Biology subjects
Publications and source records attributed to Fan, H..
SARS-CoV-2-related pangolin coronavirus GX_P2V(short_3UTR) is highly attenuated, but can cause mortality in a specifically designed human ACE2-transgenic mouse model, making it an invaluable surrogate model for evaluating the efficacy of drugs and vaccines against SARS-CoV-2.
Immunomodulatory imide drugs (IMiDs) degrade specific C2H2 zinc finger degrons in transcription factors, making them effective against certain cancers. SALL4, a cancer driver, contains seven C2H2 zinc fingers in four clusters, including an IMiD degron in zinc finger cluster two (ZFC2). Surprisingly, IMiDs do not inhibit growth of SALL4 expressing cancer cells. To overcome this limit, we focused on a non-IMiD degron, SALL4 zinc finger cluster four (ZFC4). By combining AlphaFold and the ZFC4-DNA crystal structure, we identified a potential ZFC4 drug pocket. Utilizing an in silico docking algorithm and cell viability assays, we screened chemical libraries and discovered SH6, which selectively targets SALL4-expressing cancer cells. Mechanistic studies revealed that SH6 degrades SALL4 protein through the CUL4A/CRBN pathway, while deletion of ZFC4 abolished this activity. Moreover, SH6 led to significant 62% tumor growth inhibition of SALL4+ xenografts in vivo and demonstrated good bioavailability in pharmacokinetic studies. In summary, these studies represent a new approach for IMiD independent drug discovery targeting C2H2 transcription factors in cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/574032v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@b8e4e8org.highwire.dtl.DTLVardef@bc6f01org.highwire.dtl.DTLVardef@11bbe85org.highwire.dtl.DTLVardef@791cb1_HPS_FORMAT_FIGEXP M_FIG C_FIG
The BRG/Brahma-associated factors (BAF or mSWI/SNF) family of chromatin remodeling complexes are critical regulators of gene expression and are major determinants of cancer and other diseases. Two paralog ATPases, SMARCA4 and SMARCA2 (BRG1 and BRM, respectively), provide the enzymatic activity required for chromatin remodeling. Here, we discover and characterize a novel series of compounds that potently and selectively inhibit SMARCA4/SMARCA2. Mutational and biochemical studies demonstrate that these inhibitors act through a unique mode of inhibition, distinct from reported SMARCA4/SMARCA2 inhibitors. Across a range of cancer cell lines, SMARCA4/SMARCA2 inhibition resulted in lineage-specific changes in chromatin accessibility at binding sites for key transcription factors (TFs). In uveal melanoma (UM), BAF inhibition resulted in loss of enhancer occupancy of SOX10 and MITF, two essential TFs, leading to down-regulation of the melanocytic gene expression program. In a mouse xenograft model of UM, SMARCA4/SMARCA2 inhibition was well tolerated and resulted in dose-dependent tumor regression correlating with pharmacodynamic modulation of BAF-target gene expression. These data provide the foundation for first-in-human studies of BAF ATPase inhibition as a novel therapeutic to treat TF-dependent cancers.
Dopamine significantly modulates working memory (WM)1-8, a fundamental cognitive function that maintains information during a brief delay period9-11. The temporal precision of dopaminergic modulation in WM and related neural mechanisms remain elusive. Here we unveiled the pivotal role of dopamine timing in WM performance with head-fixed mice engaged in learning an olfactory WM task. Through electrophysiology and optogenetics, we found that dopaminergic neurons in the ventral tegmental area (VTA) could encode WM information during the delay period, and manipulating dopaminergic neuronal activity bidirectionally modulated WM performance in an inverted U-shaped manner. Optogenetic manipulation of VTA activity also induced bidirectional changes in WM-related neural activity in the medial prefrontal cortex (mPFC). Imaging with a dopamine-sensitive GPCR-activation-based sensor (GRABDA)12,13 revealed transient dopamine peaks in the mPFC specifically during the early-delay period. Optogenetic stimulation of VTA-to-mPFC dopaminergic projections during the early- and late-delay period enhanced and impaired WM performance, respectively. Manipulations outside these periods or involving the nucleus accumbens had no effect. Single-unit recordings demonstrated that optogenetic excitation of VTA-to-mPFC projections could modulate mPFC neuronal activity in a manner consistent with behavioral modulation. Thus, the timing of dopamine release is critical in modulation of neuronal activity and behavior.
Seventy percent of patients with late-stage breast cancer develop distal bone metastases; however, the mechanism by which the metabolic microenvironment affects resistance to therapy remains unknown. We investigated the metabolic bone microenvironment and identified glutathione metabolism as the top pathway in osteoclasts, which provides feedback to tumor cells to help neutralize oxidative stress and generate PARP inhibitor (PARPi) therapy resistance. GPX4, the critical enzyme responsible for glutathione oxidation, was upregulated during PARPi therapy through stress-induced ATF4-dependent transcriptional programming. The increased absorption of glutamine and the upregulation of GPX4 expression work in concert to enhance glutathione metabolism in cancer cells. Human clinical sample analysis of paired primary breast tumor and bone metastasis samples revealed that GPX4 was significantly induced in bone metastases. Combination therapy utilizing PARPi and zoledronate, which blocks osteoclast activity and thereby reduces the microenvironmental glutamine supply, generates a synergistic effect in reducing bone metastasis. Thus, our results identified an essential metabolic symbiosis between bone-resident cells and metastatic cancer cells during PARPi therapy. SIGNIFICANCEOsteoclast-derived glutamine is taken up by tumor cells to synthesize glutathione and neutralize the ROS generated by PARPi. This is the first example of "metabolic symbiosis" in therapeutic resistance of bone metastasis.
Chlamydia, an obligate intracellular bacterial pathogen, has a unique developmental cycle involving the differentiation of invading elementary bodies (EBs) to noninfectious reticulate bodies (RBs), replication of RBs, and redifferentiation of RBs into progeny EBs. Progression of this cycle is regulated by three sigma factors, which direct the RNA polymerase to their respective target gene promoters. We hypothesized that the Chlamydia-specific transcriptional regulator GrgA, previously shown to activate {sigma}66 and {sigma}28, plays an essential role in chlamydial development and growth. To test this hypothesis, we applied a novel genetic tool known as dependence on plasmid-mediated expression (DOPE) to create Chlamydia trachomatis with conditional GrgA-deficiency. We show that GrgA-deficient C. trachomatis RBs have a growth rate that is approximately half of the normal rate and fail to transition into progeny EBs. In addition, GrgA-deficient C. trachomatis fail to maintain its virulence plasmid. Results of RNA-seq analysis indicate that GrgA promotes RB growth by optimizing tRNA synthesis and expression of nutrient-acquisition genes, while it enables RB-to-EB conversion by facilitating the expression of a histone and outer membrane proteins required for EB morphogenesis. GrgA also regulates numerous other late genes required for host cell exit and subsequent EB invasion into host cells. Importantly, GrgA stimulates the expression of {sigma}54, the third and last sigma factor, and its activator AtoC, and thereby indirectly upregulating the expression of {sigma}54-dependent genes. In conclusion, our work demonstrates that GrgA is a master transcriptional regulator in Chlamydia and plays multiple essential roles in chlamydial pathogenicity. IMPORTANCEHallmarks of the developmental cycle of the obligate intracellular pathogenic bacterium Chlamydia are the primary differentiation of the infectious elementary body (EB) into the proliferative reticulate body (RB) and the secondary differentiation of RBs back into EBs. The mechanisms regulating these transitions remain unclear. In this report, we developed an effective novel strategy termed DOPE that allows for the knockdown of essential genes in Chlamydia. We demonstrate that GrgA, a Chlamydia-specific transcription factor, is essential for the secondary differentiation and optimal growth of RBs. We also show that GrgA, a chromosome-encoded regulatory protein, controls the maintenance of the chlamydial virulence plasmid. Transcriptomic analysis further indicates that GrgA functions as a critical regulator of all three sigma factors that recognize different promoter sets at developmental stages. The DOPE strategy outlined here should provide a valuable tool for future studies examining chlamydial growth, development, and pathogenicity.
SARS-CoV-2 variants continue to emerge and cocirculate in humans and wild animals. The factors driving the emergence and replacement of novel variants and recombinants remain incompletely understood. Herein, we comprehensively characterized the competitive fitness of SARS-CoV-2 wild type (WT) and three variants of concern (VOCs), Alpha, Beta and Delta, by coinfection and serial passaging assays in different susceptible cells. Deep sequencing analyses revealed cell-specific competitive fitness: the Beta variant showed enhanced replication fitness during serial passage in Caco-2 cells, whereas the WT and Alpha variant showed elevated fitness in Vero E6 cells. Interestingly, a high level of neutralizing antibody sped up competition and completely reshaped the fitness advantages of different variants. More importantly, single clone purification identified a significant proportion of homologous recombinants that emerged during the passage history, and immune pressure reduced the frequency of recombination. Interestingly, a recombination hot region located between nucleotide sites 22995 and 28866 of the viral genomes could be identified in most of the detected recombinants. Our study not only profiled the variable competitive fitness of SARS-CoV-2 under different conditions, but also provided direct experimental evidence of homologous recombination between SARS-CoV-2 viruses, as well as a model for investigating SARS-CoV-2 recombination. ImportanceSARS-CoV-2 variants or subvariants keep emerging and the epidemic strains keeps changing in humans and animals. The continued replacement of the epidemic strains was attributed to higher competitive fitness evolved by the newly appeared ones than the older ones, but which factors affect the final outcomes are still not entirely clear. In this study, we performed in vitro coinfection and serial passage with three VOCs and WT under different conditions. Our results showed that the competition outcomes of these viral strains varied in different cell lines or under different immune pressure, confirming the probable effects of these two factors for the competitive fitness of different SARS-CoV-2 viral strains. Meanwhile, strikingly, we found that coinfection and serial passage with different SARS-CoV-2 viral strains can mimic the recombination process of SARS-CoV-2 occurred in coinfection individual, indicating it is a novel model to investigate the SARS-CoV-2 recombination mechanism.
Short-term memory (STM) maintains information during a short delay period. How long-range and local connections interact to support STM encoding remains elusive. Here we tackled the problem focusing on long-range projections from medial prefrontal cortex (mPFC) to anterior agranular insular cortex (aAIC) in head-fixed mice performing an olfactory delayed-response task. Optogenetic and electrophysiological experiments revealed behavioral importance of the two regions in encoding STM information. Spike-correlogram analysis revealed strong local and cross-region functional coupling (FC) between memory neurons encoding the same information. Optogenetic suppression of mPFC-aAIC projections during the delay period reduced behavioral performance, the proportion of memory neurons and memory-specific FC within aAIC, whereas optogenetic excitation enhanced all of them. MPFC-to-aAIC projections also bidirectionally modulated efficacy of STM-information transfer, measured by contribution of FC spiking pairs to coding ability of following neurons. Thus, prefrontal projections modulate functional connectivity and coding ability of insular neurons to support short-term memory.
Hallmarks of the developmental cycle of the obligate intracellular pathogenic bacterium Chlamydia are the primary differentiation of the infectious elementary body (EB) cell type into the proliferative reticulate body (RB) and the secondary differentiation of RBs back into EBs. The detailed mechanisms regulating these transitions are unclear. In this study, we developed a novel strategy termed DOPE (dependence on plasmid-mediated expression) that allows for the knockdown of essential genes in Chlamydia. Importantly, we demonstrate that GrgA, a Chlamydia-specific transcription factor, is essential for the secondary differentiation of RBs into EBs. Our development of a conditional GrgA-deficient chlamydiae should prove valuable for future studies examining chlamydial growth, development, and pathogenicity. Furthermore, because EB formation is absolutely required for chlamydial dissemination within infected individuals, and for chlamydial transmission to new hosts, our maturation-defective chlamydiae system may serve as an attractive attenuated vaccine methodology for the prevention of chlamydial diseases.
ObjectiveChondrocyte viability (CV) is an important indicator of articular cartilage health. Two-photon excitation autofluorescence (TPAF) and second harmonic generation (SHG) microscopy provide a label-free method for imaging chondrocytes. In this study, we propose an automated assessment of CV using deep learning cell segmentation and counting based on acquired TPAF/SHG images. DesignLabel-free TPAF/SHG images of cartilage samples from rats and porcine were acquired using both commercial and home-built two-photon microscopes, respectively. TPAF/SHG images were merged to form RGB color images with red, green, and blue channels assigned to TPAF (two channels) and SHG signals, respectively. To make the training datasets for the deep learning networks, individual chondrocyte areas on the RGB color images were manually circled and live or dead chondrocytes were validated by using Calcein-AM and Ethidium homodimer-1 dye labeling. We first built a chondrocyte viability network (MCV-Net) using the Mask R-CNN architecture, which could provide individual segmented cellular areas with live or dead status. Wiener deconvolution preprocessing was added before the input of MCV-Net to improve the accuracy of the CV analysis, forming the Wiener deconvolution CV network (wMCV-Net). ResultsTraining (300 images) and test (120 images) datasets were built for rats and porcine cartilage respectively. Wiener deconvolution could improve the Peak Signal-to-Noise Ratio (PSNR) for 30-40%. We demonstrated that both MCV-Net and wMCV-Net significantly improved the accuracy of the CV measurement. ConclusionA custom desktop TPAF/SHG microscope was used in collaboration with deep learning algorithm wMCV-Net based label-free method to assess the CV and get 95% accuracy with both rats and porcine samples.
The clinical success of T-cell receptor (TCR)-based immunotherapy depends on the efficacy and specificity of TCRs. Naturally occurring TCRs have limited anti-tumor potency due to their low affinity for tumor antigens. Affinity enhancement is a promising strategy to generate highly potent TCRs. However, it is concerned that affinity-enhanced TCRs are prone to lose specificity. We isolated low affinity TCRs specific for NY-ESO-1157-165/HLA-A*02:01 from peripheral blood mononuclear cells of healthy donors. An affinity-enhanced TCR candidate with optimal affinity and specificity was generated using phage display and an extensive set of in vitro and in vivo assays. Alanine scanning mutagenesis showed that the TCR candidate retained specificity by making extensive contacts to the side chains of NY-ESO-1157-165 peptide. Adoptive transfer of T cells engineered with this candidate (termed TAEST16001) significantly inhibited tumor growth in subcutaneous, metastatic, and patient-derived xenograft (PDX) mouse tumor models. This study demonstrates that sophisticated engineering and screening techniques can be utilized to generate a clinical candidate TCR with potent anti-tumor activity without losing specificity. TAEST16001 was approved by the Center for Drug Evaluation (CDE) as the first TCR-based immunotherapy clinical trial in China (ClinicalTrials.gov Identifier: NCT03159585).
Gene transcription in bacteria is carried out by the multisubunit RNA polymerase (RNAP), which is composed of a catalytic core enzyme and a promoter-recognizing {sigma} factor. RNAP core enzyme comprises two subunits, one {beta} subunit, one {beta} s subunit, and one {omega} (omega) subunit. Across multiple bacterial taxa, the RNAP {omega} subunit plays critical roles in the assembly of RNAP core enzyme and in other cellular functions, including regulation of bacterial growth, stress response, and biofilm formation. However, for several intracellular bacterium, including the obligate intracellular bacterium Chlamydia, no RNAP {omega} subunit previously has been identified. Here, we report the identification of Chlamydia trachomatis hypothetical protein CTL0286 as the chlamydial RNAP {omega} ortholog, based on sequence, synteny, and AlphaFold and AlphaFold-Multimer three-dimensional-structure predictions. We conclude that CTL0286 functions as the previously missing chlamydial {omega} ortholog. Extensions of our analysis indicate that all obligate intracellular bacteria have {omega} orthologs. IMPORTANCEChlamydiae are common mammalian pathogens. Chlamydiae have a unique developmental cycle characterized with an infectious but nondividing elementary body (EB), which can temporarily survive outside host cells, and a noninfectious reticulate body (RB), which replicates only intracellularly. Chlamydial development inside host cells can be arrested during persistence in response to adverse environmental conditions. Transcription plays a central role in the progression of the chlamydial developmental cycle as well as entry into and recovery from persistence. The identification of the elusive {omega} subunit of chlamydial RNAP makes possible future study of its regulatory roles in gene expression during chlamydial growth, development, and stress responses. This discovery also paves the way to prepare and study the intact chlamydial RNAP and its interactions with inhibitors in vitro.
The coronavirus SARS-CoV-2 has mutated quickly and caused significant global damage. This study characterizes two mRNA vaccines ZSVG-02 (Delta) and ZSVG-02-O (Omicron BA.1), and associating heterologous prime-boost strategy following the prime of a most widely administrated inactivated whole-virus vaccine (BBIBP-CorV). The ZSVG-02-O induces neutralizing antibodies that effectively cross-react with Omicron subvariants following an order of BA.1>BA.2>BA.4/5. In naive animals, ZSVG-02 or ZSVG-02-O induce humoral responses skewed to the vaccines targeting strains, but cellular immune responses cross-react to all variants of concern (VOCs) tested. Following heterologous prime-boost regimes, animals present comparable neutralizing antibody levels and superior protection across all VOCs. Single-boost only generated ancestral and omicron dual-responsive antibodies, probably by "recall" and "reshape" the prime immunity. New Omicron-specific antibody populations, however, appeared only following the second boost with ZSVG-02-O. Overall, our results support a heterologous boost with ZSVG-02-O, providing the best protection against current VOCs in inactivated virus vaccine- primed populations.
Glioblastoma multiforme (GBM) is the most prevalent malignant tumor in the central nervous system. It has diverse phenotypes, including diffuse single-cell infiltration in which the tumor cells co-opt the normal microvasculature, and the neovascularization of an expanding tumor mass. The blood-brain-tumor barrier (BBTB) is a significant obstacle to GBM treatment and restricts entry of most FDA-approved effective oncology drugs. Herein, we report that picosecond laser excitation of vascular-targeted plasmonic gold nanoparticles (AuNPs) can non-invasively and reversibly modulate the BBTB permeability (optoBBTB). OptoBBTB enhances the delivery of paclitaxel (Taxol) in two genetically engineered glioma models (GEMM) that span the spectrum of GBM phenotypes. OptoBBTB followed by Taxol delivery effectively suppresses tumor growth and prolongs the survival time of both GEMM. Moreover, our results raise the possibility that paclitaxel, which is amongst the most widely used oncology drugs because of its proven efficacy but has been abandoned for GBM following its failure to efficacy in early phase clinical trials due to poor blood-brain barrier (BBB) penetration, could now be reconsidered in combination with strategies to increase BBB permeability. In summary, optoBBTB is a novel and effective approach to increase the delivery of therapeutics with limited BBB permeability to treat neoplastic and non-neoplastic brain diseases.
Pathogen-Host adhesion is considered the first step of infection for many pathogens such as bacteria and virus. The binding of the receptor binding domain (RBD) of SARS-CoV-2 Spike protein (S protein) onto human angiotensin-converting enzyme 2 (ACE2) is considered as the first step for the SARS-CoV-2 to adhere onto the host cells during the infection. Within three years, a number of variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been found all around the world. Here, we investigated the adhesion of S Proteins from different variants and ACE2 using atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) and single-cell force spectroscopy (SCFS). We found that the unbinding force and binding probability of the S protein from Delta variant to the ACE2 was the highest among the variants tested in our study at both single-molecule and single-cell levels. Molecular dynamics simulation showed that ACE2-RBD (Omicron) complex is destabilized by the E484A and Y505H mutations and stabilized by S477N and N501Y mutations, when compared with Delta variant. In addition, a neutralizing antibody, produced by immunization with wild type RBD of S protein, could effectively inhibit the binding of S proteins from wild type, Delta and Omicron variants onto ACE2. Our results provide new insight for the molecular mechanism of the adhesive interactions between S protein and ACE2 and suggest that effective monoclonal antibody can be prepared using wild type S protein against the different variants.
The blood-brain barrier (BBB) maintains an optimal environment for brain homeostasis but excludes most therapeutics from entering the brain. Strategies that can reversibly increase BBB permeability will be essential for treating brain diseases and is the focus of significant preclinical and translational interest. Recently, we reported that picosecond laser excitation of molecular-targeted gold nanoparticles (AuNPs) induced a graded and reversible increase in BBB permeability in vivo (OptoBBB). Here we investigate how to increase the targeting efficiency and how picosecond lase stimulation of AuNP leads to an increase in endothelial paracellular permeability. Our results suggest that targeting brain endothelial glycoproteins leads to >20-fold higher targeting efficiency compared with tight junction targeting. We report that OptoBBB is associated with a transient elevation of Ca2+ that propagates to adjacent endothelial cells after laser excitation and extends the region of BBB opening. The Ca2+ response involves both internal Ca2+ depletion and Ca2+ influx. Furthermore, we demonstrate that the involvement of actin polymerization and Ca2+-dependent phosphorylation of ERK1/2 lead to cytoskeletal activation, increasing paracellular permeability. Taken together, we provide mechanistic insight into how excitation of endothelial targeted AuNPs leads to an increase in BBB permeability. These insights will be critical for guiding the future developments of this technology for brain disease treatment.
BackgroundAccurate quantification of in vivo proton magnetic resonance spectra involves modeling with a linear combination of known metabolite basis functions. Basis sets can be generated by numerical simulation using the quantum mechanical density-matrix formalism. Accurate simulations for a basis set require correct sequence timings, and pulse shapes and durations. PurposeTo present a cloud-based spectral simulation tool MRSCloud. It allows community users of MRS to simulate a vendor- and sequence-specific basis set online in a convenient and timeefficient manner. This tool can simulate basis sets for 3 major MR scanner vendors (GE, Philips, Siemens), including conventional acquisitions and spectral editing schemes (MEGA, HERMES, HERCULES) with PRESS and semi-LASER localization. Study TypeProspective. SpecimenN/A Field Strength/SequenceSimulations of 3T basis sets for conventional and spectral-editing sequences (MEGA, HERMES, HERCULES) with PRESS and sLASER localizations. AssessmentSimulated metabolite basis functions generated by MRSCloud are compared to those generated by FID-A and MARSS, and a phantom-acquired basis-set from LCModel. Statistical TestsIntraclass correlation coefficients (ICC) were calculated to measure the agreement between individual metabolite basis functions generated using different packages. Statistical analysis was performed using R in RStudio. ResultsSimulation time for a full basis set is approximately 1 hour. ICCs between MRSCloud and FID-A were at least 0.98 and ICCs between MRSCloud and MARSS were at least 0.96. ICCs between simulated MRSCloud basis spectra and acquired LCModel basis spectra were lowest for Gln at 0.68 and highest for NAA at 0.96. Data ConclusionSubstantial reductions in runtime have been achieved by implementing the 1D projection method, coherence-order filtering, and pre-calculation of propagators. High ICC values indicated that the accelerating features are running correctly and produce comparable and accurate basis sets. The generated basis set has been successfully used with LCModel.
SARS-CoV-2 related coronaviruses (SARS-CoV-2r) from Guangdong and Guangxi pangolins have been implicated in the emergence of SARS-CoV-2 and future pandemics. We previously reported the culture of a SARS-CoV-2r GX_P2V from Guangxi pangolins. Here we report the GX_P2V isolate rapidly adapted to Vero cells by acquiring two genomic mutations: an alanine to valine substitution in the nucleoprotein and a 104-nucleotide deletion in the hypervariable region (HVR) of the 3-terminus untranslated region (3-UTR). We further report the characterization of the GX_P2V variant in in vitro and in vivo infection models. In cultured Vero and BGM cells, the GX_P2V variant produced minimal cell damage and small plaques. The GX_P2V variant infected golden hamsters and BALB/c mice but was highly attenuated. Golden hamsters infected intranasally had a short duration of productive infection. These productive infections induced neutralizing antibodies against pseudoviruses of GX_P2V and SARS-CoV-2. Collectively, our data show that the GX_P2V variant is highly attenuated in in vitro and in vivo infection models. Attenuation of the variant is likely due to the 104-nt deletion in the HVR in the 3-UTR. This study furthers our understanding of pangolin coronaviruses pathogenesis and provides novel insights for the design of live attenuated vaccines against SARS-CoV-2.