Biology subjects

Zhao, R.

Publications and source records attributed to Zhao, R..

At least 127 records · Page 7Linked to original sources

bioRxiv2022

A Deep Redox Proteome Profiling Workflow and Its Application to Skeletal Muscle of a Duchene Muscular Dystrophy Model

Perturbation to the redox state accompanies many diseases and its effects are viewed through oxidation of biomolecules, including proteins, lipids, and nucleic acids. The thiol groups of protein cysteine residues undergo an array of redox post-translational modifications (PTMs) that are important for regulation of protein and pathway function. To better understand what proteins are redox regulated following a perturbation, it is important to be able to comprehensively profile protein thiol oxidation at the proteome level. Herein, we report a deep redox proteome profiling workflow and demonstrate its application in measuring the changes in thiol oxidation along with global protein expression in skeletal muscle from mdx mice, a model of Duchenne Muscular Dystrophy (DMD). In depth coverage of the thiol proteome was achieved with >18,000 Cys sites from 5608 proteins in muscle being quantified. Compared to the control group, mdx mice exhibit markedly increased thiol oxidation, where ~2% shift in the median oxidation occupancy was observed. Pathway analysis for the redox data revealed that coagulation system and immune-related pathways were among the most susceptible to increased thiol oxidation in mdx mice, whereas protein abundance changes were more enriched in pathways associated with bioenergetics. This study illustrates the importance of deep redox profiling in gaining a greater insight into oxidative stress regulation and pathways/processes being perturbed in an oxidizing environment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/504013v1_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@1917f1org.highwire.dtl.DTLVardef@1730cd0org.highwire.dtl.DTLVardef@4e4820org.highwire.dtl.DTLVardef@1615f4c_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDeep redox profiling workflow results in stoichiometric quantification of thiol oxidation for > 18,000 Cys sites in muscle C_LIO_LIThiol redox changes were much more pronounced than protein abundance changes for the overlapping set of proteins C_LIO_LIRedox changes are most significant in coagulation and immune response pathways while abundance changes on bioenergetics pathways C_LI

biochemistry↗

bioRxiv2022

Dynamic changes in the niche and transcription trigger early murine and human pluripotent stem cell-derived liver organogenesis

The shift from collective migration to differentiation is a crucial process in epithelial biology but recreating this intricate transition has thus far proved elusive. We provide experimental, mechanistic, in vivo, and bioinformatic data supporting an undoubtable link between human pluripotent stem cell (hPSC)- derived collectively migrating hepatoblasts (MHB), and transcriptionally mature, functional hPSC- hepatocytes (HEPs), which incorporates two unrecognized steps. The protocol induces FOXA-dependent induction of HBs, leading to TBX3-positive, YAP-TEAD active MHBs which provide a transcriptional match with murine liver E9.5 MHBs. Simple cultivation changes trigger MHBs to rapidly form functional day 18 HEPs, predicted by a deep-learning designed gene circuit, resulting in a [~]236% fold- increase in maturation (PACNet), on par with the highest score, but with enhanced global transcriptional shaping. Overall, incorporating the MHB to HEP transition establishes a new, unrecognized, and highly efficient mechanism for differentiation that can be cumulatively integrated with existing methods to overcome barriers to maturation.

bioengineering↗

bioRxiv2022

Spatiotemporal atlas of the fetal brain depicts cortical developmental gradient in Chinese population

The fetal brains experience rapid and complex development in utero during the second and third trimesters. MRI atlas of the fetal brain in this period enables us to quantify the normal fetal brain development in the spatiotemporal domain. In this study, we constructed a high quality spatiotemporal atlas between 23-38 weeks gestational age (GA) from 90 healthy Chinese fetuses using a pairwise and groupwise registration-based pipeline. We quantified the fetal cortical morphology and characterized the developmental trajectories of each morphological index. The cortical thickness exhibited a biphasic growth pattern; the developmental trend of curvature fitted well into the Gompertz model; sulcal depth increased linearly while surface area expanded exponentially. The cortical thickness and curvature trajectories consistently pointed to a characteristic time-point during development around 31 weeks of GA. The characteristic GA and growth rate obtained from individual cortical regions suggested a central-to-peripheral developmental gradient, with the earliest development in the parietal lobe, and we also observed within-lobe regional orders, which may be linked to biophysical events such as dendritic arborization and thalamocortical fibers ingrowth. The proposed atlas was also compared with an existing fetal atlas from a Caucasian/mixed population. Finally, we examined the structural asymmetry of the fetal brains and found extensive asymmetry that dynamically changed with development. The current study depicted a comprehensive profile of fetal cortical development and the established atlas could be used as a normative reference for neurodevelopmental and diagnostic purposes, especially in the Chinese population.

bioengineering↗

bioRxiv2022

The conservation of human functional variants and their effects across mammals

Despite the clear potential of livestock models of human functional variants to provide important insights into the biological mechanisms driving human diseases and traits, their use to date has been limited. Generating such models via genome editing is costly and time consuming, and it is unclear which variants will have conserved effects across species. In this study we address these issues by studying naturally occurring livestock models of human functional variants. We show that orthologues of over 1.6 million human variants are already segregating in domesticated mammalian species, including several hundred previously directly linked to human traits and diseases. Models of variants linked to particular phenotypes, including metabolomic disorders and height, have been preferentially maintained across species, meaning studying the genetic basis of these phenotypes is particularly tractable in livestock. Using machine learning we demonstrate it is possible to identify human variants that are more likely to have an existing livestock orthologue, and, importantly, we show that the effects of functional variants are often conserved in livestock, acting on orthologous genes with the same direction of effect. Consequently, this work demonstrates the substantial potential of naturally occurring livestock carriers of orthologues of human functional variants to disentangle their functional impacts.

genetics↗

bioRxiv2022

A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

Effective phosphoproteome of nanoscale sample analysis remains a daunting task, primarily due to significant sample loss associated with non-specific surface adsorption during enrichment of low stoichiometric phosphopeptide. We developed a novel tandem tip phosphoproteomics sample preparation method that is capable of sample cleanup and enrichment without additional sample transfer, and its integration with our recently developed SOP (Surfactant-assisted One-Pot sample preparation) and iBASIL (improved Boosting to Amplify Signal with Isobaric Labeling) approaches provides a streamlined workflow enabling sensitive, high-throughput nanoscale phosphoproteome measurements. This approach significantly reduces both sample loss and processing time, allowing the identification of >3,000 (>9,500) phosphopeptides from 1 (10) {micro}g of cell lysate using the label-free method without a spectral library. It also enabled precise quantification of [~]600 phosphopeptides from 100 cells sorted by FACS (single-cell level input for the enriched phosphopeptides) and [~]700 phosphopeptides from human spleen tissue voxels with a spatial resolution of 200 {micro}m (equivalent to [~]100 cells) in a high-throughput manner. The new workflow opens avenues for phosphoproteome profiling of mass-limited samples at the low nanogram level.

biochemistry↗

bioRxiv2021

CRISPR Cas13-based tools to track and manipulate endogenous telomeric repeat-containing RNAs in living cells

TERRA, TElomeric Repeat-containing RNA, is a long non-coding RNA transcribed from telomeres. Emerging evidence indicates that TERRA regulates telomere maintenance and chromosome end protection in normal and cancerous cells. However, the mechanism of how TERRA contributes to telomere functions is still unclear, partially owing to the shortage of approaches to track and manipulate endogenous TERRA molecules in live cells. Here, we developed a method to visualize TERRA in live cells via a combination of CRISPR Cas13 RNA labeling and Suntag technology. Single-particle tracking reveals that TERRA foci undergo anomalous diffusion in a manner that depends on the timescale and telomeric localization. Furthermore, we used a chemically-induced protein dimerization system to manipulate TERRA subcellular localization in live cells. Overall, our approaches to monitor and control TERRA locations in live cells provide powerful tools to better understand its roles in telomere maintenance and genomic integrity.

cell biology↗

bioRxiv2021

Verbena officinalis L. regulates killing efficiency of natural killer cells

Natural killer (NK) cells play a key role in eliminating pathogen-infected cells. Verbena officinalis (V. officinalis) has been used as a medical plant in traditional and modern medicine, exhibiting anti-tumor and anti-inflammation activities, but its roles in immune responses still remains largely elusive. In this work, investigated the regulation of inflammation and NK functions by V. officinalis extract (VO-extract). In an influenza virus infection mouse model, oral administration of VO-extract alleviated lung injury, promoted maturation and activation of NK cells residing in the lung, and decreased the levels of inflammatory cytokines (IL-6, TNF- and IL-1{beta}) in the serum. We further analyzed the impact of five bioactive components of VO-extract on NK killing functions. Among them, Verbenalin enhanced NK killing efficiency significantly as determined by real-time killing assays based on plate-reader or high-throughput live-cell imaging in 3D using primary human NK cells. Further investigation showed that treatment of Verbenalin accelerated killing processes by reducing the contact time of NK cells with their target cells without affecting NK proliferation, expression of cytotoxic proteins, or lytic granule degranulation. Together, our findings reveal that low doses of V. officinalis can achieve a satisfactory anti-inflammation effect against viral infection in vivo, and V. officinalis regulates activation, maturation and killing functions of NK cells. NK killing efficiency is enhanced by Verbenalin from V. officinalis, suggesting a promising potential of verbenalin to fight viral infection.

immunology↗

bioRxiv2021

A noval lightsheet scattering microscopy for long-term visualization of cell-ECM interaction

Visualizing interactions between cells and the extracellular matrix (ECM) mesh is important to understand cell behavior and regulatory mechanisms by the extracellular environment. However, long term visualization of three-dimensional (3D) matrix structures remains challenging mainly due to photobleaching or blind spots perpendicular to the imaging plane. Here, we combine label-free light-sheet scattering microcopy (LSSM) and fluorescence microscopy to solve these problems. We verified that LSSM can reliably visualize structures of collagen matrices from different origin including bovine, human and rat tail. The quality and intensity of collagen structure images acquired by LSSM did not decline with time. LSSM offers abundant wavelength choice to visualize matrix structures, maximizing combination possibilities with fluorescently-labelled cells, allowing visualizing of long-term ECM-cell interactions in 3D. Interestingly, we observed ultrathin thread-like structures between cells and matrix using LSSM, which were not observed by normal fluorescence microscopy. Transient local alignment of matrix by cell-applied forces can be observed. In summary, LSSM provides a powerful and robust approach to investigate the complex interplay between cells and ECM.

biophysics↗

bioRxiv2021

Acute silencing uncovers multiple forms of activity-dependent neuronal survival in the mature entorhinal cortex

Neurodegenerative diseases are characterized by selective vulnerability of distinct cell populations; however, the cause for this specificity remains elusive. Many circuits that degenerate in disease are shaped by neural activity during development, raising the possibility that mechanisms governing early cell loss may be misused when activity is compromised in the mature brain. Here we show that electrical activity and synaptic transmission are both required for neuronal survival in the adult entorhinal cortex, but these silencing methods trigger distinct means of degeneration in the same neuronal population. Competition between active and inactive cells drives axonal disintegration caused by synaptic inhibition, but not axon retraction due to electrical suppression. These findings suggest that activity-dependence may persist in some areas of the adult brain long after developmental critical periods have closed. We speculate that lifelong plasticity required to support memory may render entorhinal neurons vulnerable to prolonged activity changes in disease.

neuroscience↗

bioRxiv2021

Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms

Topoisomerase II (Top2) unlinks chromosomes during vertebrate DNA replication. Top2 poisons are widely-used chemotherapeutics that stabilize Top2 complexes on DNA, leading to cytotoxic DNA breaks. However, it is unclear how these drugs affect DNA replication, which is a major target of Top2 poisons. Using Xenopus egg extracts, we show that the Top2 poisons etoposide and doxorubicin both inhibit DNA replication through different mechanisms. Etoposide induces Top2-dependent DNA breaks and induces Top2-dependent fork stalling by trapping Top2 behind replication forks. In contrast, doxorubicin does not lead to appreciable break formation and instead intercalates into parental DNA to inhibit replication fork progression. In human cells, etoposide stalls replication forks in a Top2-dependent manner, while doxorubicin stalls forks independently of Top2. However, both drugs exhibit Top2-dependent cytotoxicity. Thus, despite shared genetic requirements for cytotoxicity etoposide and doxorubicin inhibit DNA replication through distinct mechanisms.

biochemistry↗

bioRxiv2021

Dynamic regulation and requirement for ribosomal RNA transcription duringmammalian development

Ribosomal RNA (rRNA) transcription by RNA Polymerase I (Pol I) is a critical rate-limiting step in ribosome biogenesis, which is essential for cell survival. Despite its global function, disruptions in ribosome biogenesis cause tissue-specific birth defects called ribosomopathies, which frequently affect craniofacial development. Here, we describe a cellular and molecular mechanism underlying the susceptibility of craniofacial development to disruptions in Pol I transcription. We show that Pol I subunits are highly expressed in the neuroepithelium and neural crest cells (NCC), which generate most of the craniofacial skeleton. High expression of Pol I subunits sustains elevated rRNA transcription in NCC progenitors, which supports their high tissue-specific levels of protein translation, but also makes NCC particularly sensitive to rRNA synthesis defects. Consistent with this model, NCC-specific deletion of Pol I subunits Polr1a, Polr1c, and associated factor Tcof1 in mice cell-autonomously diminishes rRNA synthesis, which causes an imbalance between rRNA and ribosomal proteins. This leads to increased binding of ribosomal proteins Rpl5 and Rpl11 to Mdm2 and concomitantly diminished binding between Mdm2 and p53. Consequently, p53 protein accumulates, resulting in NCC apoptosis and craniofacial anomalies. Furthermore, compound mutations in Pol I subunits and associated factors specifically exacerbates the craniofacial anomalies characteristic of the ribosomopathies Treacher Collins Syndrome and Acrofacial Dysostosis-Cincinnati Type. Altogether, our novel results demonstrate a dynamic spatiotemporal requirement for rRNA transcription during mammalian cranial NCC development and corresponding tissue-specific threshold sensitivities to disruptions in rRNA transcription in the pathogenesis of congenital craniofacial disorders. Significance statementRNA Polymerase I (Pol I) mediated rRNA transcription is required for protein synthesis in all tissues for normal growth and survival as well as for proper embryonic development. Interestingly, disruptions in Pol I mediated transcription perturb ribosome biogenesis and lead to tissue-specific birth defects, which commonly affect the head and face. Our novel results show that during mouse development, Pol I mediated rRNA transcription and protein translation is tissue-specifically elevated in neural crest cells, which give rise to bone, cartilage, and ganglia of the head and face. Using new mouse models, we further show that neural crest cells are highly sensitive to disruptions in Pol I and that when rRNA synthesis is genetically downregulated, it specifically results in craniofacial anomalies.

developmental biology↗

bioRxiv2021

Imaging erythrocyte sedimentation in whole blood

The erythrocyte sedimentation rate (ESR) is one of the oldest medical diagnostic tools. However, currently there is some debate on the structure formed by the cells during the sedimentation process. While the conventional view is that erythrocytes sediment as separate aggregates, others have suggested that they form a percolating gel, similar to other colloidal suspensions. A direct probing of the structures formed by erythrocytes in blood at stasis is then required to settle these discrepancies. Here, we report observations performed with three different optical imaging techniques: direct light transmission through thin samples, two-photon microscopy and light-sheet microscopy. All techniques revealed a dynamic structure of a channeling gel but with differences in the resolved details. A quantitative analysis of the erythrocyte related processes and interactions during the sedimentation need a further refinement of the experimental set-ups.

biophysics↗

bioRxiv2021

Metabolic reprogramming of cytotoxic T lymphocytes by high glucose enhances pancreatic beta cell apoptosis via TRAIL

Cytotoxic T lymphocytes (CTLs) are involved in development of diabetes. However, the impact of excessive glucose on CTL-mediated antigen-independent killing remains elusive. Here, we report that TNF-related apoptosis inducing ligand (TRAIL) is substantially up- regulated in CTLs in environments with high glucose (HG) both in vitro and in vivo. The PI3K- Akt-NF{kappa}B axis and non-mitochondrial reactive oxygen species are essential in HG-induced TRAIL upregulation in CTLs. TRAILhigh CTLs induce apoptosis of pancreatic beta cell line 1.4E7. Metformin and Vitamin D synergistically reduce HG-enhanced expression of TRAIL in CTLs and coherently protect 1.4E7 cells from TRAIL-mediated apoptosis. Notably, in patients with diabetes, correlation between Vitamin D concentrations in plasma and glucose levels is linked to HG-enhanced TRAIL expression on CTLs. Microarray data reveal that OXCT2, an important enzyme in ketone body catabolism, is a promising target in response to vitamin D. Our work not only reveals a novel mechanism of CTL involvement in progression of diabetes, but also establishes CTLs as a target for combined metformin and vitamin D therapy to protect pancreatic beta cells of diabetic patients.

immunology↗

bioRxiv2021

After traumatic brain injury oligodendrocytes regain a plastic phenotype and can become astrocytes

After acute brain injuries various response cascades are evoked that direct the formation of the glial scar. Here, we report that acute lesions associated with a disruption of the blood-brain barrier trigger a re-programming within the oligodendrocyte lineage. In PLP-DsRed1/GFAP-EGFP and PLP-EGFPmem/GFAP-mRFP1 transgenic mice with cortical injuries, we transiently found PLP transgene-labelled cells with activated GFAP promoter activity adjacent to the lesion site. We termed them AO cells, based on their concomitant activity of astro- and oligodendroglial genes. By fate mapping using PLP- and GFAP-split Cre complementation and NG2-CreERT2 mice we observed that major portions of AO cells surprisingly differentiated into astrocytes. Using repeated long-term in vivo two-photon laser-scanning microscopy (2P-LSM) we followed oligodendrocytes after injury. We observed their conversion into astrocytes via the AO cell stage with silencing of the PLP promoter and simultaneous activation of the GFAP promoter. In addition, we provide evidence that this oligodendrocyte-to-astrocyte conversion depends on local cues. At the lesion site higher expression levels of various glial differentiation factors were detected. And indeed, local injection of IL-6 promoted the formation of AO cells. In summary, our findings highlight the plastic potential of oligodendrocytes in acute brain trauma. An altered environmental milieu affects gene expression programs of mature oligodendrocytes and induces a plastic differentiation stage with astrogliogenic potential via transitional AO cells.

neuroscience↗

bioRxiv2021

P53 is a direct regulator of the immune co-stimulatory molecule CD80

Increasing evidence indicates oncogenes and tumor suppressors not only influence cell fitness but can also control the immunophenotype of cells. Here, we examined how 34 commonly mutated genes in colorectal cancer (CRC) may influence the expression of 8 key immunomodulatory proteins. To do this, we employed a functional genomics approach utilizing Pro-Code/CRISPR libraries for high-dimensional analysis. We introduced a library of 102 Pro-Code/gRNA combinations, targeting each of the 34 genes, in CT26 cells, a CRC cell model, and measured the expression of each of the immunomodulatory proteins by CyTOF mass cytometry. Notably, cells carrying a Pro-Code/CRISPR targeting the Trp53 lost expression of the immune co-stimulatory molecule CD80. Validation confirmed that Trp53 knockout resulted in the loss of CD80 and that activation of P53, through DNA damage or stabilization, resulted in CD80 upregulation. P53 ChIP-seq identified the CD80 promoter as a direct target of P53. CD80 regulation by P53 was identified in other cells, including normal epithelial cells and macrophages. Functionally, CD80 reduction caused by P53 loss led to a reduced capacity for CRC to prime antigen-specific T cells. These studies establish CD80, a canonical co-stimulatory molecule, as a direct target of the tumor suppressor and DNA damage response gene, P53.

immunology↗

bioRxiv2021

Impaired bidirectional communication between interneurons and oligodendrocyte precursor cells affects cognitive behavior

Cortical neural circuits are complex but very precise networks of balanced excitation and inhibition (E/I). Yet, the molecular and cellular mechanisms that form the E/I balance are just beginning to emerge. Here, using conditional GABAB receptor-deficient mice we identified a GABA/TNF-related cytokine (TNFSF12)-mediated bidirectional communication pathway between Parvalbumin-positive (PV+) fast spiking interneurons and oligodendrocyte precursor cells (OPCs) that determines the density and function of interneurons in the developing medial prefrontal cortex (mPFC). Interruption of the GABAergic signaling to OPCs resulted in reduced myelination and hypoactivity of interneurons, strong changes of cortical network activities and impaired cognitive behavior. In conclusion, glial transmitter receptors are pivotal elements in finetuning distinct brain functions.

neuroscience↗

bioRxiv2021

Collagen density defines 3D migration of CTLs and their consequent cytotoxicity against tumor cells

Solid tumors are often characterized by condensed extracellular matrix (ECM). The impact of dense ECM on cytotoxic T lymphocytes (CTL) function is not fully understood. Here, we report that CTL-mediated cytotoxicity is substantially impaired in dense collagen matrices. Although the intrinsic killing machinery including expression of cytotoxic proteins and degranulation was intact, CTL motility was substantially compromised in dense collagen. We found that for 3D CTL migration, persistence and velocity was regulated by collagen stiffness and the porosity, respectively. Interestingly, 3D CTL velocity is strongly correlated with their nuclear deformability/flexibility during migration, which is regulated by the microtubule network. Moreover, CTL migration was completely abolished by inhibition of actin polymerization and or myosin IIA. Remarkably, disruption of the microtubule-networks significantly improves the impaired migration, search efficiency, and cytotoxicity of CTLs in dense collagen. Our work suggests the microtubule network as a promising target to rescue impaired CTL killing capacity in solid tumor related scenarios.

immunology↗

bioRxiv2021

Profiling of cytokines, chemokines and growth factors in saliva and gingival crevicular fluid

In saliva and gingival crevicular fluid (GCF) soluble factors such as cytokines, chemokines and growth factors have shown a great potential serving as biomarkers for early detection and/or diagnosis of oral and systemic diseases. However, GCF and saliva, which one is a better source is still under debate. This study aimed to gain an overview of cytokines, chemokines and growth factors in saliva and GCF to pave the way for selecting suitable oral fluids for oral and systemic diseases. Multiplex cytokine assay was conducted to determine concentrations of cytokines, chemokines and growth factors in saliva and GCF samples from healthy subjects. The protocol for sample collection was carefully optimized. Stabilization, repeatability, and donor variation of the profiles were analyzed. We found that for different donors, cytokine and chemokine profiles showed unique patterns in saliva but similar patterns in GCF. In terms of growth factors, the profiles were individualized in saliva and GCF. All profiles stayed stable for the same healthy individual. In saliva, profiles of cytokines, chemokines and growth factors are individualized for different donors. In GCF, profiles of cytokines and chemokines are similar. Other factors, such as growth factors and T helper-related cytokines, are highly variable in donors. Profiles of soluble factors are not correlated in saliva and GCF. The comprehensive cytokine profiles in saliva and GCF reported in this work would serve as a good base for choosing promising cytokines for developing biomarkers in oral fluids.

immunology↗