Spatially Precise Genetic Engineering at the Electrode-Tissue Interface.

The interface between electrodes and neural tissues plays a pivotal role in determining the efficacy and fidelity of neural activity recording and modulation. While considerable efforts have been made to improve the electrode-tissue interface, the majority of studies have primarily concentrated on the development of biocompatible neural electrodes through abiotic materials and structural engineering. In this study, an approach is presented that seamlessly integrates abiotic and biotic engineering principles into the electrode-tissue interface. Specifically, ultraflexible neural electrodes with short hairpin RNAs (shRNAs) designed to silence the expression of endogenous genes within neural tissues are combined. The system facilitates shRNA-mediated knockdown of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and polypyrimidine tract-binding protein 1 (PTBP1), two essential genes associated in neural survival/growth and neurogenesis, within specific cell populations located at the electrode-tissue interface. Additionally, it is demonstrated that the downregulation of PTEN in neurons can result in an enlargement of neuronal cell bodies at the electrode-tissue interface. Furthermore, the system enables long-term monitoring of neuronal activities following PTEN knockdown in a mouse model of Parkinson's disease and traumatic brain injury. The system provides a versatile approach for genetically engineering the electrode-tissue interface with unparalleled precision, paving the way for the development of regenerative electronics and next-generation brain-machine interfaces.

Selenium maintains intestinal epithelial cells to activate M2 macrophages against deoxynivalenol injury.

Selenium (Se) is indispensable in alleviating various types of intestinal injuries. Here, we thoroughly investigated the protective effect of Se on the regulation of the epithelial cell-M2 macrophages pathway in deoxynivalenol (DON)-induced intestinal damage. In the present study, Se has positive impacts on gut health by improving gut barrier function and reducing the levels of serum DON in vivo. Furthermore, our study revealed that Se supplementation increased the abundances of GPX4, p-PI3K, and AKT, decreased the levels of 4-HNE and inhibited ferroptosis. Moreover, when mice were treated with DON and Fer-1(ferroptosis inhibitor), ferroptosis was suppressed and PI3K/AKT pathway was activated. These results indicated that GPX4-PI3K/AKT-ferroptosis was a predominant pathway in DON-induced intestinal inflammation. Interestingly, we discovered that both the number of M2 anti-inflammatory macrophages and the levels of CSF-1 decreased while the pro-inflammatory cytokine IL-6 increased in the intestine and MODE-K cells supernatant. Therefore, Se supplementation activated the CSF-1-M2 macrophages axis, resulting in a decrease in IL-6 expression and an enhancement of the intestinal anti-inflammatory capacity. This study provides novel insights into how intestinal epithelial cells regulate the CSF-1-M2 macrophage pathway, which is essential in maintaining intestinal homeostasis confer to environmental hazardous stimuli.

Self-assembled ultraflexible probes for long-term neural recordings and neuromodulation.

Ultraflexible microelectrode arrays (MEAs) that can stably record from a large number of neurons after their chronic implantation offer opportunities for understanding neural circuit mechanisms and developing next-generation brain-computer interfaces. The implementation of ultraflexible MEAs requires their reliable implantation into deep brain tissues in a minimally invasive manner, as well as their precise integration with optogenetic tools to enable the simultaneous recording of neural activity and neuromodulation. Here, we describe the process for the preparation of elastocapillary self-assembled ultraflexible MEAs, their use in combination with adeno-associated virus vectors carrying opsin genes and promoters to form an optrode probe and their in vivo experimental use in the brains of rodents, enabling electrophysiological recordings and optical modulation of neuronal activity over long periods of time (on the order of weeks to months). The procedures, including device fabrication, probe assembly and implantation, can be completed within 3 weeks. The protocol is intended to facilitate the applications of ultraflexible MEAs for long-term neuronal activity recording and combined electrophysiology and optogenetics. The protocol requires users with expertise in clean room facilities for the fabrication of ultraflexible MEAs.

Regulation of the MyD88 gene in chicken spleen inflammation induced by stress.

In order to investigate the regulatory role of the myeloid differentiation factor 88 (MyD88) gene in the stress inflammatory response to chicken spleen, the chicken stress model and macrophage (HD11) inflammation model were constructed in this study. Enzyme-linked immunosorbent assay and quantitative real-time PCR were used to investigate the effects of MyD88 on immune and inflammatory indicators. The results demonstrated that the levels of IgG, CD3+ and CD4+ in the serum of chickens in the beak trimming stress and heat stress groups decreased significantly compared to the control group without stress (P < 0.05), and the inflammation-related indices IL-1ß, TNF-α, IL-6 and NF-κB increased significantly (P < 0.05). Stress up-regulated the expression levels of MyD88, IL-1ß, NF-κB and TLR4 in the spleen, stimulated the release of inflammatory factors. Overexpression of MyD88 significantly up-regulated the expression levels of the inflammatory factors IL-1ß, TNF-α, IL-8, NF-κB and TLR4 in HD11 cells (P < 0.05). Co-treatment with lipopolysaccharide (LPS) further promoted the expression levels of the inflammatory cytokines in HD11 cells. Interference with the expression of MyD88 significantly reduced the expression level of inflammatory factors in HD11 cells (P < 0.05) and had an antagonistic effect with LPS to alleviate the inflammatory reaction. In conclusion, the MyD88 gene has a pro-inflammatory effect and is highly expressed in the beak trimming and heat stress models in chicks, regulating the inflammatory response in poultry. It was involved in regulating the expression of immune-related genes in HD11 cells and had a synergistic effect with LPS.

In this study, we constructed two chick stress models and a chicken macrophage (HD11) inflammation model to verify the potential mechanism of the myeloid differentiation factor 88 (MyD88) gene regulation of inflammatory response in poultry for the first time through in vivo and in vitro dual model tests. The results of this study preliminarily suggest that the MyD88 gene may be a reliable indicator of an inflammatory state in poultry and a key target for regulating the poultry inflammatory response.

Effect of HSPA8 gene on the proliferation, apoptosis and immune function of HD11 cells.

HSPA8 (Heat shock 70 kDa protein 8) is a molecular chaperone involved in a variety of cellular processes. This gene may affect the proliferation, apoptosis and immune function of chicken macrophages, but the specific mechanism remains unclear. The purpose of this study was to explore the effect of the HSPA8 gene on the proliferation, apoptosis and immune function of chicken macrophages. In this study, a chicken HSPA8 overexpression plasmid, interference fragment and corresponding controls were transfected into HD11 cells, and then the expression of the HSPA8 gene, cell proliferation, cell cycle, apoptosis rate and immune function of each group were detected. The results showed that transfection of the HSPA8 overexpression plasmid significantly upregulated the level of HSPA8 expression in HD11 cells compared with the control; significantly promoted the proliferation of HD11 cells and the expression of PCNA, CCND1 and CCNB3; decreased the number of cells in the G1 phase and increased the number of cells in the S phase; decreased the rate of apoptosis and upregulated the expression of Bcl-2; and promoted the expression of the LPS-induced cytokines IL-1ß, IL-6 and TNF-α. Transfection of the HSPA8 interference fragment significantly downregulated the level of HSPA8 expression in HD11 cells; significantly inhibited the proliferation of HD11 cells and the expression of PCNA, CCND1 and CDK1; increased the number of cells in the G1 phase and decreased the number of cells in the S phase; increased the rate of apoptosis, downregulated the expression of Bcl-2 and upregulated the expression levels of Fas and FasL; and inhibited the expression of the LPS-induced cytokines IL-1ß and NF-κB. The results suggested that HSPA8 promotes the proliferation of and inhibits the apoptosis of HD11 cells and has a proinflammatory effect.

PEG-mediated transduction of rAAV as a platform for spatially confined and efficient gene delivery.

BACKGROUND: Recombinant adeno-associated viruses (rAAV) are commonly used vectors for gene delivery in both basic neuroscience and clinical applications due to their nonpathogenic, minimally immunogenic, and sustained expression properties. However, several challenges remain for the wide-scale rAAV applications, including poor infection of many clinically important cell lines, insufficient expression at low titers, and diffusive transduction in vivo. METHODS: In this work, PEG, which is a safe and non-toxic polymer of ethylene oxide monomer, was applied as an auxiliary transduction agent to improve the expression of rAAV. In detail, a small dose of PEG was added into the rAAV solution for the transgene expression in cell lines in vitro, and in the central nervous system (CNS) in vivo. The biocompatibility of PEG enhancer was assessed by characterizing the immune responses, cell morphology, cell tropism of rAAV, neuronal apoptosis, as well as motor function of animals. RESULTS: The results show that small dose of PEG additive can effectively improve the gene expression characteristics of rAAV both in vitro and in vivo. Specifically, the PEG additive allows efficient transgene expression in cell lines that are difficult to be transfected with rAAV alone. In vivo studies show that the PEG additive can promote a spatially confined and efficient transgene expression of low-titer rAAV in the brain over long terms. In addition, no obvious side effects of PEG were observed on CNS in the biocompatibility studies. CONCLUSIONS: This spatially confined and efficient transduction method can facilitate the applications of rAAV in fundamental research, especially in the precise dissection of neural circuits, and also improve the capabilities of rAAV in the treatment of neurological diseases which originate from the disorders of small nuclei in the brain.

Recurrence characteristics and clinicopathological results of borderline ovarian tumors.

BACKGROUND: This study aimed to investigate the clinical and pathological characteristics, and the recurrence and prognostic factors of borderline ovarian tumors (BOTs). METHODS: The data of 286 patients admitted to hospital and followed up for more than ten months were analyzed retrospectively to study the clinicopathological characteristics and related factors of recurrence. RESULTS: The median age of the patients was 42.06 ± 14.97 years, and the duration of the follow-up ranged from 10-109 months. During the follow-up period, 40 patients had a recurrence. Of these patients, 36 were ≤ 40 years, and patients with premenopausal recurrence accounted for 20.5% (36/176). In patients undergoing conservative treatment or radical operations, the recurrence rates were 21.3% and 1.8%, respectively, and they were 13.4% (36/268) in patients at Federation International of Gynecology and Obstetrics (FIGO) stage I, and 22.2% (4/18) in patients at an advanced stage. Postoperative pathology revealed that 40 patients had micropapillary tumors, among whom ten patients (25%) had a recurrence, and 19 patients had complications with interstitial infiltration. Of these 19 patients, six had a recurrence (31.5%). Another 22 patients had complications with calcified sand bodies; among these, eight patients (36.4%) had a recurrence. All the differences were statistically significant (P < 0.05). There were four cancer-related deaths during the follow-up period. Late FIGO stage, conservative operation, and a high level of carbohydrate antigen 125 (CA125) were independent risk factors for the recurrence of BOTs. CONCLUSION: BOTs usually occur in women under 40 years, have an occult onset, and half of the patients have no obvious clinical manifestations. Serum CA125 level can be used as a tumor marker to detect BOTs and the risk of its recurrence. Operation mode and FIGO stage are important independent factors for the recurrence of BOTs.

Theanine from tea and its semi-synthetic derivative TBrC suppress human cervical cancer growth and migration by inhibiting EGFR/Met-Akt/NF-κB signaling.

Cervical cancer is the third most prevalent cancer among women worldwide. Theanine from tea and its derivatives show some anticancer activities. However, the role of theanine and its derivatives against human cervical cancer and the molecular mechanisms of action remain unclear. Thus, in this study, we aim to investigate the anticancer activities and underlying mechanisms of theanine and a theanine derivative, ethyl 6-bromocoumarin-3- carboxylyl L-theanine (TBrC), against human cervical cancer. In vitro and in vivo assays for cancer cell growth and migration have confirmed the inhibition of the cell growth and migration by TBrC and theanine in highly-metastatic human cervical cancer. TBrC displays much stronger activity than theanine on inhibition of the cell growth and migration as well as induction of apoptosis and regulation of related protein expressions in the human cervical cancer cells. TBrC and theanine greatly reduced endogenous and exogenous factors-stimulated cell migration and completely repressed HGF- and EGF+HGF-activated EGFR/Met-Akt/NF-κB signaling by reducing the phosphorylation and expressions of EGFR, Met, Akt, and NF-κB in cervical cancer cells. The enhancer of zeste homolog 2 (EZH2) knockdown decreased the cancer cell migration and NF-κB expression. The NF-κB knockdown reduced the cancer cell migration. TBrC and theanine reduced the EZH2 expression by more than 80%. In addition, TBrC and theanine significantly suppressed human cervical tumor growth in tumor-bearing nude mice without toxicity to the mice. Our results suggest that TBrC and theanine may have the potentials of the therapeutic and/or adjuvant therapeutic application in the treatment of human cervical cancer.

Repression of Human Hepatocellular Carcinoma Growth by Regulating Met/EGFR/VEGFR-Akt/NF-κB Pathways with Theanine and Its Derivative, (R)-2-(6,8-Dibromo-2-oxo-2H-chromene-3-carboxamido)-5-(ethylamino)-5-oxopentanoic Ethyl Ester (DTBrC).

To explore the potential of theanine against cancer, we have studied the anticancer activities of theanine from tea and its semisynthesized derivative, (R)-2-(6,8-dibromo-2-oxo-2H-chromene-3-carboxamido)-5-(ethylamino)-5-oxopentanoic ethyl ester (DTBrC), in in vitro, ex vivo, and in vivo models of human hepatocellular carcinoma (HHC). Theanine and DTBrC displayed inhibitory effects on the growth and migration of HHC cells in vitro, ex vivo, and in vivo. Theanine and DTBrC significantly enhanced the repression of HHC cell growth in combination with anticancer drug pirarubicin. Theanine and DTBrC completely suppressed HGF- and EGF+HGF-induced migration with a reduction of p53 tumor suppressor level and enhanced the p53 protein expression in HHC cells. The Akt and NF-κB knockdown greatly reduced cancer cell migration with a decrease in CD44 expression. DTBrC and theanine significantly repressed the protein expressions in the Met/EGFR/VEGFR-Akt/NF-κB pathways, which might be the mechanism for their biologic effects.

Hyaluronic acid and polyethylenimine self-assembled polyion complexes as pH-sensitive drug carrier for cancer therapy.

In this study, a series of polyion complexes (PICs) were prepared via electrostatic interaction between hyaluronic acid-histidine conjugate (HH) and polyethylenimine-histidine conjugate (PH). These PICs with the average size ranging from 410.5 nm to 98.5 nm at different weight ratios of HH/PH were able to encapsulate doxorubicin (DOX) as the model antitumor drug. The PICs at the weight ratio of 4:1 had negative surface charge and were of good dispersity and stability in the solution containing serum. In vitro drug release assay demonstrated that the DOX release rate were higher at acidic pH showing a controllable property. These HA coated PICs could targetedly deliver DOX to B16F10 tumor cells, showing improved antitumor activity.

Inhibition of lung tumor growth by targeting EGFR/VEGFR-Akt/NF-κB pathways with novel theanine derivatives.

The molecularly targeted agents, including anti-VEGF or anti-EGFR monoclonal antibody and some inhibitors of EGFR tyrosine kinase, are effective in the treatment of non-small-cell lung cancer (NSCLC) to a certain extent, but the benefit for a proportion of patients is still limited. Hence, it is necessary and urgent to develop more selective and effective molecular targeted agents against lung cancer. Here, we have synthesized novel theanine derivatives, methyl coumarin-3-carboxylyl L-theanine (TMC), ethyl coumarin-3-carboxylyl L-theanine (TEC), ethyl 6-fluorocoumarin- 3-carboxylyl L-theanine (TFC), and ethyl 6-nitrocoumarin-3-carboxylyl L-theanine (TNC), which are fluorescent small molecules, based on their parental compound theanine and studied their anticancer activities in vitro, ex vivo and in vivo models of human and mouse cancers. Our results show that the four theanine derivatives significantly inhibit the lung cancer cell migration and the growth of lung cancer and leukemia cell lines. TFC and TNC display enhanced effects with anticancer drugs cytarabine vincristine, andmethotrexate on inhibition of lung cancer cell growth and no toxicity to the normal human embryonic lung fibroblast and peripheral blood lymphocytes. TFC and TNC exhibit strong suppression of the highly metastatic Lewis lung cancer (LLC) and A549 tumor growth in tumor-bearing mice without toxicity to mice. TFC and TNC can effectively suppress the growth of lung cancer cells in vitro, ex vivo and in vivo by targeting EGFR/VEGFR-Akt/NF-κB pathways. Our study has suggested that TFC and TNC may have the therapeutic and/or adjuvant therapeutic applications in the treatment of lung cancers and other cancer.