[Electroacupuncture at "Zusanli"(ST36) ameliorates tau hyperphosphorylation in pancreas and hippocampus of diabetic rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Zusanli"(ST36) on the phosphorylated tau levels in pancreas and hippocampus of type 2 diabetes mellitus (T2DM) rats，so as to explore the underlying mechanism of EA in diabetic demention rats. METHODS: Forty-eight male Sprague-Dawley rats were randomly divided into control, model and EA groups, with 16 rats in each group. The T2DM model was established by 6 weeks of high-fat, high-sugar diet as well as intrape-ritoneal injection of streptozocin (STZ) solution (35 mg/kg). After that, EA (2 Hz, 0.1 mA) was applied to unilateral "Zusanli"(ST36) for 30 min, once a day, 6 times a week for 4 weeks. The survival rate was recorded every week, and the fasting blood glucose (FBG) was detected on the 1st, 6th and 11th week. The level of serum insulin (INS) was measured by using ELISA. The morphological structure of pancreas islet was observed by H.E. staining. The expressions of phosphorylated tau at the sites of Ser 396 (pS396) and Thr 231 (pT231), total tau (Tau5), phosphorylated glycogen synthase kinase-3ß (pGSK-3ß) and total glycogen synthase kinase-3ß (GSK-3ß) in pancreas and hippocampus were detected by Western blot. The expression and distribution of pS396 and pT231 in pancreas and hippocampus were assayed with immunohistochemistry. RESULTS: Compared with the control group, the survival rate presented a significant decline, the contents of FBG and INS were obviously higher(P<0.01), and the structure of the pancreas islet appeared shrunken, obscure and disordered in the model group. Furthermore, the levels of pS396, pT231 in pancreas and hippocampus were obviously higher in the model group(P<0.01)，while the level of pGSK-3ß in pancreas and hippocampus was significantly lower in the model group(P<0.01). In comparison with the model group, the survival rate of EA group was higher. Following 4 weeks' interventions, the enhanced levels of tau phosphorylation and GSK-3ß activity in pancreas and hippocampus were partly reversed in the EA group compared to the model group(P<0.05，P<0.01). CONCLUSION: EA at ST36 can reduce the level of tau phosphorylation via regulating the activity of GSK-3ß in the pancreas and hippocampus of T2DM rats, which may be related with the effect of EA on the brain function in T2DM rats.

A pathogen-like antigen-based vaccine confers immune protection against SARS-CoV-2 in non-human primates.

Activation of nucleic acid sensing Toll-like receptors (TLRs) in B cells is involved in antiviral responses by promoting B cell activation and germinal center responses. In order to take advantage of this natural pathway for vaccine development, synthetic pathogen-like antigens (PLAs) constructed of multivalent antigens with encapsulated TLR ligands can be used to activate B cell antigen receptors and TLRs in a synergistic manner. Here we report a PLA-based coronavirus disease 2019 (COVID-19) vaccine candidate designed by combining a phage-derived virus-like particle carrying bacterial RNA as TLR ligands with the receptor-binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein as the target antigen. This PLA-based vaccine candidate induces robust neutralizing antibodies in both mice and non-human primates (NHPs). Using a NHP infection model, we demonstrate that the viral clearance is accelerated in vaccinated animals. In addition, the PLA-based vaccine induces a T helper 1 (Th1)-oriented response and a durable memory, supporting its potential for further clinical development.

MBNL1 Suppressed Cancer Metastatic of Skin Squamous Cell Carcinoma Via by TIAL1/MYOD1/Caspase-9/3 Signaling Pathways.

OBJECTIVE: The incidence of skin squamous cell carcinoma (SSCC) has recently been increasing, with diverse clinical manifestations.SSCC could metastasize to lymph nodes or other organs, posing a great threat to life. The present study was designed to investigate the function and underlying mechanism of muscleblind-like protein 1 (MBNL1) in skin squamous cell carcinoma. METHODS: SCL-1 cell was used for vitro model and transfected with MBNL1 or siMBNL1 plasmids. MTT Assays, LDH activity ELISA, and Transwell chamber migration experiment were used to confirm the effects of MBNL1 on cell growth of SCL-1 cell. Western blot analysis was used to analyze the mechanism of MBNL1 in SCL-1 cell. RESULTS: Down-regulation of MBNL1 promoted cell metastasis of SSCC, while up-regulation of MBNL1 reduced cell metastasis of SSCC in vitro. Down-regulation of MBNL1 suppressed the protein expression of T cell intracellular antigen (TIAL1), myogenic determinant 1 (MyoD1) and Caspase-3 in vitro. Consistent with these observations, inhibition of TIAL1 or MYOD1 expression attenuated the effects of MBNL1 in SSCC. CONCLUSION: The present study revealed that MBNL1 suppressed thecancer metastatic capacity of SSCC via by TIAL1/MYOD1/Caspase-3 signaling pathways.

Multicentre clinicopathological study of adenoid cystic carcinoma: A report of 296 cases.

AIMS: Adenoid cystic carcinoma (ACC) is a distinctive tumour. Limited studies involving a large population have reported multicentre systematic analyses of the clinical, pathological and immunohistochemical (IHC) features of ACC as well as the potential role of IHC markers in the prognosis of ACC. METHODS AND RESULTS: The clinical, histopathological and IHC data of 296 cases obtained from two tertiary hospitals were analysed. The age at onset ranged from 12 to 87 years with a median age of 52 years. The male-to-female ratio was 1:1.3. Patients with ACC arising from the lacrimal gland were younger than those with tumours arising from other sites. Patients with tumours in the extra auditory canal and nasopharynx were older than those with tumours in other locations. Histopathologically, solid type ACC was the most frequent in the nasal cavity and paranasal sinus (6/51) group. Tumours arising from the oral cavity most commonly showed perineural invasion (10/60) and margin positivity (11/60). IHC analyses showed that CK8/18, CK7, CK14, epithelial membrane antigen and CD117 were expressed in 35/35 (100%), 87/88 (98.8%), 26/27 (96.2%), 42/43 (97.6%) and 113/120 (94.1%) patients, respectively. CK5/6, P63, smooth muscle actin, calponin and S100 were positively expressed in 73/73 (100%), 111/124 (89.5%), 38/43 (88.3%), 41/50 (82.0%) and 61/92 (66.3%) cases, respectively. S100 proteins were expressed in 54 (54/77) primary cases and two (2/9) metastatic cases (p = 0.013). CONCLUSIONS: ACC is a distinctive tumour that mainly affects middle-aged and elderly individuals, with a mild female predominance. Loss of expression of S100 proteins may be a poor prognostic factor associated with metastasis.

Methylwogonin exerts anticancer effects in A375 human malignant melanoma cells through apoptosis induction, DNA damage, cell invasion inhibition and downregulation of the mTOR/PI3K/Akt signalling pathway.

INTRODUCTION: The main purpose of the present research was to study the anticancer effects of methylwogonin in A375 human malignant melanoma cells by evaluating its effects on apoptosis, DNA fragmentation, cancer cell invasion and the mTOR/PI3K/AKT signalling pathway. MATERIAL AND METHODS: Effects on cell cytotoxicity were evaluated by MTT assay while a clonogenic assay determined the effects of methylwogonin on colony formation. Fluorescence microscopy evaluated apoptotic effects of methylwogonin in these cells using acridine orange/propidium iodide and Hoechst 33342 staining dyes. Gel electrophoresis evaluated the effects of methylwogonin on DNA fragmentation while the Matrigel invasion assay evaluated the effects of the drug on cancer cell invasion. Effects of methylwogonin on the mTOR/PI3K/AKT signalling pathway were evaluated by western blot assay. RESULTS: Methylwogonin induces concentration-dependent as well as time-dependent growth inhibitory effects inducing significant cytotoxicity in these cancer cells. Methylwogonin led to dose-dependent inhibition of colony formation in A375 human malignant melanoma cells. The number of cell colonies decreased significantly as the methylwogonin dose increased from 0, 50, 150, to 300 µM. Methylwogonin treatment of cells at lower doses led to yellow fluorescence (early apoptosis), which changed to red/orange fluorescence, indicating late apoptosis at higher doses. Similar results were obtained using Hoechst 33342 staining, revealing that 50, 150 and 300 µM doses of methylwogonin led to significant morphological changes including chromatin condensation, fragmented nuclei and cellular shrinkage. DNA ladder formation was also observed, and this effect increased with increasing doses of methylwogonin. Methylwogonin also inhibited cancer cell invasion in a dose-dependent manner. CONCLUSIONS: Different doses of methylwogonin led to concentration-dependent downregulation of phosphorylated PI3K, AKT and mTOR.

B Cell-Intrinsic MyD88 Signaling Promotes Initial Cell Proliferation and Differentiation To Enhance the Germinal Center Response to a Virus-like Particle.

Although TLR signaling in B cells has been implicated in the germinal center (GC) responses during viral infections and autoimmune diseases, the underlying mechanism is unclear. Bacterial phage Qß-derived virus-like particle (Qß-VLP) contains TLR ligands, which can enhance Qß-VLP-induced Ab response, including GC response, through TLR/MyD88 signaling in B cells. In this study, by examining Ag-specific B cell response to Qß-VLP, we found that lack of B cell MyD88 from the beginning of the immune response led to a more severe defect in the GC scale than abolishing MyD88 at later time points of the immune response. Consistently, B cell-intrinsic MyD88 signaling significantly enhanced the initial proliferation of Ag-specific B cells, which was accompanied with a dramatic increase of plasma cell generation and induction of Bcl-6+ GC B cell precursors. In addition, B cell-intrinsic MyD88 signaling promoted strong T-bet expression independent of IFN-γ and led to the preferential isotype switching to IgG2a/c. Thus, by promoting the initial Ag-specific B cell proliferation and differentiation, B cell-intrinsic MyD88 signaling enhanced both T-independent and T-dependent Ab responses elicited by Qß-VLP. This finding will provide additional insight into the role of TLR signaling in antiviral immunity, autoimmune diseases, and vaccine design.

Detection of MAPK signal transduction proteins in an ischemia/reperfusion model of mouse intestine using in vivo cryotechnique.

Intestinal ischemia and ischemia-reperfusion rapidly progress to tissue destruction and reconstruction of functional organs. To date, precise immunolocalizations and the timing of appearance of cell signaling components under such conditions have not been well visualized. Mitogen-activated protein kinase (MAPK) signal transduction pathways have been reported to be activated under various types of cell damage, and cyclic AMP response element-binding protein (CREB) was directly phosphorylated with various cellular stimuli. In this study, both the expression and the immunolocalization of ERK1/2, a member of the MAPK family, were examined in mouse intestinal tissues by in vivo cryotechnique, which is useful to retain soluble molecules including cell signaling molecules. Under normal conditions, although ERK was widely immunolocalized in the cytoplasm of epithelial cells, phosphorylated (p) ERK1/2 was slightly detected in a small amount of epithelial cells in crypt and top parts of the villi. In 5 min ischemia, more pERK1/2 immunolocalization was detected in epithelial cells of the crypt part. Up to 60 min, the pERK1/2 immunoreactivity was remarkably increased in wide areas of epithelial cells. In the 20 and 60 min ischemia groups, phosphorylated CREB was also immunostained in the nuclei of the same epithelial cell areas of pERK1/2. In 20 min ischemia with 60 min reperfusion experiments, pERK1/2 immunointensity was reduced in the crypt areas. In 60 min ischemia with 60 min reperfusion, however, it was still strongly immunolocalized in epithelial cells of the crypts. Thus, rapidly changing ERK1/2 phosphorylation was visualized in the intestinal epithelial stem cells of mouse small intestine.

Immunohistochemical detection of angiotensin II receptors in mouse cerebellum and adrenal gland using "in vivo cryotechnique".

Angiotensin II (AT) receptors, including AT receptor type 1 (AT1R) and type 2 (AT2R), are expressed in the rodent central nervous system, but their distributions and activation states are still unclear. In this study, we have performed immunohistochemical analyses of AT receptors in mouse cerebellum and adrenal gland using our "in vivo cryotechnique" (IVCT). We used antibodies against amino-terminal domains of AT receptors, which are considered to undergo conformational changes upon the binding of AT. Immunoreactivity of AT1R was detected in mouse cerebellum, and was highest in the outer tissue areas of molecular layers using IVCT. The AT1R immunostaining largely overlapped with glial fibrillary acidic protein (GFAP), a marker of Bergmann glia. Surprisingly, the AT1R immunoreactivity in the cerebellar cortex was remarkably reduced following 5 and 10 min of hypoxia or direct administration of an AT1R antagonist, losartan. By contrast, in the adrenal cortex, such AT1R immunoreactivity detected at the zona glomerulosa did not change even after 15 min of hypoxia. The correlation of localization with GFAP and also hypoxia-induced decrease of its immunoreactivity were similarly observed by immunostaining of AT2R in the cerebellar specimens. These findings demonstrated that IVCT is useful to reveal dynamically changing immunoreactivities usually affected by receptor-ligand binding as well as hypoxia, and also suggested that functional activities of AT receptors are time-dependently modulated under hypoxia in the central nervous system in comparison with the adrenal glands.

Immunolocalization of membrane skeletal protein, 4.1G, in enteric glial cells in the mouse large intestine.

4.1 family proteins are membrane skeletal proteins that interact with spectrin-actin networks and intramembraneous proteins. We reported that one of them, 4.1G, was immunolocalized in myelinated nerve fibers of the mouse peripheral nervous system, especially along cell membranes of paranodes and Schmidt-Lanterman incisures in Schwann cells. In this study, to examine 4.1G's appearance in unmyelinated peripheral nerve fibers, we focused on the enteric nervous system in mouse large intestines. In intestinal tissues prepared by an "in vivo cryotechnique" followed by freeze-substitution fixation, 4.1G was immunolocalized in Auerbach's myenteric plexus and connecting nerve fiber networks. Its immunostaining was mostly colocalized with glial fibrillar acidic protein, a marker of enteric glial cells, but not with c-Kit, a marker of interstitial cells of Cajal. Using whole-mount preparation after splitting inner and outer muscle layers, the nerve fiber networks including the plexus were clearly detected by the 4.1G immunostaining. By conventional pre-embedding immunoelectron microscopy, 4.1G was detected along cell membranes of enteric glial cells and their processes surrounding axons. These indicate that 4.1G may have some roles in adhesion and/or signal transduction in unmylinated PNS nerve fibers.