Biology subjects

Zhao, Y.

Publications and source records attributed to Zhao, Y..

At least 739 records · Page 41Linked to original sources

bioRxiv2022

Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson's Disease

Extracellular vesicles (EVs) are cell-derived nanoparticles that facilitate transport of proteins, lipids and genetic material playing important roles in intracellular communication. They have a remarkable potential as non-toxic and non-immunogenic nanocarriers for drug delivery to unreachable organs and tissues, in particular, the central nervous system (CNS). Herein, we developed a novel platform based on macrophage derived EVs to treat Parkinsons disease (PD). Specifically, we evaluated the therapeutic potential of EVs secreted by autologous macrophages that were transfected ex vivo to express glial cell line-derived neurotrophic factor (GDNF). EV-GDNF were collected from conditioned media of GDNF-transfected macrophages and characterized for GDNF content, size, charge, and expression of EV-specific proteins. The data revealed that along with the encoded neurotrophic factor, EVs released by pre-transfected macrophages carry GDNF-encoding DNA. Four months-old transgenic Parkin Q311(X)A mice were treated with EV-GDNF via intranasal administration, and the effect of this therapeutic intervention on locomotor functions was assessed over a year. Significant improvements in mobility, increase in neuronal survival, and decrease in neuroinflammation were found in PD mice treated with EV-GDNF. No offsite toxicity caused by EV-GDNF administrations was detected. Overall, EV-based approach can provide a versatile and potent therapeutic intervention for PD.

bioengineering↗

bioRxiv2022

USP11 promotes endothelial apoptosis-resistance in pulmonary hypertension by deubiquitinating HINT3

IntroductionPulmonary arterial hypertension (PAH) is a progressive, lethal, and incurable disease of the pulmonary vasculature. Evolving evidence indicates that the ubiquitin-specific proteases (USPs), play an important role in the pathogenesis of PAH by deubiquitinating key proteins involved in cell proliferation, migration, and apoptosis. Our genome-wide association study (GWAS) analysis-paired with transcriptomic profiling indicated that deubiquitinase USP11 and histidine triad nucleotide binding protein 3 (HINT3) are positively correlated and that their expression increased in lungs of PAH patients compared to control (fail donor) group, and inversely correlated with survival. However, mechanisms and function of the USP11/HNT3 axis have not been explored in PAH. Therefore, we aimed to investigate that HINT3 stabilized by USP11 activation links to endothelial apoptosis-resistance in PAH. Methods and ResultsExpression of USP11 and HINT3 was increased in the lungs of idiopathic PAH (IPAH) patients and Hypoxia/Sugen-treated mice using qRT-PCR and Western blot analyses. USP11 and HINT3 interacted physically as shown by co-immunoprecipitation (co-IP) assay in human pulmonary artery endothelial cells (HPAECs). HINT3 levels were decreased upon transfection of HA-tagged Ubi plasmid into HPAECs. Pretreatment with the potent proteasome inhibitor MG132 prolonged the half-life of HINT3 protein, indicating that HINT3 is degraded by polyubiquitination. HINT3 was stabilized and destabilized by forced overexpression or siRNA knockdown of USP11 respectively. Similarly, treatment with mitoxantrone, a USP11 antagonist, reduced HPAEC HINT3 expression. HINT3 interacted with the antiapoptotic mediator, BCL2. Overexpression of USP11 increased BCL2 content, congruent to elevated lung tissue levels seen in IPAH patients and Hypoxia/Sugen-treated mice. Conversely, knockdown of HINT3 function led to depletion of BCL2. ConclusionsThe HINT3-USP11 axis contributes to apoptosis-resistance in pulmonary artery endothelial cells, as is potentially a novel and attractive therapeutic target for ubiquitination modulators.

physiology↗

bioRxiv2022

Cachd1 is a novel Frizzled- and LRP6-interacting protein required for neurons to acquire left-right asymmetric character

Neurons on left and right sides of the nervous system frequently show asymmetric properties but how these differences arise is poorly understood. Through a forward genetic screen in zebrafish, we find that loss of function of the transmembrane protein Cachd1 results in right-sided habenula neurons adopting left-sided character. Cachd1 is expressed in habenula neuron progenitors, functions symmetrically downstream of asymmetric environmental signals that determine laterality and influences timing of the normally left-right asymmetric patterns of neurogenesis. Unbiased screening for Cachd1 partners identified the Wnt co-receptor Frizzled7 and further biochemical and structural analysis revealed Cachd1 can bind simultaneously to Fzd proteins and Lrp6, bridging between these two Wnt co-receptors. Consistent with these structural studies, lrp6 mutant zebrafish show symmetric habenulae with left-sided character and epistasis experiments with other Wnt pathway genes support an in vivo role for Cachd1 in modulating Wnt pathway activity in the brain. Together, these studies identify Cachd1 as a conserved novel Wnt-receptor interacting protein with roles in regulating neurogenesis and neuronal identity.

developmental biology↗

bioRxiv2022

Intermittent fasting mitigates cognitive deficits in Alzheimer's disease via the gut-brain axis

Intermittent fasting (IF) is suggested to mitigate cognitive impairment in Alzheimers disease (AD). We detailed the effects of IF in AD development and assessed the contribution of the gut microbiota-metabolite-brain axis in promoting the effects of fasting. A 16-week IF regimen significantly improved spatial memory and cognitive function in AD transgenic mice; this improvement was accompanied by decreased A{beta} accumulation and suppression of neuroinflammation. IF reshaped the microbiota and beneficially altered the microbial metabolites related to cognitive function. Multi-OMICs integration demonstrated a strong connection between IF induced changes in hippocampus gene expression, the composition of the gut microbes, and the serum metabolites beneficial for cognitive function. Removal of gut microbes with antibiotics abolished the neuroprotective effects of IF. These findings suggest new avenues for therapy and nutritional intervention to prevent the development of AD and other neurodegenerative diseases.

animal behavior and cognition↗

bioRxiv2022

Cardiac patch treatment alleviates ischemic cardiomyopathy correlated with reverting Piezo1/2 expression by unloading left ventricular myocardium

Pathologically elevated mechanical load promotes the adverse remodeling of left ventricle (LV) post myocardial infarction, which results in the progression from ischemic cardiomyopathy to heart failure. Cardiac patches could attenuate adverse LV remodeling by providing mechanical support to infarcted myocardium and border zone tissue. However, the mechanism of the translation from mechanical effects to favorable therapeutic outcome is still not clear. By transcriptome analysis, we found that the myocardial transcription levels of mechanosensitive ion channel proteins Piezo1 and Piezo2 significantly increased in patients with ischemic cardiomyopathy. In vitro tensile tests with local tissue information revealed a significant decrease in local strain and mechanical load in rat infarct. Cardiac function and geometry were preserved compared to non-treated control. Further, in LV myocardium of the patch-treated group, MI induced expression levels of Piezo1/2 were significantly reverted to the similar levels of the Sham group, indicating that cardiac patch beneficial effects were correlated with suppressing mechanosensitive genes, particularly Piezo1/2. These findings demonstrated the potential of cardiac patches in treating ICM patients with remodeling risks, and could provide guidance for improvement in next generation of patch devices.

pathology↗

bioRxiv2022

Space-time resolved inference-based whole-brain neurophysiological mechanism imaging: application to resting-state alpha rhythm

Neural mechanisms are complex and difficult to image. This paper presents a new space-time resolved whole-brain imaging framework, called Neurophysiological Mechanism Imaging (NMI), that identifies neurophysiological mechanisms within cerebral cortex at the macroscopic scale. By fitting neural mass models to electromagnetic source imaging data using a novel nonlinear inference method, population averaged membrane potentials and synaptic connection strengths are efficiently and accurately imaged across the whole brain at a resolution afforded by source imaging. The efficiency of the framework enables return of the augmented source imaging results overnight using high performance computing. This suggests it can be used as a practical and novel imaging tool. To demonstrate the framework, it has been applied to resting-state magnetoencephalographic source estimates. The results suggest that endogenous inputs to cingulate, occipital, and inferior frontal cortex are essential modulators of resting-state alpha power. Moreover, endogenous input and inhibitory and excitatory neural populations play varied roles in mediating alpha power in different resting-state sub-networks. The framework can be applied to arbitrary neural mass models and has broad applicability to image neural mechanisms in different brain states. HighlightsO_LIThe whole-brain imaging framework can disclose the neurophysiological substrates of complicated brain functions in a spatiotemporal manner. C_LIO_LIDeveloped a semi-analytical Kalman filter to estimate neurophysiological variables in the nonlinear neural mass model efficiently and accurately from large-scale electromagnetic time-series. C_LIO_LIThe semi-analytical Kalman filter is 7.5 times faster and 5% more accurate in estimating model parameters than the unscented Kalman filter. C_LIO_LIProvided several group-level statistical observations based on neurophysiological variables and visualised them in a whole-brain manner to show different perspectives of neurophysiological mechanisms. C_LIO_LIApplied the framework to study resting-state alpha oscillation and found novel relationships between local neurophysiological variables in specific brain regions and alpha power. C_LI

neuroscience↗

bioRxiv2022

Downregulating PTBP1 fails to convert astrocytes into hippocampal neurons and to alleviate symptoms in Alzheimer's mouse models

Conversion of astroglia into functional neurons has been considered as a promising therapeutic strategy for neurodegenerative diseases. Recent studies reported that downregulation of the RNA binding protein, PTBP1, converts astrocytes into neurons in situ in multiple mouse brain regions, consequently improving pathological phenotypes associated with Parkinsons disease, RGC loss, and aging. Here, we demonstrate that PTBP1 downregulation using antisense oligonucleotides or an astrocyte specific AAV-mediated shRNA system fails to convert hippocampal astrocytes into neurons in WT, and {beta}-amyloid (5xFAD) and tau (PS19) Alzheimers disease (AD) mouse models, and fails to reverse synaptic/cognitive deficits and AD-associated pathology. Similarly, PTBP1 downregulation cannot convert astrocytes into neurons in the striatum and substantia nigra. Together, our study suggests that cell fate conversion strategy for neurodegenerative disease therapy through manipulating one single gene, such as PTBP1, warrants more rigorous scrutiny.

neuroscience↗

bioRxiv2022

Extensive diversity of RNA viruses in ticks revealed by metagenomics in northeastern China

Recently, several emerging tick-borne viruses have been identified to be associated with human diseases in northeastern China. Here, we used metagenomics to investigate the virome diversity in Haemaphysalis japonica, H. conicinna, Dermacentor silvarum, and Ixodes persulcatus ticks in northeastern China. A total of 22 RNA viruses were identified and belonged to more eight viral families, including four each in Nairoviridae and Phenuiviridae, three each in Flaviviridae, Rhabdoviridae, and Solemoviridae, two in Chuviridae, and one each in Partitiviridae, Tombusviridae, and unclassified. Of them, eight viruses were novel species, belonging to Nairoviridae (Jian nairovirus and Yichun nairovirus), Phenuiviridae (Mudanjiang phlebovirus), Rhabdoviridae (Tahe rhabdovirus 1-3), Chuviridae (Yichun mivirus), and Tombusviridae (Yichun tombus-like virus), and five members were established human pathogens, including Alongshan virus, tick-borne encephalitis virus, Songling virus, Beiji nairovirus, and Nuomin virus. I. persulcatus ticks had significant higher viral species than those in H. japonica, H. concinna, and D. silvarum ticks. Significant differences in tick viromes were observed among Daxingan, Xiaoxingan and Changbai mountains. These findings showed an extensive diversity of RNA viruses in ticks in northeastern China, revealed potential public health threats from the emerging tick-borne viruses. Further studies are needed to explain the natural circulation and pathogenicity of these viruses.

microbiology↗

bioRxiv2022

Integrative analysis of the 3D genome and epigenome in mouse embryonic tissues

While a rich set of putative cis-regulatory sequences involved in mouse fetal development has been annotated recently based on chromatin accessibility and histone modification patterns, delineating their role in developmentally regulated gene expression continues to be challenging. To fill this gap, we mapped chromatin contacts between gene promoters and distal sequences genome-wide in seven mouse fetal tissues, and for one of them, across six developmental stages. We identified 248,620 long-range chromatin interactions centered at 14,138 protein-coding genes and characterized their tissue-to-tissue variations as well as developmental dynamics. Integrative analysis of the interactome with previous epigenome and transcriptome datasets from the same tissues revealed a strong correlation between the chromatin contacts and chromatin state at distal enhancers, as well as gene expression patterns at predicted target genes. We predicted target genes of 15,098 candidate enhancers, and used them to annotate target genes of homologous candidate enhancers in the human genome that harbor risk variants of human diseases. We present evidence that schizophrenia and other adult disease risk variants are frequently found in fetal enhancers, providing support for the hypothesis of fetal origins of adult diseases.

developmental biology↗

bioRxiv2022

Flavivirus prM interacts with MDA5 and MAVS to inhibit RLR antiviral signaling

Vector-borne flaviviruses, including tick-borne encephalitis virus (TBEV), Zika virus (ZIKA), West Nile virus (WNV), yellow fever virus (YFV), dengue virus (DENV), and Japanese encephalitis virus (JEV), pose a growing threat to public health worldwide, and have evolved complex mechanisms to overcome host antiviral innate immunity. However, the underlying mechanisms of flavivirus structural proteins to evade host immune response remain elusive. Here we show that TBEV structural proteins, including pre-membrane (prM), envelope, and capsid proteins, could inhibit type I interferon (IFN-I) production. Mechanically, TBEV prM interacted with both MDA5 and MAVS and interfered with the formation of MDA5-MAVS complex, thereby impeded the nuclear translocation and dimerization of IRF3 to inhibit RLR antiviral signaling. ZIKA and WNV prM was also demonstrated to interact with both MDA5 and MAVS, while dengue virus serotype 2 (DENV2) and YFV prM associated only with MDA5 or MAVS to suppress IFN-I production. In contrast, JEV prM could not suppress IFN-I production. Overexpression of TBEV and ZIKA prM significantly promoted the replication of Sendai virus. Our findings reveal the immune evasion mechanisms of flavivirus prM, which may contribute to understanding flavivirus pathogenicity, therapeutic intervention and vaccine development. Author SummaryAll flaviviruses must overcome the antiviral innate immunity to infect vertebrate host. The non-structural proteins of flaviviruses are mainly responsible for viral replication and host innate immune escape, and the structural proteins are involved in the virus assembly, which are potential targets for prevention and treatment of flaviviral infections. Whether flavivirus structural proteins participate in host innate immune escape remains to be determined. Here, we found that tick-borne encephalitis virus structural proteins precursor membrane (prM), capsid, and envelope proteins can antagonize type I interferon production, in which prM interacts with both MDA5 and MAVS to inhibit RLR antiviral signaling. Additionally, Zika virus and West Nile virus prMs also interact with both MDA5 and MAVS, while dengue virus serotype 2 and yellow fever virus prMs associate only with MDA5 or MAVS to suppress type I interferon production. In contrast, Japanese encephalitis virus prM cannot antagonize type I interferon production. Our findings reveal that flavivirus prM inhibits type I interferon production via interacting with MDA5 and/or MAVS in a species-dependent manner, which may contribute to understanding flavivirus pathogenicity, therapeutic intervention, and vaccine development.

microbiology↗

bioRxiv2022

Inducible lncRNA transgenic mice reveal continual role of HOTAIR in promoting breast cancer metastasis

HOTAIR is a 2.2 kb long noncoding RNA (lncRNA) whose dysregulation has been linked to oncogenesis, defects in pattern formation during early development, and irregularities during the process of epithelial-to-mesenchymal transition (EMT). However, the oncogenic transformation determined by HOTAIR in vivo and its impact on chromatin dynamics are incompletely understood. Here we generate a transgenic mouse model with doxycycline-inducible expression of human HOTAIR in the context of the MMTV-PyMT breast cancer-prone background to systematically interrogate the cellular mechanisms by which human HOTAIR lncRNA acts to promote breast cancer progression. We show that sustained high levels of HOTAIR over time increased breast metastatic capacity and invasiveness in breast cancer cells, promoting migration and subsequent metastasis to the lung. Subsequent withdrawal of HOTAIR overexpression reverted the metastatic phenotype, indicating oncogenic lncRNA addiction. Furthermore, HOTAIR overexpression altered both the cellular transcriptome and chromatin accessibility landscape of multiple metastasis-associated genes and promoted epithelial to mesenchymal transition. These alterations are abrogated within several cell cycles after HOTAIR expression is reverted to basal levels, indicating an erasable lncRNA-associated epigenetic memory. These results suggest that a continual role for HOTAIR in programming a metastatic gene regulatory program. Targeting HOTAIR lncRNA may potentially serve as a therapeutic strategy to ameliorate breast cancer progression.

cancer biology↗

bioRxiv2022

Circular mRNA encoded PROTAC (RiboPROTAC) as a new platform for the degradation of intracellular therapeutic targets

Although proteolysis targeting chimera (PROTAC) technology that hijacking the ubiquitin-proteasome system (UPS) to artificially degrade protein is an emerging promising technology for many undruggable targets, it still faces several challenges, such as the difficulty to select high specificity molecule to protein of interest (POI), and only few of the E3-ligase are suitable for PROTAC mediated protein degradation. Protein-based PROTAC, termed BioPROTAC, owns the advantage of specificity but lacks membrane permeability. Here, we develop a new targeted protein degrading platform, which we termed RiboPROTAC, by encoding BioPROTAC protein degraders with circular mRNA (cmRNA) and delivering intracellularly. In this way, intracellular protein GFP and nuclear protein PCNA were successfully degraded. For PCNA targeting, not only the level of target protein is strongly decreased, but also the cell function related is strongly interfered, such as cell proliferation, apoptosis, as well as the cell cycle. What is more, for the first time, we observed that degrading PCNA with RiboPROTAC causes immunogenic cell death (ICD), although not strong, but was verified in vivo. Importantly, our in vivo results demonstrated that cmRNA encoded PCNA degrader exhibited significant tumor suppression effect, thus we provide the first proof of concept for the application of RiboPROTAC as potential mRNA therapeutic. We consider that RiboPROTAC is a new and superior PROTAC technology for targeting the undruggable targets.

molecular biology↗

bioRxiv2022

Importance of Mobile Genetic Element Immunity in Numerically Abundant Trichodesmium Clades

The colony-forming cyanobacteria Trichodesmium spp. are considered one of the most important nitrogen-fixing genera in the warm, low nutrient, open ocean. Despite this central biogeochemical role, many questions about their evolution, physiology, and trophic interactions remain unanswered. To address these questions, we describe the genetic potential of the genus via significantly improved genomic assemblies of strains Trichodesmium thiebautii H94, Trichodesmium erythraeum 2175, and 17 new Trichodesmium metagenome-assembled genomes (MAGs, >50% complete) from hand-picked, Trichodesmium colonies spanning the Atlantic Ocean. Phylogenomics identified [~]four N2 fixing clades of Trichodesmium across the transect, with T. thiebautii dominating the colony-specific reads. Pangenomic analyses showed that all T. thiebautii MAGs are enriched in defense mechanisms and encode a vertically inherited Type III-B Clustered Regularly Interspaced Short Palindromic Repeats and associated protein-based immunity system (CRISPR-Cas hereafter). Surprisingly, this CRISPR-Cas system was absent in all T. erythraeum genomes and MAGs, vertically inherited by T. thiebautii, and correlated with increased signatures of horizontal gene transfer. Multiple lines of evidence indicate that the CRISPR-Cas system is functional in situ: 1. Trichodesmium CRISPR spacer sequences with 100% identical hits to field-assembled, putative phage genome fragments were identified, 2. High Trichodesmium spacer sequence variability indicating rapid adaptation, and 3. metaproteomic and transcriptomic expression analyses detecting the CRISPR-Cas system components in Trichodesmium colonies from the Atlantic and Pacific Oceans. These data suggest that phage or mobile genetic element immunity in T. thiebautii could contribute to their success, gene diversity, and numerical dominance over T. erythraeum in the oceans, thus warranting further Trichodesmium virome investigations. Significance statementOur work identifies CRISPR-Cas immunity as a phylogenetically distinct, environmentally expressed factor in the speciation of closely related N2-fixing Trichodesmium clades. These findings suggest that differential phage predation and resistance could be a previously overlooked selective pressure in the genus, potentially leading to the current numerical dominance of T. thiebautii over T. erythraeum in the oceans. Furthermore, while the currently CO2-limited T. erythraeum is expected to be a winner of anthropogenic climate change, their predicted higher phage sensitivity than T. thiebautii could challenge this outcome.

microbiology↗

bioRxiv2022

RNA Pol II pausing facilitates phased pluripotency transitions by buffering transcription

Promoter-proximal RNA Pol II pausing is a critical step in transcriptional control. Pol II pausing has been studied predominantly in tissue culture systems. While Pol II pausing has been shown to be required for mammalian development, the phenotypic and mechanistic details of this requirement are unknown. Here, we find that loss of RNA Pol II pausing stalls pluripotent state transitions in the epiblast of the early mouse embryo. Using Nelfb-/- mice and a novel NELFB- degron mouse embryonic stem cells, we show that mouse ES cells (mESCs) representing the naive state of pluripotency successfully initiate a transition program, but fail to balance levels of induced and repressed genes and enhancers in the absence of NELF. Consistently, we find an increase in chromatin-associated NELF during pluripotency transitions. Overall, our work reveals the molecular and phenotypic roles of Pol II pausing in pluripotency and introduces Pol II pausing as a modulator of cell state transitions.

molecular biology↗

bioRxiv2022

Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions

The Sc2.0 project is building a eukaryotic synthetic genome from scratch, incorporating thousands of designer features. A major milestone has been achieved with the assembly of all individual Sc2.0 chromosomes. Here, we describe the consolidation of multiple synthetic chromosomes using endoreduplication intercross to generate a strain with 6.5 synthetic chromosomes. Genome-wide chromosome conformation capture and long-read direct RNA sequencing were performed on this strain to evaluate the effects of designer modifications, such as loxPsym site insertion, tRNA relocation, and intron deletion, on 3D chromosome organization and transcript isoform profiles. To precisely map "bugs", we developed a method, CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS", exploiting directed mitotic recombination in heterozygous diploids. Using this method, we first fine-mapped a synII defect resulting from two loxPsym sites in the 3 UTR of SHM1. This approach was also used to map a combinatorial bug associated with synIII and synX, revealing a highly unexpected genetic interaction that links transcriptional regulation, inositol metabolism and tRNASerCGA abundance. "Starvation" for tRNASerCGA leads to insufficient levels of the key positive inositol biosynthesis regulator, Swi3, which contains tandem UCG codons. Finally, to expedite consolidation, we employed a new method, chromosome swapping, to incorporate the largest chromosome (synIV), thereby consolidating more than half of the Sc2.0 genome in a single strain.

genomics↗

bioRxiv2022

Manipulating the 3D organization of the largest synthetic yeast chromosome

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field, especially when the synthetic genomes are extensively modified with thousands of designer features. Here we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp Saccharomyces cerevisiae chromosome resulting from extensive genome streamlining and modification. During the construction of synIV, we developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction and facilitated chromosome debugging. In addition to the drastic sequence changes made to synIV by rewriting it, we further manipulated the three-dimensional structure of synIV in the yeast nucleus to explore spatial gene regulation within the nuclear space. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of the largest synthetic yeast chromosome shed light on higher-order architectural design of the synthetic genomes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/487066v2_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@18bf586org.highwire.dtl.DTLVardef@1a471d5org.highwire.dtl.DTLVardef@12d7690org.highwire.dtl.DTLVardef@1c32510_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDe novo synthesis of the largest eukaryotic chromosome, synIV C_LIO_LISynIV shows similar 3D structure to wild-type IV, despite thousands of changes made to it C_LIO_LI"Inside-out" repositioning of synIV in nucleus shows minor transcriptional changes C_LIO_LIMultipoint tethering synIV to inner nuclear membrane represses transcription of whole chromosome C_LI

synthetic biology↗

bioRxiv2022

Platelet response to influenza vaccination reflects effects of aging

Platelets are uniquely positioned as mediators of not only hemostasis but also innate immunity, but how age and alterations in functional status such as frailty influence platelet function during an immune response remains unclear. We assessed the platelet transcriptome in younger (age 21-35) and older (age [≥] 65) adults (including frail and non-frail individuals) following influenza vaccination. Prior to vaccination, we identified an age- and frailty-associated increase in expression of platelet activation and mitochondrial RNAs and decrease in RNAs encoding proteins mediating translation. Using tensor decomposition analysis, we also elucidated dynamic post-vaccination platelet activation and translation signatures associated with age and frailty. At the protein level, enhanced platelet activation was found in non-frail older adults, compared to young individuals both prior to and post-vaccine; but frail adults showed decreased platelet activation compared to non-frail that could reflect the influence of decreased translation RNA expression. Our results reveal an age-dependent alteration in platelet function prior to and post-vaccination that may contribute to age-associated chronic inflammation.

immunology↗

bioRxiv2022

Probing Notch1-Dll4 Signaling in Regulating Osteogenic Differentiation of Human Mesenchymal Stem Cells using Single Cell Nanobiosensor

Human mesenchymal stem cells (hMSCs) have great potential in cell-based therapies for tissue engineering and regenerative medicine due to their self-renewal and multipotent properties. Recent studies indicate that Notch1-Dll4 signaling is an important pathway in regulating osteogenic differentiation of hMSCs. However, the fundamental mechanisms that govern osteogenic differentiation are poorly understood due to a lack of effective tools to detect gene expression at single cell level. Here, we established a double-stranded locked nucleic acid (LNA)/DNA (LNA/DNA) nanobiosensor for gene expression analysis in single hMSC in both 2D and 3D microenvironments. We first characterized this LNA/DNA nanobiosensor and demonstrated the Dll4 mRNA expression dynamics in hMSCs during osteogenic differentiation. By incorporating this nanobiosensor with live hMSCs imaging during osteogenic induction, we performed dynamic tracking of hMSCs differentiation and Dll4 mRNA gene expression profiles of individual hMSC during osteogenic induction. Our results showed the dynamic expression profile of Dll4 during osteogenesis, indicating the heterogeneity of hMSCs during this dynamic process. We further investigated the role of Notch1-Dll4 signaling in regulating hMSCs during osteogenic differentiation. Pharmacological perturbation is applied to disrupt Notch1-Dll4 signaling to investigate the molecular mechanisms that govern osteogenic differentiation. In addition, the effects of Notch1-Dll4 signaling on hMSCs spheroids differentiation were also investigated. Our results provide convincing evidence supporting that Notch1-Dll4 signaling is involved in regulating hMSCs osteogenic differentiation. Specifically, Notch1-Dll4 signaling is active during osteogenic differentiation. Our results also showed that Dll4 is a molecular signature of differentiated hMSCs during osteogenic induction. Notch inhibition mediated osteogenic differentiation with reduced Alkaline Phosphatase (ALP) activity. Lastly, we elucidated the role of Notch1-Dll4 signaling during osteogenic differentiation in a 3D spheroid model. Our results showed that Notch1-Dll4 signaling is required and activated during osteogenic differentiation in hMSCs spheroids. Inhibition of Notch1-Dll4 signaling mediated osteogenic differentiation and enhanced hMSCs proliferation, with increased spheroid sizes. Taken together, the capability of LNA/DNA nanobiosensor to probe gene expression dynamics during osteogenesis, combined with the engineered 2D/3D microenvironment, enables us to study in detail the role of Notch1-Dll4 signaling in regulating osteogenesis in 2D and 3D microenvironment. These findings will provide new insights to improve cell-based therapies and organ repair techniques.

bioengineering↗