CRISPR-based gene expression platform for precise regulation of bladder cancer.

The development of compact CRISPR systems has facilitated delivery but has concurrently reduced gene editing efficiency, thereby limiting the further utilization of CRISPR systems. Enhancing the efficiency of CRISPR systems poses a challenging task and holds significant implications for the advancement of biotechnology. In our work, we report a synthetic dual-antibody system that can stably exist in the intracellular environment, specifically inhibiting the functions of NF-κB and ß-catenin. This not only elevates the transgenic expression of the CRISPR system by suppressing the innate immune response within cells to enhance the gene editing efficiency but also demonstrates a notable tumor inhibitory effect. Based on the specific output expression regulation of CRISPR-CasΦ, we constructed a CRISPR-based gene expression platform, which includes sensor modules for detecting intracellular ß-catenin and NF-κB, as well as an SDA module to enhance overall efficiency. In vitro experiments revealed that the CRISPR-based gene expression platform exhibited superior CDK5 expression inhibition efficiency and specific cytotoxicity towards tumor cells. In vitro experiments, we found that CRISPR-based gene expression platforms can selectively kill bladder cancer cells through T cell-mediated cytotoxicity. Our design holds significant assistant potential of transgene therapy and may offer the capability to treat other diseases requiring transgene therapy.

Physcion Protects Against Ethanol-Induced Liver Injury by Reprogramming of Circadian Clock.

The circadian clock plays a key role in our daily physiology and metabolism. Alcohol consumption disrupts the circadian rhythm of metabolic genes in the liver; however, the potential contribution of circadian clock modulation to alcoholic liver disease (ALD) is unknown. We identified a novel liver protective agent, physcion, which can alleviate fat accumulation and inflammation in ALD mice via reprogramming the hepatic circadian clock. The model of alcoholic hepatitis was established by intragastrically administering ethanol. In vitro, physcion was investigated by treating HepG2 cells with ethanol. The role of circadian clock in Physcion caused liver protection was tested by knocking down the core circadian gene Bmal1. Physcion application caused reduced lipogenesis and alleviated inflammation in alcohol-induced mice. In alcoholic hepatosteatosis models, physcion upregulated the core circadian genes. And the circadian misalignment triggered by ethanol was efficiently reversed by physcion. Physcion attenuated lipogenesis via reprogramming the circadian clock in HepG2 cells. Suppression of Bmal1 by RNA interference abolished the protective of physcion. In addition, Physcion binds to the active pocket of BMAL1 and promotes its expression. The study identified the novel liver protective effects of physcion on alcohol-induced liver injury, and modulation of the core circadian clock regulators contributes to ALD alleviation. More importantly, strategies targeting the circadian machinery, for example, Bmal1, may prove to be beneficial treatment options for this condition.

Rotating magnetic field ameliorates experimental autoimmune encephalomyelitis by promoting T cell peripheral accumulation and regulating the balance of Treg and Th1/Th17.

Multiple sclerosis (MS) is an autoimmune disease characterized by T cell infiltration and demyelination of the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) is a classical preclinical animal model of MS. In this study, we found that rotating magnetic field (RMF) treatment exerts potential preventive effects on the discovery of EAE, including reducing the severity of the disease and delaying the onset of the disease. The results indicated that RMF (0.2 T, 4 Hz) treatment increases the accumulation of CD4+ cells in the spleen and lymph nodes by downregulating the expression of CCL-2, CCL-3 and CCL-5, but has no significant effect on myelin oligodendrocyte glycoprotein (MOG) specific T cell responses. Simultaneously, RMF treatment adjusted the imbalance between regulatory T (Treg) cell and T helper 1 (Th1) cells or T helper 17 (Th17) cells by increasing the proportion of Treg cells and inhibiting the ratio of Th1 and Th17 cell subsets. These findings suggest that exposure to RMF may improve EAE disease by promoting CD4+ cell accumulation into peripheral lymphoid tissue, improving the imbalance between Treg and Th1/Th17 cells. Therefore, as a mild physical therapy approach, RMF, is likely to be a potential way to alter the development of EAE.

Synthetic and immunological studies on the OCT4 immunodominant motif antigen-based anti-cancer vaccine.

Objective: Cancer stem cell is one of the important causes of tumorigenesis as well as a drug target in the treatment of malignant tumor. However, at present, there is no immune vaccine targeting these cells. Octamer-binding transcription factor 4 (OCT4), a marker of embryonic stem cells and germ cells, often highly expresses in the early stages of tumorigenesis and is therefore a good candidate for cancer vaccine development. Methods: To identify the optimal carrier and adjuvant combination, we chemically synthesized and linked three different OCT4 epitope antigens to a carrier protein, keyhole limpet hemocyanin (KLH), combined with Toll-like receptor 9 agonist (TLR9). Results: Immunization with OCT4-3 + TLR9 produced the strongest immune response in mice. In prevention assays, significant tumor growth inhibition was achieved in BABL/c mice treated with OCT4-3 + TLR9 (P < 0.01). Importantly, the results showed that cytotoxic T lymphocyte activity and the inhibition of tumor growth were enhanced in mice immunized with OCT4-3 combined with TLR9. Meanwhile, multiple cytokines [such as interferon (IFN)-γ (P < 0.05), interleukin (IL)-12 (P < 0.05), IL-2 (P < 0.01), and IL-6 (P < 0.05)] promoting cellular immune responses were shown to be greatly enhanced in mice immunized with OCT4-3 + TLR9. Moreover, we considered safety considerations in terms of the composition of the vaccines to help facilitate the development of effective next-generation vaccines. Conclusions: Collectively, these experiments demonstrated that combination therapy with TLR9 agonist induced a tumor-specific adaptive immune response, leading to the suppression of primary tumor growth in testis embryonic carcinoma.

Rotating magnetic field delays human umbilical vein endothelial cell aging and prolongs the lifespan of Caenorhabditis elegans.

The biological effects of magnetic fields are a research hotspot in the field of biomedical engineering. In this study, we further investigated the effects of a rotating magnetic field (RMF; 0.2 T, 4 Hz) on the growth of human umbilical vein endothelial cells (HUVECs) and Caenorhabditis elegans. The results showed that RMF exposure prolonged the lifespan of C. elegans and slowed the aging of HUVECs. RMF treatment of HUVECs showed that activation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) was associated with decreased mitochondrial membrane potential (MMP) due to increased intracellular Ca2+ concentrations induced by endoplasmic reticulum stress in anti-aging mechanisms. RMF also promoted the health status of C. elegans by improving activity, reducing age-related pigment accumulation, delaying Aß-induced paralysis and increasing resistance to heat and oxidative stress. The prolonged lifespan of C. elegans was associated with decreased levels of daf-16 which related to the insulin/insulin-like growth factor signaling pathway (IIS) activity and reactive oxygen species (ROS), whereas the heat shock transcription factor-1 (hsf-1) pathway was not involved. Moreover, the level of autophagy was increased after RMF treatment. These findings expand our understanding of the potential mechanisms by which RMF treatment prolongs lifespan.

Inhibition of NLRP3 inflammasome-mediated pyroptosis in macrophage by cycloastragenol contributes to amelioration of imiquimod-induced psoriasis-like skin inflammation in mice.

Psoriasis is a common chronic inflammatory skin disease, and the infiltrated macrophages in psoriatic skin lesions play a key role in the progression of this uncontrolled cutaneous inflammation. However, the current therapeutic strategies for patients with psoriasis are not satisfactory. Here, we report that cycloastragenol (CAG), a natural active small compound isolated from Astragalus membranaceus, significantly ameliorated imiquimod (IMQ)-induced psoriasiform dermatitis in mice by targeting proinflammatory macrophages. CAG significantly reduced the clinical scores, decreased the epidermal thickness, and ameliorated the deteriorating histopathology observed in IMQ-induced mice. CAG treatment specifically reduced the dermal infiltration of macrophages, rather than of dendritic cells, neutrophils, or T lymphocytes, into psoriatic skin. CAG dose-dependently decreased the level of proinflammatory cytokines, including IL-1ß, TNF-α and IL-6, in murine psoriatic skin and serum, as well as in IMQ-stimulated, bone-marrow-derived macrophages. When compared to the control group, CAG significantly decreased IMQ-triggered NLRP3 inflammasome activation and gasdermin D-mediated cell pyroptosis in these proinflammatory macrophages. CAG also suppressed the assembly of the NLRP3 inflammasome complex. Taken together, the results show that CAG selectively modulates macrophage function by inhibiting NLRP3 inflammasome-mediated pyroptosis to ameliorate IMQ-induced psoriasis-like skin inflammation in mice. Our findings also identify an effective drug candidate for the treatment of psoriasis.