Biology subjects

Qian, C.

Publications and source records attributed to Qian, C..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Geometry-based BERT: an experimentally validated deep learning model for molecular property prediction in drug discovery

Various deep learning based methods have significantly impacted the realm of drug discovery. The development of deep learning methods for identifying novel structural types of active compounds has become an urgent challenge. In this paper, we introduce a self-supervised representation learning framework, i.e., GEO-BERT. GEO-BERT considers the information of atoms and chemical bonds in chemical structures as the input, and integrates the positional information of the three-dimensional conformation of the molecule for training. Specifically, GEO-BERT enhances its ability to characterize molecular structures by introducing three different positional relationships: atom-atom, bond-bond, and atom-bond. By benchmarking study, GEO-BERT has demonstrated optimal performance on multiple benchmarks. We also performed prospective study to validate the GEO-BERT model, with screening for DYRK1A inhibitors as a case. Two potent and novel DYRK1A inhibitors (IC50: <1 M) were ultimately discovered at a hit rate of 10%. Taken together, we have developed the Geometry-based BERT model for molecular property prediction and proved its practical utility in early-stage drug discovery. Graphical Abstract GEO-BERT is a model pretrained on large-scale drug molecule data, while improving the accuracy of property prediction by using three-dimensional structural information within the molecule. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/630211v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1e9bc54org.highwire.dtl.DTLVardef@4b6832org.highwire.dtl.DTLVardef@a5d8eeorg.highwire.dtl.DTLVardef@44f7be_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2024

Influenza Virus Enhances Bacterial Growth in the Lungs through Weakening Capsule Receptor-Mediated Bacterial Capture by Neutrophils

Certain serotypes of encapsulated bacteria are more associated with severe pneumonia than others, but the biological reason behind this association has remained largely unknown. This study has uncovered a causal relationship between capsule serotype and virulence phenotype of Streptococcus pneumoniae, a leading cause of community-acquired pneumonia, in mice using isogenic bacteria. Certain high-virulence (HV) serotypes persist in the lungs and lead to severe pneumonia, whereas the low-virulence (LV) serotypes are effectively cleared. The capsule type-dependent difference in virulence is determined by the capsule-binding receptors, which recognize the capsular polysaccharides of the LV serotypes (but not the HV serotypes) and enable pathogen capture by neutrophils through complement activation. The capsule receptor-driven immunity of lung neutrophils is significantly compromised by pre-infection of influenza virus, which enhances the survival and virulence of the LV S. pneumoniae serotypes, and recurs in the heightened pathogenicity of Haemophilus influenzae. This study not only unravels the long-sought mechanistic mystery of serotype-dependent virulence in bacterial lung infections, but also provides an explanation for the well-known pathogenesis synergism between influenza virus and respiratory bacteria. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/622589v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1464164org.highwire.dtl.DTLVardef@16f34e5org.highwire.dtl.DTLVardef@1f9ad46org.highwire.dtl.DTLVardef@df104d_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Spatially global effects of feature-based attention in functional subdivisions of human subcortical nuclei

Attention can prioritize the processing of non-spatial features throughout the visual field. While human subcortical nuclei are known to play important roles in spatial attention, the subcortical mechanisms of feature-based attention remain elusive. Using high-resolution 7T fMRI, we investigated the spatially global effects of color-based attention in functional subdivisions of human subcortical nuclei. Paying attention to a color matching the unattended stimulus across the visual field selectively modulated BOLD signals in the parvocellular (P) layers of the lateral geniculate nucleus (LGN) of the thalamus, enhancing the response to the unattended stimulus while reducing the response to the attended stimulus. The spatially global effects of color-based attention were also found in the deeper layers of the superior colliculus (SC), early visual cortices, and the intraparietal sulcus (IPS) of the parietal lobe. Effective connectivity analyses further revealed enhanced feedforward and feedback connectivity between LGN and V1, along with top-down modulation from IPS through the SC and ventral pulvinar. Finally, attended color can be decoded from multi-voxel response patterns in frontoparietal regions. These findings demonstrate that color-based attention selectively modulates color processing in P subdivisions of the LGN of the thalamus throughout the visual field, controlled by top-down signals from the parietal cortex through the SC and pulvinar. HighlightsO_LIColor-based attention selectively modulates parvocellular activity in the LGN throughout the visual field. C_LIO_LISpatially global effect of feature-based attention significantly modulates activity in the deeper layers of the SC. C_LIO_LIFeature-based attention enhances both feedforward and feedback connectivity between the LGN and V1. C_LIO_LITop-down signals from IPS through the SC and pulvinar controls feature-based attention. C_LI

neuroscience↗

bioRxiv2024

Celastrol binds to pyruvate kinase and imitates the metabolic reprogramming of ATP

ATP is the final product of glycolysis and the TCA cycle. However, the counter-regulation of glycosis and TCA by ATP is poorly understood. Here we show that ATP analog celastrol (CLT) binds to the ATP binding pocket on pyruvate kinase PKM (PKM) while inducing allosteric regulation of PKM. Quantum calculation predicts hydrogen bond formation between CLT and asparagine. Liquid chromatography-mass spectrometry further identifies PKM as CLTs target. The inhibition of PKM is stronger and longer for CLT while weak and short for ATP. Notably, CLT-PKM interaction perfectly underlies the hypoglycemic effects by forming glucose carbon flux U turn before PKM. Besides, the PKM allostery induces a tricarboxylic acid (TCA) vortex which could promote amino acid and lipid degradation as the energy compensation, leading to a significant weight loss. Additionally, CLT exerts efficient antioxidant effects by altering the glucose flux to strengthen the pentose phosphate pathway. Consequently, the CLT-PKM interaction vividly reproduces the ATP-PKM interaction named "ATP resistance" in the diabetes pathogenesis.

pharmacology and toxicology↗

bioRxiv2024

A machine learning approach reveals that spore levels in organic bulk tank milk are dependent on farm characteristics and meteorological factors

Bacterial spores in raw milk can lead to quality issues in milk and milk derived products. Since these spores originate from farm environments, it is important to understand contributions of farm-level factors to spore levels in raw milk. Identifying highly influential factors will guide interventions to control the transmission of spores from farm environments into bulk tank raw milk and therefore minimize spoilage in the finished products. The objective of this study was to investigate the impact of farm management practices and meteorological factors on levels of different spore types in organic raw milk by leveraging machine learning models. In this study, raw milk from certified organic dairy farms (n = 102) located across 11 states was collected 6 times over a year and tested for standard plate count, psychrotolerant spore count, mesophilic spore count, thermophilic spore count, and butyric acid bacteria. At each sampling date, a survey was collected from each farm to obtain structured data about farm management practices. Meteorological factors related to temperature, precipitation, solar radiation, and wind were obtained on the date of sampling as well as 1, 2, and 3 days prior to the date of sampling from an open-source website. The dataset was stratified separately based on the use of a parlor for milking, number of years since organic certification, and whether the lactating herd was exposed to pasture time into sub-datasets to address the potential confounders. Using the entire datasets and 6 sub-datasets respectively, we constructed random forest regression models to predict log10 mesophilic spore count, log10 thermophilic spore count, and log10 butyric acid bacteria most probable number as well as a random forest classification model to classify the presence of psychrotolerant spores in each raw milk sample. The summary statistics showed that spore levels vary considerably between certified organic farms but were only slightly higher than spore levels previously reported from conventional dairy farms. The variable importance plots from the random forest models suggest that herd size, certification year, employee-related variables (e.g., number of people milking cows per week), clipping and flaming udders, stocking density, and principal component representing air temperatures are among the top variables influencing the spore levels in organic raw milk, despite the limitation in the model performance (the highest performance for regression and classification is R2 of 0.36 for predicting TSC for farms with a parlor and accuracy of 0.73 for classifying positive PSC for farms without a parlor, respectively). The relatively small effects of top variables as demonstrated by the partial dependence plots suggest that an individualized approach that synergistically considers multiple farm and environmental factors is needed to enable a risk-based approach for managing spore levels. While at the current stage, these models were insufficiently accurate to be used as predictive tools, incorporating novel data streams such as video surveillance and daily farm observations with computer vision and natural language processing, respectively, has the potential to enhance the performance of the model as a real-time monitoring tool for spores as an indicator of milk microbiological quality.

microbiology↗

bioRxiv2024

A novel hybrid Wireless Integrated Sensing Detector for simultaneous EEG and MRI (WISDEM)

Concurrent recording of EEG/fMRI signals reveals cross-scale neurovascular dynamics that are crucial for elucidating fundamental linkage between function and behaviors. However, MRI scanners generate tremendous artifacts for EEG detection. Despite existing denoising methods, cabled connections to EEG receivers are susceptible to environmental fluctuations inside MRI scanners, creating baseline drifts that complicate EEG signal retrieval from the noisy background. Here, a Wireless Integrated Sensing Detector for simultaneous EEG and MRI (WISDEM) is developed to encode fMRI and EEG signals on distinct sidebands of the detectors oscillation carrier wave for detection by a standard MRI console over the entire duration of fMRI sequence. Local field potential (LFP) and fMRI maps are retrieved through low-pass and high-pass filtering of frequency-demodulate signals. From optogenetically-stimulated somatosensory cortex, the positive correlation between evoked LFP and fMRI signals validates strong neurovascular coupling, enabling cross-scale brain mapping with this 2-in-1 transducer as a research and diagnostic tool.

bioengineering↗

bioRxiv2024

Amblyopic deficits in monocular processing and binocular interactions revealed by submillimeter 7T fMRI and EEG frequency tagging

Disruption of retinal input early in life can lead to amblyopia, a condition characterized by reduced visual acuity despite corrected optics. Although extensive losses of neural activity have been found in the early visual cortex, it remains unclear whether they reflect deficits in feedforward or feedback processing, or abnormal binocular interactions. Combining submillimeter 7T fMRI and EEG frequency tagging, our study revealed the precise neural deficits in monocular processing and binocular interactions in human adults with unilateral amblyopia. Cortical depth-dependent fMRI revealed monocular response deficits in cortical layers of the primary visual cortex (V1) receiving thalamic input, which carried over to the downstream areas (V2-V4) in feedforward processing. Binocular stimulation produced a greater signal loss in the superficial layers of V1, consistent with suppression from the fellow eye by lateral inhibition. EEG data further demonstrate reduced suppression from the amblyopic eye, weakened binocular integration, and delayed monocular and binocular processing. Our results support attenuated and delayed monocular processing in V1 layers receiving thalamic input in human amblyopia, followed by imbalanced binocular suppression and weakened binocular integration in the superficial layers, which further reduced signal strength and processing speed. These precise neural deficits can help developing more targeted and effective treatments for the vision disorder. SignificanceCortical depth-dependent 7T fMRI and EEG frequency tagging revealed attenuated and delayed neural activity in monocular processing and binocular interactions in human amblyopia. Neural deficits in monocular processing arise from the input layers of V1, followed by imbalanced binocular suppression and weakened binocular integration in the superficial layers. The precise neural deficits at high spatiotemporal resolution can help developing more targeted and effective treatment for amblyopia. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/591076v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@c174d8org.highwire.dtl.DTLVardef@de51eborg.highwire.dtl.DTLVardef@1d443c0org.highwire.dtl.DTLVardef@ac267a_HPS_FORMAT_FIGEXP M_FIG C_FIG This figure illustrates the precise neural deficits of amblyopia in monocular processing and binocular interactions based on the main findings of the current study. Attenuated and delayed neural activity arises from V1 cortical layers receiving thalamic input in monocular processing, followed by imbalanced binocular suppression and weakened binocular integration in the superficial layers, further reducing visual signal strength and processing speed.

neuroscience↗

bioRxiv2024

Exposure assessment suggests some cytotoxic Bacillus cereus group genotypes can grow over 3 logs in HTST milk throughout the shelf life at temperature abuse conditions

Cytotoxic Bacillus cereus group strains are common causes of foodborne illness including diarrhea. However, our ability to assess food safety risks associated with the exposure to cytotoxic B. cereus group strains via contaminated food is limited due to the lack of predictive tools. In this study, we experimentally quantified the growth of 17 cytotoxic B. cereus group strains, representing six phylogenetic groups, in skim milk broth and used the growth data to develop an exposure assessment model. While none of the tested strains showed detectable growth in HTST milk at 4 or 6{degrees}C, 15 of the 17 strains showed growth at 10{degrees}C, 1 of the 17 strains showed growth at 8{degrees}C, and all strains grew at [≥]14{degrees}C. Growth data for 16 strains allowed us to generate linear secondary growth models, which were then used to develop the exposure assessment model. We simulated a five-stage supply chain with up to 35 consumer storage days, as that was the timing when distinguishable variations in percent milk containers over 105 CFU/m with different B. cereus genotypes were observed. When the initial contamination level of the HTST milk is set at an average of 100 CFU/mL, the model predicts that, on consumer home storage day 21 and 35, 2.81{+/-}0.66 and 4.13{+/-}2.53 % (mean {+/-} standard deviation) of the milk containers would exceed B. cereus group concentrations of 105 CFU/mL; these data represent the average across all strains. Sensitivity analysis showed that variation in the input parameter Q0, the initial physiological state of cells, has the largest effect on models prediction for 1 of 4 group II isolates, 1 of 6 group IV isolates and both group V isolates, suggesting the need to better characterize the growth parameters of these isolates. What-if scenario analysis showed that increased mean and variability in storage temperature at the consumers home both have substantial influence on final predicted B. cereus group concentration in milk containers. This model introduces an initial tool designed to facilitate risk-based food safety decision making for products that are contaminated with low B. cereus group levels.

microbiology↗

bioRxiv2024

Single dopaminergic neuron DAN-c1 in Drosophila larval brain mediates aversive olfactory learning through D2-like receptors

The intricate relationship between the dopaminergic system and olfactory associative learning in Drosophila has been an intense scientific inquiry. Leveraging the formidable genetic tools, we conducted a screening of 57 dopaminergic drivers, leading to the discovery of DAN-c1 driver, uniquely targeting a pair of dopaminergic neurons (DAN) in the larval brain. While the involvement of excitatory D1-like receptors is well-established, the role of D2-like receptors (D2Rs) remains underexplored. Our investigation reveals the expression of D2Rs in both DANs and the mushroom body (MB) of third instar larval brains. Silencing D2Rs in DAN-c1 via microRNA disrupts aversive learning, further supported by optogenetic activation of DAN-c1 during training, affirming the inhibitory role of D2R autoreceptor. Intriguingly, D2R knockdown in the MB impairs both appetitive and aversive learning. These findings elucidate the distinct contributions of D2Rs in diverse brain structures, providing novel insights into the molecular mechanisms governing associative learning in Drosophila larvae.

neuroscience↗

bioRxiv2023

Magnetogenetic stimulation inside MRI induces spontaneous and evoked changes in neural circuits activity in rats

The ability to modulate specific neural circuits and simultaneously visualize and measure brain activity with MRI would greatly impact understanding brain function in health and disease. The combination of neurostimulation methods and MRI in animal models have already shown promise in elucidating fundamental mechanisms associated with brain activity. We developed an innovative magnetogenetics neurostimulation technology that can trigger neural activity through magnetic fields. Similar to other genetic-based neuromodulation methods, magnetogenetics offers cell-, area- and temporal- specific control of neural activity. However, the magnetogenetics protein (Electromagnetic Preceptive Gene (EPG)) are activated by non-invasive magnetic fields, providing a unique way to target neural circuits by the MRI gradients while simultaneously measure their effect on brain activity. EPG was expressed in rats visual cortex and the amplitude of low-frequency fluctuation (fALFF), resting-state functional connectivity (FC), and sensory activation was measured using a 7T MRI. The results demonstrate that EPG-expressing rats had significantly higher signal fluctuations in the visual areas and stronger FC in sensory areas consistent with known anatomical visuosensory and visuomotor connections. This new technology complements the existing neurostimulation toolbox and provides a mean to study brain function in a minimally-invasive way which was not possible previously.

neuroscience↗

bioRxiv2023

Discovery of a Novel Chemotype as DYRK1A Inhibitors against Alzheimer's disease: Computational Modeling and Biological Evaluation

Dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) plays an essential role in tau and A{beta} pathology closely related to Alzheimers disease (AD). Accumulative evidence has demonstrated DYRK1A inhibition is able to reduce the pathological features of AD. Nevertheless, there is no approved DYRK1A inhibitors for clinical use as anti-AD drugs. This is somewhat the lack of effective and safe chemotypes of DYRK1A inhibitors. To address this issue, we carried out in silico screening, in vitro assays and in vivo efficacy evaluation with the aim to discover a new class of DYRK1A inhibitors for potential treatment of AD. By in silico screening, we selected and purchased 16 potential DYRK1A inhibitors from the Specs chemical library. Among them, compound Q17 (Specs ID: AO-476/40829177) potently inhibited DYRK1A. The hydrogen bonds between compound Q17 and each of three amino acid residues named GLU239, LEU241 and LYS188, were uncovered by molecular docking and molecular dynamics simulation. The cell-based assays showed that compound Q17 could protect SH-SY5Y cells from okadaic acid (OA)-induced injury by targeting DYRK1A. More importantly, compound Q17 significantly improved cognitive dysfunction in 3xTg-AD mice, ameliorated pathological changes, and reduced the expression of DYRK1A, GSK-3{beta} and GSK-3{beta} (pSer9), attenuated tau hyperphosphorylation and A{beta} deposition as well. In summary, our computational modeling strategy is effective to identify novel chemotypes of DYRK1A inhibitors with great potential to treat AD, and the identified compound Q17 in this study is worthy of further study. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/565431v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@75abecorg.highwire.dtl.DTLVardef@16a3611org.highwire.dtl.DTLVardef@39aeborg.highwire.dtl.DTLVardef@afc8ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2023

ONECUT2 Activates Diverse Resistance Drivers of Androgen Receptor-Independent Heterogeneity in Prostate Cancer

Significance StatementONECUT2 (OC2) is a master transcription factor that alters lineage identity by activating gene networks associated with both neuroendocrine prostate cancer and prostate adenocarcinoma. A small molecule inhibitor of OC2 represses the lineage plasticity program activated by enzalutamide, suggesting OC2 inhibition as a novel therapeutic strategy to prevent emergence of treatment-resistant variants. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/560025v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@13278a4org.highwire.dtl.DTLVardef@8e82aeorg.highwire.dtl.DTLVardef@25a115org.highwire.dtl.DTLVardef@728eb4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphic AbstractC_FLOATNO C_FIG Androgen receptor-(AR-) indifference is a mechanism of resistance to hormonal therapy in prostate cancer (PC). Here we demonstrate that the HOX/CUT transcription factor ONECUT2 (OC2) activates resistance through multiple drivers associated with adenocarcinoma, stem-like and neuroendocrine (NE) variants. Direct OC2 targets include the glucocorticoid receptor and the NE splicing factor SRRM4, among others. OC2 regulates gene expression by promoter binding, enhancement of chromatin accessibility, and formation of novel super-enhancers. OC2 also activates glucuronidation genes that irreversibly disable androgen, thereby evoking phenotypic heterogeneity indirectly by hormone depletion. Pharmacologic inhibition of OC2 suppresses lineage plasticity reprogramming induced by the AR signaling inhibitor enzalutamide. These results demonstrate that OC2 activation promotes a range of drug resistance mechanisms associated with treatment-emergent lineage variation in PC. Our findings support enhanced efforts to therapeutically target this protein as a means of suppressing treatment-resistant disease.

cancer biology↗

bioRxiv2023

X chromosome encoded histone demethylase UTX regulates mammalian axon regeneration via microRNA-124

Epigenetic regulation of neuronal transcriptomic landscape is emerging to be a key coordinator of mammalian neural regeneration. Here we investigated roles of two histone 3 lysine 27 (H3K27) demethylases Kdm6a/b in controlling neuroprotection and axon regeneration. Deleting either Kdm6a or Kdm6b led to enhanced sensory axon regeneration in the PNS, whereas in the CNS only deleting Kdm6a in retinal ganglion cells (RGCs) significantly enhanced optic nerve regeneration. Moreover, both Kdm6a and Kdm6b functioned to regulate RGC survival but with different mechanisms. Mechanistically, Kdm6a regulates RGC regeneration via distinct pathway from that of Pten and co-deleting Kdm6a and Pten resulted in long distance optic nerve regeneration passing the optic chiasm. In addition, RNA-seq profiling revealed that Kdm6a deletion switched the RGC transcriptomics into a developmental-like state and suppressed several known repressors of neural regeneration. Klf4 was identified as a direct downstream target of Kdm6a-H3K27me3 signaling in both sensory neurons and RGCs to regulate axon regeneration. These findings not only revealed different roles of Kdm6a and Kdm6b in regulation of neural regeneration and their underlying mechanisms, but also identified Kdm6a- mediated histone demethylation signaling as a novel epigenetic target for supporting CNS neural regeneration.

neuroscience↗

bioRxiv2023

Targeting pioneer transcription factor Ascl1 to promote optic nerve regeneration

In adult mammalian central nervous system (CNS) neurons, axon regeneration after injury remains limited due to unfavorable gene regulatory programs. Factors enabling comprehensive epigenetic and transcriptional transitions, for instance, pivotal transcription factors that mediate neurogenesis and morphogenesis may be sufficient to promote CNS axon regeneration. Based on the analyses of multiple public whole-genome RNA and chromatin accessibility sequencing dataset of mouse retina development, as well as previous functional studies on the regeneration-capable dorsal root ganglion neurons, we hypothesize that the overexpression of pioneer transcription factor Achaete-Scute homolog 1 (Ascl1) would promote axon regeneration in the adult mammalian CNS neurons. We employed the optic nerve crush in mice, a common model for studying CNS axon regeneration, neuron survival and glaucoma, to investigate the effect of Ascl1 overexpression on the post-injury optic nerve regeneration. We found that Ascl1 could sufficiently promote regenerated axons past the crush site and significantly preserve the survival of retinal ganglion cells. Mechanistically, we revealed that effects of Ascl1 was mediated by known pro-regeneration factor Sox11 but not others. Together, our study established an effective workflow combined with the integrated computational inference and experimental validation for discovering functionally important target for promoting CNS neuron axon regeneration and survival.

neuroscience↗

bioRxiv2023

Intercellular communication atlas reveals Oprm1 as a neuroprotective factor for retinal ganglion cells

The progressive death of mature neurons often results in neurodegenerative diseases. While the previous studies have mostly focused on identifying intrinsic mechanisms controlling neuronal survival, the extracellular environment also plays a critical role in regulating cell viability. Here we explore how intercellular communication contributes to the survival of retinal ganglion cells (RGCs) following the optic nerve crush (ONC). Although the direct effect of the ONC is restricted to the RGCs, we observed transcriptomic responses in other retinal cells to the injury based on the single-cell RNA-seq, with astrocytes and Muller glia having the most interactions with RGCs. By comparing the RGC subclasses with distinct resilience to ONC-induced cell death, we found that the high-survival RGCs tend to have more ligand-receptor interactions with other retinal cells, suggesting that these RGCs are intrinsically programmed to foster more communication with their surroundings. Furthermore, we identified the top 47 interactions that are stronger in the high-survival RGCs, likely representing neuroprotective interactions. We performed functional assays on one of the receptors, -opioid receptor (Oprm1), a receptor known to play roles in regulating pain, reward, and addictive behavior. Although Oprm1 is preferentially expressed in intrinsically photosensitive retinal ganglion cells (ipRGC), its neuroprotective effect could be transferred to multiple RGC subclasses by selectively overexpressing Oprm1 in pan-RGCs in ONC, excitotoxicity, and glaucoma models. Lastly, manipulating Oprm1 activity improved visual functions or altered pupillary light response in mice. Our study provides an atlas of cell-cell interactions in intact and post-ONC retina, and a strategy to predict molecular mechanisms controlling neuroprotection, underlying the principal role played by extracellular environment in supporting neuron survival.

neuroscience↗

bioRxiv2023

The homeodomain drives favorable DNA binding energetics of prostate cancer target ONECUT2

The ONECUT transcription factors feature a CUT and a homeodomain, evolutionarily conserved elements that bind DNA cooperatively, but the process remains mechanistically enigmatic. Using an integrative DNA binding analysis of ONECUT2, a driver of aggressive prostate cancer, we show that the homeodomain energetically stabilizes the ONECUT2-DNA complex through allosteric modulation of CUT. Further, evolutionarily conserved base-interactions in both the CUT and homeodomain are necessary for the favorable thermodynamics. We have identified a novel arginine pair unique to the ONECUT family homeodomain that can adapt to DNA sequence variations. Base interactions in general, including by this arginine pair, are critical for optimal DNA binding and transcription in a prostate cancer model. These findings provide fundamental insights into DNA binding by CUT-homeodomain proteins with potential therapeutic implications. One-Sentence SummaryBase-specific interactions regulate homeodomain-mediated stabilization of DNA binding by the ONECUT2 transcription factor.

biophysics↗

bioRxiv2023

Particle-based phasor-FLIM-FRET resolves protein-protein interactions inside single viral particles

Fluorescence lifetime imaging microscopy (FLIM) is a popular modality to create additional contrast in fluorescence images. By carefully analyzing pixel-based nanosecond lifetime patterns, FLIM allows studying complex molecular populations. At the single molecule or single particle level, however, image series often suffer from low signal intensities per pixel, rendering it difficult to quantitatively disentangle different lifetime species, such as during FRET analysis in the presence of a significant donor-only fraction. To address this problem, we combined particle localization with phasor-based FLIM analysis. Using simulations, we first showed that an average of [~]300 photons, spread over the different pixels encompassing single fluorescing particles and without background, is enough to determine a correct phasor signature (standard deviation <5% for a 4 ns lifetime). For immobilized single- or double-labeled dsDNA molecules, we next validated that particle-based phasor-FLIM-FRET readily allows estimating fluorescence lifetimes and FRET from single molecules. Thirdly, we applied particle-based phasor-FLIM-FRET to investigate protein-protein interactions in sub diffraction HIV-1 viral particles. To do this, we first quantitatively compared the fluorescence brightness, lifetime and photostability of different popular fluorescent protein-based FRET probes when genetically fused to the HIV-1 integrase enzyme (IN) in viral particles, and conclude that eGFP, mTurquoise2 and mScarlet perform best. Finally, for viral particles co-expressing FRET-donor/acceptor labeled IN, we determined the absolute FRET efficiency of IN oligomers. Available in a convenient open-source graphical user interface, we believe that particle-based phasor-FLIM-FRET is a promising tool to provide detailed insights in samples suffering from low overall signal intensities. Why it mattersPhasor-FLIM is an extraordinarily popular tool for fluorescence lifetime imaging analysis. However, it remains susceptible for low signal intensities, operational challenges and therefore required informed users and a clear analysis understanding. In this work we developed a convenient all-graphical workflow for quantitative phasor-FLIM in heterogenous and low-signal samples and applied it to quantifying absolute FRET efficiencies from protein-protein interactions inside single viral particles. Moreover, containing a well-illustrated theoretical introduction to time-domain phasor-FLIM, our paper helps novice users to correctly implement phasor-FLIM in standard microscopy practice.

biophysics↗

bioRxiv2023

Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen

The cytoplasmic dynein-1 (dynein) motor plays a key role in cellular organisation by transporting a wide variety of cellular constituents towards the minus ends of microtubules. However, relatively little is known about how the biosynthesis, assembly and functional diversity of the motor is orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a guide RNA library targeting 18,253 genes, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints generated from multiplexed images revealed co-functional genes involved in many cellular processes, including several candidate novel regulators of core dynein functions. Mechanistic analysis of one of these proteins, the RNA-binding protein SUGP1, provides evidence that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our dataset represents a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organisation that were captured by our high-content imaging.

cell biology↗