Synergistic hyperactivation of both mTORC1 and mTORC2 underlies the neural abnormalities of PTEN-deficient human neurons and cortical organoids.

Mutations in the phosphatase and tensin homolog (PTEN) gene are associated with severe neurodevelopmental disorders. Loss of PTEN leads to hyperactivation of the mechanistic target of rapamycin (mTOR), which functions in two distinct protein complexes, mTORC1 and mTORC2. The downstream signaling mechanisms that contribute to PTEN mutant phenotypes are not well delineated. Here, we show that pluripotent stem cell-derived PTEN mutant human neurons, neural precursors, and cortical organoids recapitulate disease-relevant phenotypes, including hypertrophy, electrical hyperactivity, enhanced proliferation, and structural overgrowth. PTEN loss leads to simultaneous hyperactivation of mTORC1 and mTORC2. We dissect the contribution of mTORC1 and mTORC2 by generating double mutants of PTEN and RPTOR or RICTOR, respectively. Our results reveal that the synergistic hyperactivation of both mTORC1 and mTORC2 is essential for the PTEN mutant human neural phenotypes. Together, our findings provide insights into the molecular mechanisms that underlie PTEN-related neural disorders and highlight novel therapeutic targets.

Incorporation of poly(γ-glutamic acid) in lipid nanoparticles for enhanced mRNA delivery efficiency in vitro and in vivo.

Messenger RNA (mRNA)-based therapy shows immense potential for broad biomedical applications. However, the development of safe and efficacious mRNA delivery vectors remains challenging due to delivery barriers and inefficient intracellular payload release. Herein, we presented a simple strategy to boost the mRNA intracellular release by incorporation of anionic poly(γ-glutamic acid) (PGA) into an ionizable lipid-based LNP/mRNA. We systematically investigated the impact of PGA incorporation on mRNA transfection both in vitro and in vivo. The molecular weights and formulation ratios of PGA greatly affected the transfection efficacy of LNP/mRNA. From in vitro study, the optimized LNP/mRNA/PGA was formulated by incorporation of PGA with the molecular weight of 80 kDa or 200 kDa and the charge ratio (N/P/C) of 25/1/1. The optimized formulation achieved around 3-fold mRNA expression in HeLa cells compared to the bare LNP/mRNA. The intracellular releasing study using specific DNA probe revealed that this enhancement of transfection efficacy was attributed to the elevated mRNA release into cytoplasm. Moreover, the optimized LNP/mRNA/PGA achieved up to 5-fold or 3-fold increase of luciferase mRNA expression in vivo after being injected into mice systematically or intramuscularly, respectively. In addition, the incorporation of PGA did not significantly alter the biodistribution profile of the complexes on both organ and cellular levels. Therefore, our work provides a simple strategy to boost mRNA delivery, which holds great promise to improve the efficacy of mRNA therapeutics for various biomedical applications. STATEMENT OF SIGNIFICANCE: The process of designing and screening potent mRNA carriers is complicated and time-consuming, while the efficacy is not always satisfying due to the delivery barriers and inefficient mRNA release. This work presented an alternative strategy to boost the mRNA delivery efficacy by incorporating an anionic natural polymer poly(γ-glutamic acid) (PGA) into LNP/mRNA complexes. The optimized LNP/mRNA/PGA achieved up to 3-fold and 5-fold increase in transfection efficacy in vitro and in vivo, respectively. Intracellular releasing analysis revealed that the enhancement of transfection efficacy was mainly attributed to the elevated intracellular release of mRNA. In addition, the incorporation of PGA did not alter the biodistribution or the biosafety profile of the complexes. These findings indicate that PGA incorporation is a promising strategy to improve the efficacy of mRNA therapeutics.

Chitosan functionalized gold nanostars as a theranostic platform for intracellular microRNA detection and photothermal therapy.

The development of a theranostic platform that integrates both diagnostic and therapeutic capabilities is in great need for precise and personalized medicine. Here, we present a novel nanoplatform (AuNS@CS-hpDNA) formulated by chitosan functionalized gold nanostar composites and further complexed with fluorescent hairpin DNA (hpDNA) probes for tumor-related miRNA imaging and photothermal therapy (PTT). The optimized AuNS@CS-hpDNA nanoplatform mediated efficient hpDNA probe loading and intracellular delivery. Subsequently, the cytosol transfer of the hpDNA probe enabled specific hybridization using the targeted miRNA, which triggered the recovery of fluorescence for the precise detection of biomarker miR21 in living cells and realized the distinguishing cancer cell line MCF-7 and normal cells. Meanwhile, the AuNS@CS-hpDNA nanoplatform exhibited excellent photothermal conversion properties, which induced efficient cancer cell killing under laser irradiation. Thus, the developed AuNS@CS-hpDNA nanoplatform could simultaneously realize the precise detection of cancer cells and accurately initiate efficient PTT, which represents a promising strategy for precise cancer therapy.

Ogt-mediated O-GlcNAcylation inhibits astrocytes activation through modulating NF-κB signaling pathway.

Previous studies have shown that Ogt-mediated O-GlcNAcylation is essential for neuronal development and function. However, the function of O-GlcNAc transferase (Ogt) and O-GlcNAcylation in astrocytes remains largely unknown. Here we show that Ogt deficiency induces inflammatory activation of astrocytes in vivo and in vitro, and impairs cognitive function of mice. The restoration of O-GlcNAcylation via GlcNAc supplementation inhibits the activation of astrocytes, inflammation and improves the impaired cognitive function of Ogt deficient mice. Mechanistically, Ogt interacts with NF-κB p65 and catalyzes the O-GlcNAcylation of NF-κB p65 in astrocytes. Ogt deficiency induces the activation of NF-κB signaling pathway by promoting Gsk3ß binding. Moreover, Ogt depletion induces the activation of astrocytes derived from human induced pluripotent stem cells. The restoration of O-GlcNAcylation inhibits the activation of astrocytes, inflammation and reduces Aß plaque of AD mice in vitro and in vivo. Collectively, our study reveals a critical function of Ogt-mediated O-GlcNAcylation in astrocytes through regulating NF-κB signaling pathway.

Ogt Deficiency Induces Abnormal Cerebellar Function and Behavioral Deficits of Adult Mice through Modulating RhoA/ROCK Signaling.

Previous studies have shown the essential roles of O-GlcNAc transferase (Ogt) and O-GlcNAcylation in neuronal development, function and neurologic diseases. However, the function of Ogt and O-GlcNAcylation in the adult cerebellum has not been well elucidated. Here, we have found that cerebellum has the highest level of O-GlcNAcylation relative to cortex and hippocampus of adult male mice. Specific deletion of Ogt in granule neuron precursors (GNPs) induces abnormal morphology and decreased size of the cerebellum in adult male Ogt deficient [conditional knock-out (cKO)] mice. Adult male cKO mice show the reduced density and aberrant distribution of cerebellar granule cells (CGCs), the disrupted arrangement of Bergman glia (BG) and Purkinje cells. In addition, adult male cKO mice exhibit aberrant synaptic connection, impaired motor coordination, and learning and memory abilities. Mechanistically, we have identified G-protein subunit α12 (Gα12) is modified by Ogt-mediated O-GlcNAcylation. O-GlcNAcylation of Gα12 facilitates its binding to Rho guanine nucleotide exchange factor 12 (Arhgef12) and consequently activates RhoA/ROCK signaling. RhoA/ROCK pathway activator LPA can rescue the developmental deficits of Ogt deficient CGCs. Therefore, our study has revealed the critical function and related mechanisms of Ogt and O-GlcNAcylation in the cerebellum of adult male mice.SIGNIFICANCE STATEMENT Cerebellar function are regulated by diverse mechanisms. To unveil novel mechanisms is critical for understanding the cerebellar function and the clinical therapy of cerebellum-related diseases. In the present study, we have shown that O-GlcNAc transferase gene (Ogt) deletion induces abnormal cerebellar morphology, synaptic connection, and behavioral deficits of adult male mice. Mechanistically, Ogt catalyzes O-GlcNAcylation of Gα12, which promotes the binding to Arhgef12, and regulates RhoA/ROCK signaling pathway. Our study has uncovered the important roles of Ogt and O-GlcNAcylation in regulating cerebellar function and cerebellum-related behavior. Our results suggest that Ogt and O-GlcNAcylation could be potential targets for some cerebellum-related diseases.

Niosome-Assisted Delivery of DNA Fluorescent Probe with Optimized Strand Displacement for Intracellular MicroRNA21 Imaging.

MicroRNAs play a vital role in cancer development and are considered as potential biomarkers for early prognostic assessment. Here, we propose a novel biosensing system to achieve fluorescence imaging of miRNA21 (miR21) in cancer cells. This system consists of two components: an optimized "off-on" double-stranded DNA (dsDNA) fluorescent for miR21 sensing by efficient strand-displacement reaction and a potent carrier vesicle, termed niosome (SPN), to facilitate the efficient intracellular delivery of the dsDNA probe. A series of dsDNA probes based on fluorescence energy resonance transfer (FRET) was assembled to target miR21. By optimizing the appropriate length of the reporter strand in the dsDNA probe, high accuracy and sensitivity for miR21 recognition are ensured. To overcome the cellular barrier, we synthesized SPN with the main components of a nonionic surfactant Span 80 and a cationic lipid DOTAP, which could efficiently load dsDNA probes via electrostatic interactions and potently deliver the dsDNA probes into cells with good biosafety. The SPN/dsDNA achieved efficient miR21 fluorescent imaging in living cells, and could discriminate cancer cells (MCF-7) from normal cells (L-02). Therefore, the proposed SPN/dsDNA system provides a powerful tool for intracellular miRNA biosensing, which holds great promise for early cancer diagnosis.

Soil microbial communities of dryland legume plantations are more complex than non-legumes.

Soil microorganisms play a crucial role in the vegetation restoration of dryland plantations and participating in biogeochemical cycles. However, how the co-occurrence networks of soil microbial communities respond to dryland legume and non-legume plantations is unclear. Here, we conducted a comparative analysis of legume (13-, 35-, and 55-years Caragana korshinskii) and non-legume (13- and 55-years Platycladus orientalis) plantations, including plant communities, soil physicochemical properties, and soil microbial communities, in the west of the Loess Plateau, China. The results showed higher richness and diversity, more keystone taxa and positive relationships, and larger connectivity and potential functions existed in soil bacterial and fungal communities of legume plantations. Meanwhile, richer plant communities and higher soil nutrients in legume plantations were found than those in non-legume plantations. We revealed that legume plantations shaped a more complex co-occurrence network, forming a virtuous cycling of "plant-soil-microbe" continuum in legume plantation ecosystems. Our results provided a new perspective on evaluating the ecological value and plantation stability of legume tree species in the vegetation restoration engineering of drylands.

The clinical value of long noncoding RNA GAS5 in acute ischemic stroke: Correlation with disease risk, inflammation, severity, and risk of recurrence.

BACKGROUND: Long noncoding RNA growth arrest-specific 5 (lnc-GAS5) is involved in the pathophysiology of acute ischemic stroke (AIS) by regulating vascular stenosis, inflammation, and neurocyte apoptosis. This study aimed to explore the clinical value of lnc-GAS5 in patients with AIS. METHODS: Plasma samples were collected from 120 patients with AIS at admission and 60 controls after enrollment, and lnc-GAS5 expression in the plasma of all participants was assessed by reverse transcription quantitative polymerase chain reaction. In patients with AIS, disease severity was evaluated using National Institute of Health Stroke Scale (NIHSS) score, and plasma inflammatory cytokine levels were measured by enzyme-linked immunosorbent assay. Recurrence-free survival (RFS) was calculated during a 36-month follow-up period. RESULTS: Lnc-GAS5 expression levels were higher in patients with AIS than in the controls (p < 0.001), and it had the potential to discriminate the controls from patients with AIS (area under the curve: 0.893, 95% confidence interval: 0.849-0.938). In patients with AIS, elevated lnc-GAS5 levels were positively correlated with NIHSS score (r = 0.397, p < 0.001), diabetes mellitus (p = 0.046), and higher levels of tumor necrosis factor alpha (TNF-α; r = 0.374, p < 0.001), interleukin-6 (IL-6; r = 0.223, p < 0.001), and interleukin-17A (IL-17A; r = 0.222, p = 0.015). The expression levels of lnc-GAS5 were also negatively correlated with the levels of interleukin-10 (IL-10; r = -0.350, p < 0.001) and RFS (p = 0.036). CONCLUSION: Lnc-GAS5 is correlated with higher susceptibility to AIS, inflammation, and severity, and can predict an increased risk of AIS recurrence, indicating that monitoring of lnc-GAS5 might improve the management of AIS.

Ogt controls neural stem/progenitor cell pool and adult neurogenesis through modulating Notch signaling.

Ogt catalyzed O-linked N-acetylglucosamine (O-GlcNAcylation, O-GlcNAc) plays an important function in diverse biological processes and diseases. However, the roles of Ogt in regulating neurogenesis remain largely unknown. Here, we show that Ogt deficiency or depletion in adult neural stem/progenitor cells (aNSPCs) leads to the diminishment of the aNSPC pool and aberrant neurogenesis and consequently impairs cognitive function in adult mice. RNA sequencing reveals that Ogt deficiency alters the transcription of genes relating to cell cycle, neurogenesis, and neuronal development. Mechanistic studies show that Ogt directly interacts with Notch1 and catalyzes the O-GlcNAc modification of Notch TM/ICD fragment. Decreased O-GlcNAc modification of TM/ICD increases the binding of E3 ubiquitin ligase Itch to TM/ICD and promotes its degradation. Itch knockdown rescues neurogenic defects induced by Ogt deficiency in vitro and in vivo. Our findings reveal the essential roles and mechanisms of Ogt and O-GlcNAc modification in regulating mammalian neurogenesis and cognition.

Fto-modulated lipid niche regulates adult neurogenesis through modulating adenosine metabolism.

Adult neurogenesis is regulated by diverse factors including the local environment, i.e. the neurogenic niche. However, whether the lipid in the brain regulates adult neurogenesis and related mechanisms remains largely unknown. In the present study, we found that lipid accumulates in the brain during postnatal neuronal development. Conditional knockout of Fto (cKO) in lipid not only reduced the level of lipid in the brain but also impaired the learning and memory of mice. In addition, Fto deficiency in lipid did not affect the proliferation of adult neural stem cells (aNSCs), but it did inhibit adult neurogenesis by inducing cell apoptosis. Mechanistically, specific deleting Fto in lipid altered gene expression and increased adenosine secretion of adipocytes. The treatment of adenosine promoted the apoptosis of newborn neurons. As a whole, these results reveal the important function of the lipid niche and its associated mechanism in regulating adult neurogenesis.

Direct Powering a Real Cardiac Pacemaker by Natural Energy of a Heartbeat.

Implantable medical devices are widely used for monitoring and treatment of severe diseases. In particular, an implantable cardiac pacemaker is the most effective therapeutic device for treating bradyrhythmia, however its surgical replacement is inevitable every 5-12 years due to the limited life of the built-in battery. Although several approaches of energy harvesting have been explored in this decade for powering cardiac pacemakers, the modern, commercial, and full-function pacemaker has never been powered effectively yet. Here, we report an integrated strategy for directly powering a modern and full-function cardiac pacemaker, which can pace the porcine heart in vivo by harvesting the natural energy of a heartbeat, without using any external energy storage element. The generator includes an elastic skeleton and two piezoelectric composites, which could generate a high-output current of 15 µA in vivo over state-of-the-art performance. This study makes an impressive step toward fabricating a self-powered cardiac pacemaker and resolving the power issue of implantable medical devices by piezoelectric harvesting technology.