Antidiabetic Close Loop Based on Wearable DNA-Hydrogel Glucometer and Implantable Optogenetic Cells.

Diabetes mellitus and its associated secondary complications have become a pressing global healthcare issue. The current integrated theranostic plan involves a glucometer-tandem pump. However, external condition-responsive insulin delivery systems utilizing rigid glucose sensors pose challenges in on-demand, long-term insulin administration. To overcome these challenges, we present a novel model of antidiabetic management based on printable metallo-nucleotide hydrogels and optogenetic engineering. The conductive hydrogels were self-assembled by bioorthogonal chemistry using oligonucleotides, carbon nanotubes, and glucose oxidase, enabling continuous glucose monitoring in a broad range (0.5-40 mM). The optogenetically engineered cells were enabled glucose regulation in type I diabetic mice via a far-red light-induced transgenic expression of insulin with a month-long avidity. Combining with a microchip-integrated microneedle patch, a prototyped close-loop system was constructed. The glucose levels detected by the sensor were received and converted by a wireless controller to modulate far-infrared light, thereby achieving on-demand insulin expression for several weeks. This study sheds new light on developing next-generation diagnostic and therapy systems for personalized and digitalized precision medicine.

Mutual regulation of CD4+ T cells and intravascular fibrin in infections.

Innate myeloid cells especially neutrophils and their extracellular traps are known to promote intravascular coagulation and thrombosis formation in infections and various other conditions. Innate myeloid cell dependent fibrin formation can support systemic immunity while its dysregulation enhances the severity of infectious diseases. Less is known about the immune mechanisms preventing dysregulation of fibrin homeostasis in infection. During experimental systemic infections local fibrin deposits in the liver microcirculation cause rapid arrest of CD4+ T cells. Arrested T helper cells mostly represent Th17 cells that partially originate from the small intestine. Intravascular fibrin deposits activate mouse and human CD4+ T cells which can be mediated by direct fibrin - CD4+ T cell interactions. Activated CD4+ T cells suppress fibrin deposition and microvascular thrombosis by directly counteracting coagulation activation by neutrophils and classical monocytes. T cell activation, which is initially triggered by IL- 12p40- and MHC-II dependent mechanisms, enhances intravascular fibrinolysis via LFA-1. Moreover, CD4+ T cells disfavor the association of the fibrinolysis inhibitor TAFI with fibrin whereby fibrin deposition is increased by TAFI in the absence but not presence of T cells. In human infections thrombosis development is inversely related to microvascular levels of CD4+ T cells. Thus, fibrin promotes LFA-1 dependent T helper cell activation in infections which drives a negative feedback cycle that rapidly restricts intravascular fibrin and thrombosis development.

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.

Sonogenetics-controlled synthetic designer cells for cancer therapy in tumor mouse models.

Bacteria-based therapies are powerful strategies for cancer therapy, yet their clinical application is limited by a lack of tunable genetic switches to safely regulate the local expression and release of therapeutic cargoes. Rapid advances in remote-control technologies have enabled precise control of biological processes in time and space. We developed therapeutically active engineered bacteria mediated by a sono-activatable integrated gene circuit based on the thermosensitive transcriptional repressor TlpA39. Through promoter engineering and ribosome binding site screening, we achieved ultrasound (US)-induced protein expression and secretion in engineered bacteria with minimal noise and high induction efficiency. Specifically, delivered either intratumorally or intravenously, engineered bacteria colonizing tumors suppressed tumor growth through US-irradiation-induced release of the apoptotic protein azurin and an immune checkpoint inhibitor, a nanobody targeting programmed death-ligand 1, in different tumor mouse models. Beyond developing safe and high-performance designer bacteria for tumor therapy, our study illustrates a sonogenetics-controlled therapeutic platform that can be harnessed for bacteria-based precision medicine.

Ultra-Broadband Ultraviolet-Visible Light-Short Wavelength Infrared InGaAs Focal Plane Arrays via n-InP Contact Layer Removal.

PIN InGaAs short wavelength infrared (SWIR) focal plane array (FPA) detectors have attracted extensive attention due to their high detectivity, high quantum efficiency, room temperature operation, low dark current, and good radiation resistance. Furthermore, InGaAs FPA detectors have wide applications in many fields, such as aviation safety, biomedicine, camouflage recognition, and infrared night vision. Recently, extensive research has been conducted on the extension of the response spectrum from short wavelength infrared (SWIR) to visible light (VIS) through InP substrate removal and reserving the n-InP contact layer. However, there is little research on the absorption of InGaAs detectors in the ultraviolet (UV) band. In this paper, we present an ultra-broadband UV-VIS-SWIR 640 × 512 15 µm InGaAs FPA detector by removing the n-InP contact layer in the active area and reserving the InP contact layer around the pixels for n contact, creating incident light to be directly absorbed by the In0.53Ga0.47As absorption layer. In addition, the optical absorption characteristics of InGaAs infrared detectors with and without an n-InP contact layer are studied theoretically. The test results show that the spectral response is extended to the range of 200-1700 nm. The quantum efficiency is higher than 45% over a broad wavelength range of 300-1650 nm. The operability is up to 99.98%, and the responsivity non-uniformity is 3.28%. The imaging capability of InGaAs FPAs without the n-InP contact layer has also been demonstrated, which proves the feasibility of simultaneous detection for these three bands.

A programmable targeted protein-degradation platform for versatile applications in mammalian cells and mice.

Myriad physiological and pathogenic processes are governed by protein levels and modifications. Controlled protein activity perturbation is essential to studying protein function in cells and animals. Based on Trim-Away technology, we screened for truncation variants of E3 ubiquitinase Trim21 with elevated efficiency (ΔTrim21) and developed multiple ΔTrim21-based targeted protein-degradation systems (ΔTrim-TPD) that can be transfected into host cells. Three ΔTrim-TPD variants are developed to enable chemical and light-triggered programmable activation of TPD in cells and animals. Specifically, we used ΔTrim-TPD for (1) red-light-triggered inhibition of HSV-1 virus proliferation by degrading the packaging protein gD, (2) for chemical-triggered control of the activity of Cas9/dCas9 protein for gene editing, and (3) for blue-light-triggered degradation of two tumor-associated proteins for spatiotemporal inhibition of melanoma tumor growth in mice. Our study demonstrates that multiple ΔTrim21-based controllable TPD systems provide powerful tools for basic biology research and highlight their potential biomedical applications.

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.

Synthesis, Performance, Mechanism: A Hyperbranched Phase Reverse Nano-Demulsifier for Condensate Emulsion.

Organic amine and nanosilica were combined to create a nano-demulsifier, which was employed in the oil-water separation process of a condensate emulsion. The nano-demulsifier has the structure of hyperbranched polymers and the skeleton structure of hyperbranched nanomaterials, and displays the demulsification impact of organic amine polymers as well as the synergistic effect of nanomaterials. This nano-demulsifier has the potential to drastically reduce the quantity of condensate demulsifiers utilized in the gathering station. The dehydration rate of the condensate lotion in the gas gathering station can reach more than 95% only at a concentration of 1.0 wt.%. Its application can significantly increase the separation efficiency of the condensate emulsion as well as the quality of condensate oil. It has a positive impact on cost reduction and efficiency in gas well production. The mechanism of action of the demulsifier was also studied, and the results show that the demulsifier is a phase reverse demulsifier.

CD201+ fascia progenitors choreograph injury repair.

Optimal tissue recovery and organismal survival are achieved by spatiotemporal tuning of tissue inflammation, contraction and scar formation1. Here we identify a multipotent fibroblast progenitor marked by CD201 expression in the fascia, the deepest connective tissue layer of the skin. Using skin injury models in mice, single-cell transcriptomics and genetic lineage tracing, ablation and gene deletion models, we demonstrate that CD201+ progenitors control the pace of wound healing by generating multiple specialized cell types, from proinflammatory fibroblasts to myofibroblasts, in a spatiotemporally tuned sequence. We identified retinoic acid and hypoxia signalling as the entry checkpoints into proinflammatory and myofibroblast states. Modulating CD201+ progenitor differentiation impaired the spatiotemporal appearances of fibroblasts and chronically delayed wound healing. The discovery of proinflammatory and myofibroblast progenitors and their differentiation pathways provide a new roadmap to understand and clinically treat impaired wound healing.

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.

Engineering natural molecule-triggered genetic control systems for tunable gene- and cell-based therapies.

The ability to precisely control activities of engineered designer cells provides a novel strategy for modern precision medicine. Dynamically adjustable gene- and cell-based precision therapies are recognized as next generation medicines. However, the translation of these controllable therapeutics into clinical practice is severely hampered by the lack of safe and highly specific genetic switches controlled by triggers that are nontoxic and side-effect free. Recently, natural products derived from plants have been extensively explored as trigger molecules to control genetic switches and synthetic gene networks for multiple applications. These controlled genetic switches could be further introduced into mammalian cells to obtain synthetic designer cells for adjustable and fine tunable cell-based precision therapy. In this review, we introduce various available natural molecules that were engineered to control genetic switches for controllable transgene expression, complex logic computation, and therapeutic drug delivery to achieve precision therapy. We also discuss current challenges and prospects in translating these natural molecule-controlled genetic switches developed for biomedical applications from the laboratory to the clinic.

Immunity and reproduction protective effects of Chitosan Oligosaccharides in Cyclophosphamide/Busulfan-induced premature ovarian failure model mice.

Introduction: Premature ovarian failure (POF) is a major cause of infertility among women of reproductive age. Unfortunately, there is no effective treatment available currently. Researchers have shown that immune disorders play a significant role in the development of POF. Moreover, growing evidence suggest that Chitosan Oligosaccharides (COS), which act as critical immunomodulators, may have a key role in preventing and treating a range of immune related reproductive diseases. Methods: KM mice (6-8 weeks) received a single intraperitoneal injection of cyclophosphamide (CY, 120mg/kg) and busulfan (BUS, 30mg/kg) to establish POF model. After completing the COS pre-treatment or post-treatment procedures, peritoneal resident macrophages (PRMs) were collected for neutral erythrophagocytosis assay to detect phagocytic activity. The thymus, spleen and ovary tissues were collected and weighed to calculate the organ indexes. Hematoxylin-eosin (HE) staining was performed to observe the histopathologic structure of those organs. The serum levels of estrogen (E2) and progesterone (P) were measured via the enzyme-linked immunosorbent assay (ELISA). The expression levels of immune factors including interleukin 2 (IL-2), interleukin 4 (IL-4), and tumor necrosis factor α (TNF-α), as well as germ cell markers Mouse Vasa Homologue (MVH) and Fragilis in ovarian tissue, were analyzed by Western blotting and qRT-PCR. In addition, ovarian cell senescence via p53/p21/p16 signaling was also detected. Results: The phagocytic function of PRMs and the structural integrity of thymus and spleen were preserved by COS treatment. The levels of certain immune factors in the ovaries of CY/BUS- induced POF mice were found to be altered, manifested as IL-2 and TNF-α experiencing a significant decline, and IL-4 presenting a notable increase. Both pre-treatment and post-treatment with COS were shown to be protective effects against the damage to ovarian structure caused by CY/BUS. Senescence-associated ß-galactosidase (SA-ß-Gal) staining results showed that COS prevents CY/BUS-induced ovarian cell senescence. Additionally, COS regulated estrogen and progesterone levels, enhanced follicular development, and blocked ovarian cellular p53/p21/p16 signaling which participating in cell senescence. Conclusion: COS is a potent preventative and therapeutic medicine for premature ovarian failure by enhancing both the ovarian local and systemic immune response as well as inhibiting germ cell senescence.

Chito-oligosaccharides and macrophages have synergistic effects on improving ovarian stem cells function by regulating inflammatory factors.

BACKGROUND: Chronic low-grade inflammation and ovarian germline stem cells (OGSCs) aging are important reasons for the decline of ovarian reserve function, resulting in ovarian aging and infertility. Regulation of chronic inflammation is expected to promote the proliferation and differentiation of OGSCs, which will become a key means for maintaining and remodeling ovarian function. Our previous study demonstrated that Chitosan Oligosaccharides (Cos) promoted the OGSCs proliferation and remodelled the ovarian function through improving the secretion of immune related factors,but the mechanism remains unclear, and the role of macrophages, the important source of various inflammatory mediators in the ovary needs to be further studied. In this study, we used the method of macrophages and OGSCs co-culture to observe the effect and mechanism of Cos on OGSCs, and explore what contribution macrophages give during this process. Our finding provides new drug treatment options and methods for the prevention and treatment of premature ovarian failure and infertility. METHODS: We used the method of macrophages and OGSCs co-culture to observe the effect and mechanism of Cos on OGSCs, and explore the important contribution of macrophages in it. The immunohistochemical staining was used to locate the OGSCs in the mouse ovary. Immunofluorescent staining, RT-qPCR and ALP staining were used to identify the OGSCs. CCK-8 and western blot were used to evaluate the OGSCs proliferation. ß-galactosidase(SA-ß-Gal) staining and western blot were used to detect the changing of cyclin-dependent kinase inhibitor 1A(P21), P53, Recombinant Sirtuin 1(SIRT1) and Recombinant Sirtuin 3(SIRT3). The levels of immune factors IL-2, IL-10, TNF-α and TGF-ß were explored by using Western blot and ELISA. RESULTS: We found that Cos promoted OGSCs proliferation in a dose-and time-dependent manner, accompanied by IL-2, TNF-α increase and IL-10, TGF-ß decrease. Mouse monocyte-macrophages Leukemia cells(RAW) can also produce the same effect as Cos. When combined with Cos, it can enhance the proliferative effect of Cos in OGSCs, and further increase IL-2, TNF-α and further decrease IL-10, TGF-ß. The macrophages can enhance the proliferative effect of Cos in OGSCs is also associated with the further increase in IL-2, TNF-α and the further decrease in IL-10, TGF-ß. In this study, we determined that the anti-aging genes SIRT-1 and SIRT-3 protein levels were increased by Cos and RAW respectively, whereas the senescence-associated SA-ß-Gal and aging genes P21 and P53 were decreased. Cos and RAW had a protective effect on OGSCs delaying aging. Furthermore, RAW can further decrease the SA-ß-Gal and aging genes P21 and P53 by Cos, and further increase SIRT1 and SIRT3 protein levels in OGSCs by Cos. CONCLUSION: In conclusion, Cos and macrophages have synergistic effects on improving OGSCs function and delaying ovarian aging by regulating inflammatory factors.

UnVELO: Unsupervised Vision-Enhanced LiDAR Odometry with Online Correction.

Due to the complementary characteristics of visual and LiDAR information, these two modalities have been fused to facilitate many vision tasks. However, current studies of learning-based odometries mainly focus on either the visual or LiDAR modality, leaving visual-LiDAR odometries (VLOs) under-explored. This work proposes a new method to implement an unsupervised VLO, which adopts a LiDAR-dominant scheme to fuse the two modalities. We, therefore, refer to it as unsupervised vision-enhanced LiDAR odometry (UnVELO). It converts 3D LiDAR points into a dense vertex map via spherical projection and generates a vertex color map by colorizing each vertex with visual information. Further, a point-to-plane distance-based geometric loss and a photometric-error-based visual loss are, respectively, placed on locally planar regions and cluttered regions. Last, but not least, we designed an online pose-correction module to refine the pose predicted by the trained UnVELO during test time. In contrast to the vision-dominant fusion scheme adopted in most previous VLOs, our LiDAR-dominant method adopts the dense representations for both modalities, which facilitates the visual-LiDAR fusion. Besides, our method uses the accurate LiDAR measurements instead of the predicted noisy dense depth maps, which significantly improves the robustness to illumination variations, as well as the efficiency of the online pose correction. The experiments on the KITTI and DSEC datasets showed that our method outperformed previous two-frame-based learning methods. It was also competitive with hybrid methods that integrate a global optimization on multiple or all frames.

Measurement of Secreted Embryonic Alkaline Phosphatase.

Secreted reporters have been demonstrated to be simple and useful tools for analyzing transcriptional regulation in mammalian cells. The distinctive feature of these assays is the ability to detect reporter gene expression in the culture supernatant without affecting the cell physiology or leading to cell lysis, which allows repeated experimentation and sampling of the culture medium using the same cell cultures. Secreted embryonic alkaline phosphatase (SEAP) is one of the most widely used reporter, which can be easily detected using colorimetry following incubation with a substrate, such as p-nitrophenol phosphate. In this report, we present detailed procedures for detection and quantification of the SEAP reporter. We believe that this step-by-step protocol can be easily used by researchers to monitor and measure molecular genetic events in a variety of mammalian cells due to its simplicity and ease of handling. Graphical abstract Schematic overview of the workflow described in this protocol.

Fascia Layer-A Novel Target for the Application of Biomaterials in Skin Wound Healing.

As the first barrier of the human body, the skin has been of great concern for its wound healing and regeneration. The healing of large, refractory wounds is difficult to be repaired by cell proliferation at the wound edges and usually requires manual intervention for treatment. Therefore, therapeutic tools such as stem cells, biomaterials, and cytokines have been applied to the treatment of skin wounds. Skin microenvironment modulation is a key technology to promote wound repair and skin regeneration. In recent years, a series of novel bioactive materials that modulate the microenvironment and cell behavior have been developed, showing the ability to efficiently facilitate wound repair and skin attachment regeneration. Meanwhile, our lab found that the fascial layer has an indispensable role in wound healing and repair, and this review summarizes the research progress of related bioactive materials and their role in wound healing.

Therapeutic Silencing of p120 in Fascia Fibroblasts Ameliorates Tissue Repair.

Deep skin wounds rapidly heal by mobilizing extracellular matrix and cells from the fascia, deep beneath the dermal layer of the skin, to form scars. Despite wounds being an extensively studied area and an unmet clinical need, the biochemistry driving this patch-like repair remains obscure. Lacking also are efficacious therapeutic means to modulate scar formation in vivo. In this study, we identify a central role for p120 in mediating fascia mobilization and wound repair. Injury triggers p120 expression, largely within engrailed-1 lineage-positive fibroblasts of the fascia that exhibit a supracellular organization. Using adeno-associated virus‒mediated gene silencing, we show that p120 establishes the supracellular organization of fascia engrailed-1 lineage-positive fibroblasts, without which fascia mobilization is impaired. Gene silencing of p120 in fascia fibroblasts disentangles their supracellular organization, reducing the transfer of fascial cells and extracellular matrix into wounds and augmenting wound healing. Our findings place p120 as essential for fascia mobilization, opening, to our knowledge, a previously unreported therapeutic avenue for targeted intervention in the treatment of a variety of skin scar conditions.

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.