Engineering Material Properties of Transcription Factor Condensates to Control Gene Expression in Mammalian Cells and Mice.

Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. However, the impact of the material properties of biomolecular condensates on important processes, such as the control of gene expression, remains largely elusive. Here, the material properties of optogenetically induced transcription factor condensates are systematically tuned, and probed for their impact on the activation of target promoters. It is demonstrated that transcription factors in rather liquid condensates correlate with increased gene expression levels, whereas stiffer transcription factor condensates correlate with the opposite effect, reduced activation of gene expression. The broad nature of these findings is demonstrated in mammalian cells and mice, as well as by using different synthetic and natural transcription factors. These effects are observed for both transgenic and cell-endogenous promoters. The findings provide a novel materials-based layer in the control of gene expression, which opens novel opportunities in optogenetic engineering and synthetic biology.

Characterization of the proteome of stable and unstable carotid atherosclerotic plaques using data-independent acquisition mass spectrometry.

BACKGROUND: Currently, noninvasive imaging techniques and circulating biomarkers are still insufficient to accurately assess carotid plaque stability, and an in-depth understanding of the molecular mechanisms that contribute to plaque instability is still lacking. METHODS: We established a clinical study cohort containing 182 patients with carotid artery stenosis. After screening, 39 stable and 49 unstable plaques were included in the discovery group, and quantitative proteomics analysis based on data independent acquisition was performed for these plaque samples. Additionally, 35 plaques were included in the validation group to validate the proteomics results by immunohistochemistry analysis. RESULTS: A total of 397 differentially expressed proteins were identified in stable and unstable plaques. These proteins are primarily involved in ferroptosis and lipid metabolism-related functions and pathways. Plaque validation results showed that ferroptosis- and lipid metabolism-related proteins had different expression trends in stable plaques versus unstable fibrous cap regions and lipid core regions. Ferroptosis- and lipid metabolism-related mechanisms in plaque stability were discussed. CONCLUSIONS: Our results may provide a valuable strategy for revealing the mechanisms affecting plaque stability and will facilitate the discovery of specific biomarkers to broaden the therapeutic scope.

Epidermal ET-1 signal induces activation of resting hair follicles by upregulating the PI3K/AKT pathway in the dermis.

The prevalence of alopecia has increased recently. Hair loss is often accompanied by the resting phase of hair follicles (HFs). Dermal papilla (DP) plays a crucial role in HF development, growth, and regeneration. Activating DP can revive resting HFs. Augmenting WNT/ß-catenin signaling stimulates HF growth. However, the factors responsible for activating resting HFs effectively are unclear. In this study, we investigated epidermal cytokines that can activate resting HFs effectively. We overexpressed ß-catenin in both in vivo and in vitro models to observe its effects on resting HFs. Then, we screened potential epidermal cytokines from GEO DATASETs and assessed their functions using mice models and skin-derived precursors (SKPs). Finally, we explored the molecular mechanism underlying the action of the identified cytokine. The results showed that activation of WNT/ß-catenin in the epidermis prompted telogen-anagen transition. Keratinocytes infected with Ctnnb1-overexpressing lentivirus enhanced SKP expansion. Subsequently, we identified endothelin 1 (ET-1) expressed higher in hair-growing epidermis and induced the proliferation of DP cells and activates telogen-phase HFs in vivo. Moreover, ET-1 promotes the proliferation and stemness of SKPs. Western blot analysis and in vivo experiments revealed that ET-1 induces the transition from telogen-to-anagen phase by upregulating the PI3K/AKT pathway. These findings highlight the potential of ET-1 as a promising cytokine for HF activation and the treatment of hair loss.

AAV-delivered muscone-induced transgene system for treating chronic diseases in mice via inhalation.

Gene therapies provide treatment options for many diseases, but the safe and long-term control of therapeutic transgene expression remains a primary issue for clinical applications. Here, we develop a muscone-induced transgene system packaged into adeno-associated virus (AAV) vectors (AAVMUSE) based on a G protein-coupled murine olfactory receptor (MOR215-1) and a synthetic cAMP-responsive promoter (PCRE). Upon exposure to the trigger, muscone binds to MOR215-1 and activates the cAMP signaling pathway to initiate transgene expression. AAVMUSE enables remote, muscone dose- and exposure-time-dependent control of luciferase expression in the livers or lungs of mice for at least 20 weeks. Moreover, we apply this AAVMUSE to treat two chronic inflammatory diseases: nonalcoholic fatty liver disease (NAFLD) and allergic asthma, showing that inhalation of muscone-after only one injection of AAVMUSE-can achieve long-term controllable expression of therapeutic proteins (ΔhFGF21 or ΔmIL-4). Our odorant-molecule-controlled system can advance gene-based precision therapies for human diseases.

A programmable protease-based protein secretion platform for therapeutic applications.

Cell-based therapies represent potent enabling technologies in biomedical science. However, current genetic control systems for engineered-cell therapies are predominantly based on the transcription or translation of therapeutic outputs. Here we report a protease-based rapid protein secretion system (PASS) that regulates the secretion of pretranslated proteins retained in the endoplasmic reticulum (ER) owing to an ER-retrieval signal. Upon cleavage by inducible proteases, these proteins are secreted. Three PASS variants (chemPASS, antigenPASS and optoPASS) are developed. With chemPASS, we demonstrate the reversal of hyperglycemia in diabetic mice within minutes via drug-induced insulin secretion. AntigenPASS-equipped cells recognize the tumor antigen and secrete granzyme B and perforin, inducing targeted cell apoptosis. Finally, results from mouse models of diabetes, hypertension and inflammatory pain demonstrate light-induced, optoPASS-mediated therapeutic peptide secretion within minutes, conferring anticipated therapeutic benefits. PASS is a flexible platform for rapid delivery of therapeutic proteins that can facilitate the development and adoption of cell-based precision therapies.

Cardiomyocytic cyclic GMP-AMP synthase is critical for the induction of experimental cardiac graft rejection.

OBJECTIVE: During cardiac transplantation, cellular injury and DNA damage can result in the accumulation of cytosolic double-stranded DNA (dsDNA), which can activate the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon gene (STING) signaling pathway and thus induce multiple proinflammatory responses. However, the role of the cGAS-STING pathway in cardiac transplantation remains unclear. This study explored the role of cardiomyocytic cGAS in mouse heart transplantation during the ischemia/reperfusion and rejection processes. METHODS AND RESULTS: Cytosolic dsDNA accumulation and cGAS-STING signaling pathway component upregulation were observed in the grafts posttransplantation. The use of cGAS-deficient donor tissues led to significantly prolonged graft survival. The underlying mechanisms involved decreased expression and phosphorylation of downstream proteins, including TANK binding kinase 1 and nuclear factor κB. In parallel, notably diminished expression levels of various proinflammatory cytokines were observed. Accordingly, substantially decreased proportions of macrophages (CD11b+F4/80+) and CD8+ T cells were observed in the spleen. The activation of CD8+ T cells (CD8+CD69+) within the graft and the proportion of effector memory (CD44highCD62Llow) lymphocytes in the spleen were notably decreased. Treatment with the cGAS inhibitor Ru.521 led to significantly prolonged graft survival. CONCLUSIONS: Cardiomyocytic cGAS plays a critical role by sensing cytosolic dsDNA during cardiac transplantation and could serve as a potential therapeutic target to prevent graft rejection.

The dynamic cellular landscape of grafts with acute rejection after heart transplantation.

BACKGROUND: Acute cellular rejection (ACR) is a major barrier to the long-term survival of cardiac allografts. Although immune cells are well known to play critical roles in ACR, the dynamic cellular landscape of allografts with ACR remains obscure. METHODS: Single-cell RNA sequencing (scRNA-seq) was carried out for mouse cardiac allografts with ACR. Bioinformatic analysis was performed, and subsequent transplant experiments were conducted to validate the findings. RESULTS: Despite an overall large depletion of cardiac fibroblasts (CFBs), highly expanded cytotoxic T lymphocytes and a CXCL10+Gbp2+ subcluster of CFBs were enriched within grafts at the late stage. CXCL10+Gbp2+ CFBs featured strong interferon responsiveness and high expression of chemokines and major histocompatibility complex molecules, implying their involvement in the recruitment and activation of immune cells. Cell‒cell communication analysis revealed that CXCL9/CXCL10-CXCR3 might contribute to regulating CXCL10+Gbp2+ CFB-induced chemotaxis and immune cell recruitment. In vivo transplant studies revealed the therapeutic potential of CXCR3 antagonism in transplant rejection. CONCLUSIONS: The findings of our study unveiled a novel CFB subcluster that might mediate acute cardiac rejection. Targeting CXCR3 could prolong allograft survival.

An Optogenetic-Controlled Cell Reprogramming System for Driving Cell Fate and Light-Responsive Chimeric Mice.

Pluripotent stem cells (PSCs) hold great promise for cell-based therapies, disease modeling, and drug discovery. Classic somatic cell reprogramming to generate induced pluripotent stem cells (iPSCs) is often achieved based on overexpression of transcription factors (TFs). However, this process is limited by side effect of overexpressed TFs and unpredicted targeting of TFs. Pinpoint control over endogenous TFs expression can provide the ability to reprogram cell fate and tissue function. Here, a light-inducible cell reprogramming (LIRE) system is developed based on a photoreceptor protein cryptochrome system and clustered regularly interspaced short palindromic repeats/nuclease-deficient CRISPR-associated protein 9 for induced PSCs reprogramming. This system enables remote, non-invasive optogenetical regulation of endogenous Sox2 and Oct4 loci to reprogram mouse embryonic fibroblasts into iPSCs (iPSCLIRE ) under light-emitting diode-based illumination. iPSCLIRE cells can be efficiently differentiated into different cells by upregulating a corresponding TF. iPSCLIRE cells are used for blastocyst injection and optogenetic chimeric mice are successfully generated, which enables non-invasive control of user-defined endogenous genes in vivo, providing a valuable tool for facile and traceless controlled gene expression studies and genetic screens in mice. This LIRE system offers a remote, traceless, and non-invasive approach for cellular reprogramming and modeling of complex human diseases in basic biological research and regenerative medicine applications.

Engineering antiviral immune-like systems for autonomous virus detection and inhibition in mice.

The ongoing COVID-19 pandemic has demonstrated that viral diseases represent an enormous public health and economic threat to mankind and that individuals with compromised immune systems are at greater risk of complications and death from viral diseases. The development of broad-spectrum antivirals is an important part of pandemic preparedness. Here, we have engineer a series of designer cells which we term autonomous, intelligent, virus-inducible immune-like (ALICE) cells as sense-and-destroy antiviral system. After developing a destabilized STING-based sensor to detect viruses from seven different genera, we have used a synthetic signal transduction system to link viral detection to the expression of multiple antiviral effector molecules, including antiviral cytokines, a CRISPR-Cas9 module for viral degradation and the secretion of a neutralizing antibody. We perform a proof-of-concept study using multiple iterations of our ALICE system in vitro, followed by in vivo functionality testing in mice. We show that dual output ALICESaCas9+Ab system delivered by an AAV-vector inhibited viral infection in herpetic simplex keratitis (HSK) mouse model. Our work demonstrates that viral detection and antiviral countermeasures can be paired for intelligent sense-and-destroy applications as a flexible and innovative method against virus infection.

Lactobacillus rhamnosus HN001 Ameliorates BEZ235-Induced Intestinal Dysbiosis and Prolongs Cardiac Transplant Survival.

Cardiac allograft rejection remains a major factor limiting long-term engraftment after transplantation. A novel phosphoinositide 3-kinase (PI3K)/mTOR dual inhibitor, BEZ235, prolonged cardiac allograft survival by effectively suppressing activation of the PI3K/serine/threonine kinase (AKT)/mTOR pathway. However, long-term usage of pharmacological immunosuppressant drugs can cause intestinal microbiota dysbiosis. We established mouse models of allogeneic heterotopic heart transplantation with different treatments. Fecal samples were collected and subjected to 16S rRNA sequencing and targeted fecal metabolomic analysis. Graft samples were taken for immune cell detection by flow cytometry. Inflammatory cytokines in serum were quantified by enzyme-linked immunosorbent assay (ELISA). Compared to single-target approaches (IC-87114 and rapamycin), BEZ235 more efficiently prolongs cardiac transplant survival. Interestingly, BEZ235 reduces the diversity and abundance of the intestinal microbiota community. We demonstrated that Lactobacillus rhamnosus HN001 rescues the intestinal microbiota imbalance induced by BEZ235. IMPORTANCE Our data confirmed that the combination of BEZ235 and Lactobacillus rhamnosus HN001 significantly prolongs cardiac transplant survival. A main metabolic product of Lactobacillus rhamnosus HN001, propionic acid (PA), enriches regulatory T (Treg) cells and serves as a potent immunomodulatory supplement to BEZ235. Our study provides a novel and efficient therapeutic strategy for transplant recipients.

A small and highly sensitive red/far-red optogenetic switch for applications in mammals.

Optogenetic technologies have transformed our ability to precisely control biological processes in time and space. Yet, current eukaryotic optogenetic systems are limited by large or complex optogenetic modules, long illumination times, low tissue penetration or slow activation and deactivation kinetics. Here, we report a red/far-red light-mediated and miniaturized Δphytochrome A (ΔPhyA)-based photoswitch (REDMAP) system based on the plant photoreceptor PhyA, which rapidly binds the shuttle protein far-red elongated hypocotyl 1 (FHY1) under illumination with 660-nm light with dissociation occurring at 730 nm. We demonstrate multiple applications of REDMAP, including dynamic on/off control of the endogenous Ras/Erk mitogen-activated protein kinase (MAPK) cascade and control of epigenetic remodeling using a REDMAP-mediated CRISPR-nuclease-deactivated Cas9 (CRISPR-dCas9) (REDMAPcas) system in mice. We also demonstrate the utility of REDMAP tools for in vivo applications by activating the expression of transgenes delivered by adeno-associated viruses (AAVs) or incorporated into cells in microcapsules implanted into mice, rats and rabbits illuminated by light-emitting diodes (LEDs). Further, we controlled glucose homeostasis in type 1 diabetic (T1D) mice and rats using REDMAP to trigger insulin expression. REDMAP is a compact and sensitive tool for the precise spatiotemporal control of biological activities in animals with applications in basic biology and potentially therapy.

A far-red light-inducible CRISPR-Cas12a platform for remote-controlled genome editing and gene activation.

The CRISPR-Cas12a has been harnessed as a powerful tool for manipulating targeted gene expression. The possibility to manipulate the activity of CRISPR-Cas12a with a more precise spatiotemporal resolution and deep tissue permeability will enable targeted genome engineering and deepen our understanding of the gene functions underlying complex cellular behaviors. However, currently available inducible CRISPR-Cas12a systems are limited by diffusion, cytotoxicity, and poor tissue permeability. Here, we developed a far-red light (FRL)­inducible CRISPR-Cas12a (FICA) system that can robustly induce gene editing in mammalian cells, and an FRL-inducible CRISPR-dCas12a (FIdCA) system based on the protein-tagging system SUperNova (SunTag) that can be used for gene activation under light-emitting diode­based FRL. Moreover, we show that the FIdCA system can be deployed to activate target genes in mouse livers. These results demonstrate that these systems developed here provide robust and efficient platforms for programmable genome manipulation in a noninvasive and spatiotemporal fashion.

BET protein inhibition evidently enhances sensitivity to PI3K/mTOR dual inhibition in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC), the second most common primary liver cancer, is a fatal malignancy with a poor prognosis and only very limited therapeutic options. Although molecular targeted therapy is emerged as a promising treatment strategy, resistance to molecular-targeted therapy occurs inevitably, which represents a major clinical challenge. In this study, we confirmed that mammalian target of rapamycin (mTOR) signaling is the most significantly affected pathways in ICC. As a novel phosphoinositide 3-kinase (PI3K)/mTOR dual inhibitor, BEZ235, exerts antitumour activity by effectively and specifically blocking the dysfunctional activation of the PI3K/serine/threonine kinase (AKT)/mTOR pathway. We generate the orthotopic ICC mouse model through hydrodynamic transfection of AKT and yes-associated protein (YAP) plasmids into the mouse liver. Our study confirmed that BEZ235 can suppress the proliferation, invasion and colony conformation abilities of ICC cells in vitro but cannot effectively inhibit ICC progression in vivo. Inhibition of PI3K/mTOR allowed upregulation of c-Myc and YAP through suppressed the phosphorylation of LATS1. It would be a novel mechanism that mediated resistance to PI3K/mTOR dual inhibitor. However, Bromo- and extraterminal domain (BET) inhibition by JQ1 downregulates c-Myc and YAP transcription, which could enhance the efficacy of PI3K/mTOR inhibitors. The efficacy results of combination therapy exhibited effective treatment on ICC in vitro and in vivo. Our data further confirmed that the combination of PI3K/mTOR dual inhibitor and BET inhibition induces M1 polarization and suppresses M2 polarization in macrophages by regulating the expression of HIF-1α. Our study provides a novel and efficient therapeutic strategy in treating primary ICC.

Tadalafil enhances the therapeutic efficacy of BET inhibitors in hepatocellular carcinoma through activating Hippo pathway.

Bromodomain and extraterminal (BET) proteins are promising therapeutic targets for hematological and solid tumors. However, BET inhibitor monotherapy did not show a significant therapeutic benefit for hepatocellular carcinoma (HCC) in preclinical trials. Here, we identified YAP/TAZ genes, as determinants for sensitivity to BET inhibitors. YAP/TAZ expression, especially TAZ, promote resistance to BET inhibitor. In addition, we analyzed that the mRNA level of PDE5 was positively correlated with YAP/TAZ based on TCGA database and demonstrated tadalafil, a PDE5 inhibitor, could block YAP/TAZ protein expression by activating Hippo pathway. Cotreatment with tadalafil and JQ-1 synergistically reduced YAP/TAZ protein expression, suppressed proliferation and induced G0-G1 arrest of cultured HCC cells. JQ-1 alone does not show significant benefits in a mouse model of HCC induced by c-Myc/N-Ras plasmids. In contrast, the combination, tadalafil and JQ-1, successfully suppressed tumor progression, enhanced antitumor immunity by improving the ratio of activated CD8 and extended the survival time of mice. Our data define the key role of YAP/TAZ in mediating resistance to BET inhibitor, described the PDE5/PKG/Hippo/YAP/TAZ axis and identified a common clinical drug that can be developed as an effective combined strategy to overcome BET inhibitor resistance in MYC/Ras-driven HCC.

Low-frequency multi-order acoustic absorber based on spiral metasurface.

In this work, we propose a spiral metasurface for multi-order sound absorption in the low-frequency range (<1000 Hz). By dividing the long channel of the spiral metasurface into a series of tunable sub-cavities and employing recessed necks, the metasurface can quasi-perfectly (>0.95 in experiments) absorb airborne sound at multiple low-frequency orders without being limited by the number of equivalent cavities. Owing to the superior impedance manipulation provided by the spiral metasurface, each absorption order can be tuned flexibly with a constant external shape. By suitably modulating the sub-cavities and the recessed necks, we obtained multi-order high-absorption metasurfaces with dual-chamber, tri-chamber, and four-chamber designs. The ratio of the lowest resonant wavelength to the thickness is as high as 78. The samples, which are fabricated by three-dimensional printing technology, were measured to verify the theoretical results. We also investigate the relationship between the geometric parameters of the recessed necks and the sound absorption performance, which facilitates the more feasibly designed multi-order metasurfaces. The concept can be further applied to broadband absorption with ultra-thin thickness and has potential applications for noise reduction.

Amphiregulin promotes hair regeneration of skin-derived precursors via the PI3K and MAPK pathways.

OBJECTIVES: There are significant clinical challenges associated with alopecia treatment, including poor efficiency of related drugs and insufficient hair follicles (HFs) for transplantation. Skin-derived precursors (SKPs) exhibit great potential as stem cell-based therapies for hair regeneration; however, the proliferation and hair-inducing capacity of SKPs gradually decrease during culturing. MATERIALS AND METHODS: We describe a 3D co-culture system accompanied by kyoto encyclopaedia of genes and genomes and gene ontology enrichment analyses to determine the key factors and pathways that enhance SKP stemness and verified using alkaline phosphatase assays, Ki-67 staining, HF reconstitution, Western blot and immunofluorescence staining. The upregulated genes were confirmed utilizing corresponding recombinant protein or small-interfering RNA silencing in vitro, as well as the evaluation of telogen-to-anagen transition and HF reconstitution in vivo. RESULTS: The 3D co-culture system revealed that epidermal stem cells and adipose-derived stem cells enhanced SKP proliferation and HF regeneration capacity by amphiregulin (AREG), with the promoted stemness allowing SKPs to gain an earlier telogen-to-anagen transition and high-efficiency HF reconstitution. By contrast, inhibitors of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways downstream of AREG signalling resulted in diametrically opposite activities. CONCLUSIONS: By exploiting a 3D co-culture model, we determined that AREG promoted SKP stemness by enhancing both proliferation and hair-inducing capacity through the PI3K and MAPK pathways. These findings suggest AREG therapy as a potentially promising approach for treating alopecia.

Liquid-liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice.

Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity.

Anti-cytokine therapy and small molecule agents for the treatment of inflammatory bowel disease.

Inflammatory bowel disease (IBD), including Crohn disease and ulcerative colitis, with multifactorial etiologies has led to a global health-associated burden in many countries. Substantial efforts are devoted to understand the pathogenesis, behavioral and environmental triggers, which may be specifically valuable for the treatment of IBD. The specific pathogenesis underlying IBD is as yet incompletely understood. The use of anti-cytokine therapy and small molecule agents targeting the immune system is thought to restore the body's intestinal barrier function and relieve inflammation with manageable adverse effects. In this review, we report recent advances in anti-cytokine therapy and treatment with small molecule agents for the management of IBD.

Immobilization of thermophilic lipase in inorganic hybrid nanoflower through biomimetic mineralization.

Thermophilic lipase QLM from Alcaligenes sp. was successfully immobilized in Cu3(PO4)2-based inorganic hybrid nanoflower through biomimetic mineralization. The morphology, structure and element composition of immobilized enzyme were systemically characterized to elucidate the successful loading of enzyme molecules. The optimal temperature (65 °C) and pH (8.0) of immobilized enzyme were then determined by monitoring the hydrolysis of p-nitrophenyl caprylate. Moreover, compared with free enzyme, immobilized enzyme in inorganic hybrid nanoflower exhibited enhanced stability against thermal, pH and metal ions, attributing to the protective effect of nanoflower shell. Additionally, the immobilized enzyme possessed excellent reusability and long-term storage stability, with slightly decreased activity after being repeatedly used for 8 cycles or stored in water at room temperature for 4 weeks. Overall, the immobilization in inorganic hybrid nanoflower provided a facile and effective approach for the preparation of immobilized enzymes with favorable activity, stability and reusability, and thus the strategy showed great potential in developing ideal catalysts for future biocatalytic applications.