2024

Determination of metal ion transport rate of human ZIP4 using stable zinc isotopes

The essential microelement zinc is absorbed in the small intestine mainly by the zinc transporter ZIP4, a representative member of the Zrt/Irt-like protein (ZIP) family. ZIP4 is reportedly upregulated in many cancers, making it a promising oncology drug target. To date, there have been no reports on the turnover number of ZIP4, which is a crucial missing piece of information needed to better understand the transport mechanism. In this work, we used a non-radioactive zinc isotope, 70Zn, and inductively coupled plasma mass spectrometry (ICP-MS) to study human ZIP4 (hZIP4) expressed in HEK293 cells. Our data showed that 70Zn can replace the radioactive 65Zn as a tracer in kinetic evaluation of hZIP4 activity. This approach, combined with the quantification of the cell surface expression of hZIP4 using biotinylation or surface-bound antibody, allowed us to estimate the apparent turnover number of hZIP4 to be in the range of 0.08-0.2 s-1. The turnover numbers of the truncated hZIP4 variants are significantly smaller than that of the full-length hZIP4, confirming a crucial role for the extracellular domain in zinc transport. Using 64Zn and 70Zn, we measured zinc efflux during the cell-based transport assay and found that it has little effect on the zinc import analysis under these conditions. Finally, we demonstrated that use of laser ablation (LA) ICP-TOF-MS on samples applied to a solid substrate significantly increased the throughput of the transport assay. We envision that the approach reported here can be applied to the studies of metal transporters beyond the ZIP family.

biochemistry↗

bioRxiv2024

Roles and mechanisms of leptomeningeal collaterals in human distal MCA territory ischemia

BackgroundLeptomeningeal collaterals (LMCs) provided hemodynamic support and reperfusion during acute ischemic stroke (AIS). Although regulation details have been deeply acquired from animal models, human cortex may share limited hemodynamic patterns due to larger physical scale that needs further investigation. MethodsWe performed sequential hierarchical blocking during awake craniotomy on fourteen human subjects to mimic AIS in middle cerebral artery (MCA) M4 segment territory. Widefield microscope was applied to reveal microcirculation of LMCs. Relative flow rate (RFR) was measured to evaluate the impact of LMCs hierarchy on microcirculation maintenance. Single-cell sequencing and spatial transcriptomics were further performed to analyze the mechanism of different LMCs recruitment abilities. ResultsLMCs RFR decreased to 30.75% immediately (95% CI, 6.64 - 54.86%, P < 0.001), and about 80.25% RFR was finally recovered within 5 minutes (95% CI, 55.2 - 105.3%, P = 0.0034) during M4 occlusion. For M5 and pial branches occlusion, RFR decreased to 55.78% with no further recovery (95% CI, 22.50-89.05%, P = 0.014), and 4 in 11 subjects had no valid microcirculation. For subjects with better recruitment ability, the proportion of arteriole is much higher in leptomeninx (76 - 79% vs. 36 - 56%). There were more significant interaction pairs associated with key signaling pathways between arteriole and neuron/astrocyte (TGF-beta signaling pathway, ECM-receptor interaction) ConclusionsAbundant LMCs supported hemodynamic stability during AIS in human subjects. The proportion of arteriole is much higher in leptomeninx for subjects with better recruitment ability. The Active neuron/astrocyte - arteriole interaction may improve LMCs recruitment. GRAPH ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/594043v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@eb7fb8org.highwire.dtl.DTLVardef@10e10aforg.highwire.dtl.DTLVardef@6954c7org.highwire.dtl.DTLVardef@13ceb98_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

CRISPR screens reveal convergent targeting strategies against evolutionarily distinct chemoresistance in cancer

Resistance to chemotherapy has been a major hurdle that limits therapeutic benefits for many types of cancer. Here we systematically identify genetic drivers underlying chemoresistance by performing 30 genome-scale CRISPR knockout screens for seven chemotherapeutic agents in multiple cancer cells. Chemoresistance genes vary between conditions primarily due to distinct genetic background and mechanism of action of drugs, manifesting heterogeneous and multiplexed routes towards chemoresistance. By focusing on oxaliplatin and irinotecan resistance in colorectal cancer, we unravel that evolutionarily distinct chemoresistance can share consensus vulnerabilities identified by 26 second-round CRISPR screens with druggable gene library. We further pinpoint PLK4 as a therapeutic target to overcome oxaliplatin resistance in various models via genetic ablation or pharmacological inhibition, highlighting a single-agent strategy to antagonize evolutionarily distinct chemoresistance. Our study not only provides resources and insights into the molecular basis of chemoresistance, but also proposes potential biomarkers and therapeutic strategies against such resistance.

cancer biology↗

bioRxiv2024

Regulation of Chromatin Modifications through Coordination of Nucleus Size and Epithelial Cell Morphology Heterogeneity

Cell morphology heterogeneity is pervasive in epithelial collectives, yet the underlying mechanisms driving such heterogeneity and its consequential biological ramifications remain elusive. Here, we observed a consistent correlation between the epithelial cell morphology and nucleus morphology during crowding, revealing a persistent log-normal probability distribution characterizing both cell and nucleus areas across diverse epithelial model systems. We further showed that this morphological diversity arises from asymmetric partitioning during cell division. Moreover, we provide insights into the impact of nucleus morphology on chromatin modifications. We demonstrated that constraining nucleus leads to downregulation of the euchromatic mark H3K9ac and upregulation of the heterochromatic mark H3K27me3. Furthermore, we showed that nucleus size regulates H3K27me3 levels through histone demethylase UTX. These findings highlight the significance of cell morphology heterogeneity as a driver of chromatin state diversity, shaping functional variability within epithelial tissues.

biophysics↗

bioRxiv2024

Image-based Strain Analysis Reveals Intracellular Strain Controlled by Nucleo-Cytoskeletal Coupling

Cells can sense and transduce mechanical forces, such as stretching, and convert these signals into diverse cell biological events. While much effort has been devoted to identifying the downstream biochemical and cellular responses, it is equally crucial to pinpoint the mechanical stimuli within a cell driving these responses. Specifically, much remains unknown about how intracellular strains are distributed and controlled during mechanical deformation. In this study, we developed a microscopy-based intracellular strain measurement technique. Utilizing the intrapopulation mechanical heterogeneity of epithelial monolayers, we observed an inverse relationship between cytoplasmic and nuclear strains. We found that this anti-correlation is abolished by the inhibition of Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, suggesting that nucleo-cytoskeletal coupling controls intracellular strain distribution. We discovered a direct connection between cytoplasmic strain and stretch-induced nucleus size changes, implying that molecular events arising from cytoplasmic deformation may drive nuclear remodeling during stretching. By conducting multivariable analyses, we found that the intracellular strain can be inferred from cell morphology. Overall, our experimental platform and findings provide a foundation for unraveling the relationship between mechanotransduction pathways and upstream intracellular strain. SIGNIFICANCEMechanical stimuli exert influence on epithelial cells, not only orchestrating embryogenesis and regeneration, but also regulating cancer progression and inflammatory conditions. Despite efforts to identify mechanically activated molecular events, understanding how deformation is distributed within cells to induce subcellular responses remains limited. Specifically, the control of subcellular strain distribution during mechanical stretch is unclear. In this study, we developed a microscopy-based method to measure subcellular strain and observed an inverse relationship between cytoplasmic and nuclear strains. Disrupting nucleo-cytoplasmic coupling abolished this relationship, suggesting its role in controlling strain distribution. Additionally, we found that cytoplasmic strain correlates with nucleus size changes during stretching, indicating cytoplasmic events influence nucleus remodeling.

biophysics↗

bioRxiv2024

Dissociable memory modulation mechanisms facilitate fear amnesia at different timescales

Memory reactivation renders consolidated memory fragile and thereby opens the window for memory updates, such as memory reconsolidation. However, whether memory retrieval facilitates update mechanisms other than memory reconsolidation remains unclear. We tested this hypothesis in three experiments with healthy human participants. First, we demonstrate that memory retrieval-extinction protocol prevents the return of fear expression shortly after extinction training and this short-term effect is memory reactivation dependent (Study 1, N = 57 adults). Furthermore, across different timescales, the memory retrieval-extinction paradigm triggers distinct types of fear amnesia in terms of cue-specificity and cognitive control dependence, suggesting that the short-term fear amnesia might be caused by different mechanisms from the cue-specific amnesia at a longer and separable timescale (Study 2, N = 79 adults). Finally, using continuous theta-burst stimulation (Study 3, N = 75 adults), we directly manipulated brain activity in the dorsolateral prefrontal cortex, and found that both memory reactivation and intact prefrontal cortex function were necessary for the short-term fear amnesia after the retrieval-extinction protocol. The differences in temporal scale, cue-specificity, and cognitive control ability dependence between the short- and long-term amnesia suggest that memory retrieval and extinction training trigger distinct underlying memory update mechanisms. These findings suggest the potential involvement of coordinated memory modulation processes upon memory retrieval and may inform clinical approaches for addressing persistent maladaptive memories.

neuroscience↗

bioRxiv2024

InstructPLM: Aligning Protein Language Models to Follow Protein Structure Instructions

Large language models are renowned for their efficacy in capturing intricate patterns, including co-evolutionary relationships, and underlying protein languages. However, current methodologies often fall short in illustrating the emergence of genomic insertions, duplications, and insertion/deletions (indels), which account for approximately 14% of human pathogenic mutations. Given that structure dictates function, mutated proteins with similar structures are more likely to persist throughout biological evolution. Motivated by this, we leverage crossmodality alignment and instruct fine-tuning techniques inspired by large language models to align a generative protein language model with protein structure instructions. Specifically, we present a method for generating variable-length and diverse proteins to explore and simulate the complex evolution of life, thereby expanding the repertoire of options for protein engineering. Our proposed protein LM-based approach, InstructPLM, demonstrates significant performance enhancements both in silico and in vitro. On native protein backbones, it achieves a perplexity of 2.68 and a sequence recovery rate of 57.51, surpassing Protein-MPNN by 39.2% and 25.1%, respectively. Furthermore, we validate the efficacy of our model by redesigning PETase and L-MDH. For PETase, all fifteen designed variable-length PETase exhibit depolymerization activity, with eleven surpassing the activity levels of the wild type. Regarding L-MDH, an enzyme lacking an experimentally determined structure, InstructPLM is able to design functional enzymes with an AF2-predicted structure. Code and model weights of InstructPLM are publicly available*.

bioinformatics↗

bioRxiv2024

Quantifying Leaf Symptoms of Sorghum Charcoal Rot in Images of Field-Grown Plants 9 Using Deep Neural Networks

Charcoal rot of sorghum (CRS) is a significant disease affecting sorghum crops, with limited genetic resistance available. The causative agent, Macrophomina phaseolina (Tassi) Goid, is a highly destructive fungal pathogen that targets over 500 plant species globally, including essential staple crops. Utilizing field image data for precise detection and quantification of CRS could greatly assist in the prompt identification and management of affected fields and thereby reduce yield losses. The objective of this work was to implement various machine learning algorithms to evaluate their ability to accurately detect and quantify CRS in red-green-blue (RGB) images of sorghum plants exhibiting symptoms of infection. EfficientNet-B3 and a fully convolutional network (FCN) emerged as the top-performing models for image classification and segmentation tasks, respectively. Among the classification models evaluated, EfficientNet-B3 demonstrated superior performance, achieving an accuracy of 86.97%, a recall rate of 0.71, and an F1 score of 0.73. Of the segmentation models tested, FCN proved to be the most effective, exhibiting a validation accuracy of 97.76%, a recall rate of 0.68, and an F1 score of 0.66. As the size of the image patches increased, both models validation scores increased linearly, and their processing time decreased exponentially. The models, in addition to being immediately useful for breeders and growers of sorghum, advance the domain of automated plant phenotyping and may serve as a base for drone-based or other automated field phenotyping efforts. Additionally, the models presented herein can be accessed through a web-based application where users can easily analyze their own images. Core ideasO_LIAutomated phenotyping tools are required for the efficient detection and quantification of charcoal rot of sorghum. C_LIO_LIClassification and segmentation models can distinguish between concurrent plant stresses with similar symptoms. C_LIO_LILarger image patch sizes generally improve model performance and reduce processing time. C_LI

plant biology↗

bioRxiv2024

Overcoming the preferred orientation problem in cryoEM with self-supervised deep-learning

While advances in single-particle cryoEM have enabled the structural determination of macromolecular complexes at atomic resolution, particle orientation bias (the so-called "preferred" orientation problem) remains a complication for most specimens. Existing solutions have relied on biochemical and physical strategies applied to the specimen and are often complex and challenging. Here, we develop spIsoNet, an end-to-end self-supervised deep-learning-based software to address the preferred orientation problem. Using preferred-orientation views to recover molecular information in under-sampled views, spIsoNet improves both angular isotropy and particle alignment accuracy during 3D reconstruction. We demonstrate spIsoNets capability of generating near-isotropic reconstructions from representative biological systems with limited views, including ribosomes, {beta}-galactosidases, and a previously intractable hemagglutinin trimer dataset. spIsoNet can also be generalized to improve map isotropy and particle alignment of preferentially oriented molecules in subtomogram averaging. Therefore, without additional specimen-preparation procedures, spIsoNet provides a general computational solution to the preferred orientation problem.

biophysics↗

bioRxiv2024

The Class III peroxidase OsPrx20 is a key regulator of stress response and growth in rice

In plants, reactive oxygen species maintain strictly low intracellular levels that is prerequisite for their function as second messengers. However, abiotic and biotic stresses are also long-term processes, requiring rapid scavenging of excess intracellular ROS. Plant Class III peroxidases, as multifunctional enzymes, are core enzymes in regulating intracellular ROS homeostasis and are key to regulating the complicated signaling network. Here, we found a rice Class III peroxidase OsPrx20 maintains intracellular ROS homeostasis in different conditions. OsPrx20 is a target of Ca2+/calmodulin-dependent protein kinase (OsDMI3) and phosphorylates its Thr-244 site by OsDMI3. Overexpression of OsPrx20 enhances osmotic stress tolerance but reduces blast resistance, while lack of OsPrx20 is opposite, suggesting OsPrx20 positively regulates osmotic stress tolerance but negatively regulates blast resistance in rice. Meanwhile, overexpression of OsPrx20 enlarges spike size and grain fullness, whereas lack of OsPrx20 leads to dwarfism and smaller spike. In ABA signaling, OsPrx20 Thr-244 phosphorylation is specifically dependent on OsDMI3 to reduce the sensitivity of ABA to seed germination and root growth and to enhance osmotic stress tolerance without affecting spike and grain development. Our study reveals an essential regulatory mechanism that directly activates OsPrx20 in ABA signaling, highlighting the multi-functionality of OsPrx20 in different biological processes.

plant biology↗

bioRxiv2024

Enhancing altruism by electrically augmenting frontoparietal gamma-band phase coupling

Cooperation, productivity, and cohesion in human societies depend on altruism, the tendency to share resources with others even though this is costly. While altruism is a widely shared social norm, people vary strongly in their inclination to behave altruistically, in particular across situations with different types of inequality in resource distribution. What neurobiological factors underlie this variability? And can these be targeted by interventions to enhance altruistic behavior? Here, we build on recent EEG findings that altruistic choices during disadvantageous inequality correlate with oscillatory gamma-band coherence between frontal regions (representing others interest) and parietal regions (representing neural evidence accumulation). We apply a transcranial alternating current stimulation (tACS) protocol designed to exogenously enhance this fronto-parietal coherence and find that this leads to increased altruism, specifically during disadvantageous inequality as hypothesized based on the EGG findings. Computational modeling reveals that this transcranial entrainment does not just add noise to the decision process but specifically increases the weight individuals assign to other-regarding concerns during choices. Our findings show that altruism can be enhanced by neurostimulation designed to enhance oscillatory synchronization between frontal and parietal areas. This establishes a neural basis for altruism and identifies a neural target for interventions aimed at improving prosocial behavior.

neuroscience↗

bioRxiv2024

Regulatory Functional Landscape of the HMX1 Gene for Normal Ear Development

Enhancers, through the combinatorial action of transcription factors (TFs), dictate both the spatial specificity and the levels of gene expression, and their aberrations can result in diseases. The association between HMX1 and its downstream enhancer with ear deformities has been previously documented. However, the pathogenic variations and molecular mechanisms underlying the bilateral constricted ear (BCE) malformations remain unclear. This study identifies a copy number variation (CNV) encompassing three enhancers that induces BCE. These enhancers, collectively termed the positional identity hierarchical enhancer cluster (PI-HEC), co-regulate spatiotemporal expression of the HMX1 gene in a coordinated and synergistic mode, each displaying unique activity-location-structure characteristics. A thorough exploration of this regulatory locus reveals that specific motif clusters within the PI-HEC variably modulate its activity and specificity, with the high mobility group (HMG) box combined with Coordinator and homeodomain (HD)-TFs notably influencing them respectively. Our findings based on various types of mouse models, reveal that both aberrant Hmx1 expression in the fibroblasts of the basal pinna, originating from neural crest cells, and ectopic expression in the distal pinna structures contribute to the abnormal development of the outer ear, including the cartilage, muscle, and epidermis tissues. This study deepens our understanding of mammalian ear morphogenesis and sheds light on the complexity of gene expression regulation by enhancers and specific sequence motifs.

developmental biology↗

bioRxiv2024

A spectral framework to map QTLs affecting joint differential networks of gene co-expression

Studying the mechanisms underlying the genotype-phenotype association is crucial in genetics. Gene expression studies have deepened our understanding of the genotype [->] expression [->] phenotype mechanisms. However, traditional expression quantitative trait loci (eQTL) methods often overlook the critical role of gene co-expression networks in translating genotype into phenotype. This gap highlights the need for more powerful statistical methods to analyze genotype [->] network [->] phenotype mechanism. Here, we develop a network-based method, called snQTL, to map quantitative trait loci affecting gene co-expression networks. Our approach tests the association between genotypes and joint differential networks of gene co-expression via a tensor-based spectral statistics, thereby overcoming the ubiquitous multiple testing challenges in existing methods. We demonstrate the effectiveness of snQTL in the analysis of three-spined stickleback (Gasterosteus aculeatus) data. Compared to conventional methods, our method snQTL uncovers chromosomal regions affecting gene co-expression networks, including one strong candidate gene that would have been missed by traditional eQTL analyses. Our framework suggests the limitation of current approaches and offers a powerful network-based tool for functional loci discoveries. Significance statementThis work addresses a key gap in understanding the mechanistic foundations for genotype-phenotype associations. While existing expression quantitative trait loci (eQTL) methods identify candidate loci affecting gene expression variants, they often neglect the crucial role of gene co-expression networks. Here, we develop a network-based QTL framework to map genetic loci affecting the gene co-expression network. Utilizing a tensor-based spectral approach, our snQTL method estimates the differential co-expression patterns and effectively identifies the associated genetic loci. Application of snQTL to three-spined sticklebacks revealed candidate loci missed by standard methods. This work suggests the limitations of current approaches and highlights the potential of network-based functional loci discovery.

genetics↗

bioRxiv2024

Targeting PIKfyve-driven lipid homeostasis as a metabolic vulnerability in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) subsists in a nutrient-deregulated microenvironment, making it particularly susceptible to treatments that interfere with cancer metabolism1 2. For example, PDAC utilizes and is dependent on high levels of autophagy and other lysosomal processes3-5. Although targeting these pathways has shown potential in preclinical studies, progress has been hampered by the challenge of identifying and characterizing favorable targets for drug development6. Here, we characterize PIKfyve, a lipid kinase integral to lysosomal functioning7, as a novel and targetable vulnerability in PDAC. In human patient and murine PDAC samples, we discovered that PIKFYVE is overexpressed in PDAC cells compared to adjacent normal cells. Employing a genetically engineered mouse model, we established the essential role of PIKfyve in PDAC progression. Further, through comprehensive metabolic analyses, we found that PIKfyve inhibition obligated PDAC to upregulate de novo lipid synthesis, a relationship previously undescribed. PIKfyve inhibition triggered a distinct lipogenic gene expression and metabolic program, creating a dependency on de novo lipid metabolism pathways, by upregulating genes such as FASN and ACACA. In PDAC, the KRAS-MAPK signaling pathway is a primary driver of de novo lipid synthesis, specifically enhancing FASN and ACACA levels. Accordingly, the simultaneous targeting of PIKfyve and KRAS-MAPK resulted in the elimination of tumor burden in a syngeneic orthotopic model and tumor regression in a xenograft model of PDAC. Taken together, these studies suggest that disrupting lipid metabolism through PIKfyve inhibition induces synthetic lethality in conjunction with KRAS-MAPK-directed therapies for PDAC.

cancer biology↗

bioRxiv2024

Nine high-quality Anas genomes provide new insights into Anas evolution and domestication

The evolutionary origin and genetic architecture of domestic animals are becoming more tractable as the availability of more domestic species genomes. Evolutionary studies of wild and domestic organisms have yielded many fascinating discoveries, while the stories behind the species diversity of Anas or the domestication of duck were largely unknown. Here, we assembled eight chromosome-level Anas genomes. Together with our recently available Pekin duck genome, we investigated Anas phylogeny, genetic differentiation, and gene flow. Extensive phylogenetic inconsistencies were observed in Anas genomes, particularly two phylogeny conflicts between autosome and Z-chromosome. However, the Z chromosome was less impacted by introgression and more suitable to elucidate phylogenetic relationships than autosomes. From the Z-chromosome perspective, we found that the speciation of Anas platyrhynchos and Anas zonorhyncha accompanied with female-biased gene flow, and remodeled duck domestication history. Moreover, we constructed an Anas pan-genome and identified several differentiated SVs between domestic and wild ducks. These SVs might act as repressors/enhancers to regulate their neighboring genes (i.e., GHR and FER), which represented the promising "domestication genes". Additionally, Anas genomes were found being presented LTR retrotransposon bursts, which might largely contribute to functional shifts of genes involved in duck domestication (i.e., MITF and IGF2BP1). This study opens a new window to unravel avian speciation and domestication from Z chromosome.

genomics↗

bioRxiv2024

High-throughput Screening of Compounds against Autoluminescent Nonreplicating Mycobacterium tuberculosis under Diverse Conditions

The screening of new anti-mycobacterial chemicals is primarily focused on inhibiting the active growing bacteria. However, a major challenge in tuberculosis control is the ability of Mycobacterium tuberculosis to enter a nonreplicating state for extended periods, rendering it resistant to many clinical drugs and complicating eradication efforts. Existing low-oxygen-recovery assays designed for screening compounds targeting nonreplicating M. tuberculosis have limitations, including the colony-forming unit counting for non-luminous M. tuberculosis and the instability of the free plasmid carrying luxAB genes in luminescent M. tuberculosis, along with exogenous substrate requirements for light producing. Moreover, these assays fail to accurately replicate the growth conditions of nonreplicating M. tuberculosis in vitro, thus resulting in less convincing results. To address these challenges, we have developed an autoluminescence-based, cholesterol-enriched culture evaluation model to assess 17 anti-tuberculosis drugs of different classes against nonreplicating M. tuberculosis. Our findings indicate that the relative light unit, measured in real-time, serves as a reliable surrogate marker for colony-forming unit, which typically becomes available one month later. This suggests the utility of our model for the rapid determination of drug susceptibility dynamically. The autoluminescent M. tuberculosis, harbouring luxCDABE gene cluster within its genome, can emit blue-green light stably and autonomously without requiring an external substrate supplement. The minimal inhibitory concentrations of all the drugs tested under anaerobic conditions are significantly different from that detected in aerobic environment. Our model allows for rapid, precise, and efficient assessment of drug activity under anaerobic conditions, thereby enabling a more comprehensive evaluation of anti-mycobacterial efficacy. Overall, our model represents a significant advancement in anti-tuberculosis drug discovery and pharmaceutical development.

microbiology↗

bioRxiv2024

Dark-based Optical Sectioning assists Background Removal in Fluorescence Microscopy

A fundamental challenge in fluorescence microscopy is the defocused background caused by scattering light, optical aberration, or limited axial resolution. Severe defocus backgrounds will submerge the in-focus information and cause artifacts in the following processing. Here, we leverage a priori knowledge about dark channels of biological structures and dual frequency separation to develop a single-frame defocus removal algorithm. It stably improves the signal-to-background ratio and structural similarity index measure of images by approximately 10-fold, and recovers in-focus signal with 85% accuracy, even when the defocus background is 50 times larger than in-focus information. Our Dark-based optical sectioning approach (Dark sectioning) is fully compatible with various microscopy techniques, such as wide-filed microscopy, polarized microscopy, laser-scanning / spinning-disk confocal microscopy, stimulated emission depletion microscopy, lightsheet microscopy, and light-field microscopy. It also complements reconstruction or processing algorithms such as deconvolution, structure illumination microscopy, and super-resolution optical fluctuation imaging.

bioinformatics↗