Double Fluorescent Staining Research Articles

Circ-HOMER1 enhances the inhibition of miR-1322 on CXCL6 to regulate the growth and aggressiveness of hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a common liver malignancy worldwide accompanying with the high rate of recurrence. Accumulating reports have documented the significance of circular RNAs (circRNAs) in carcinogenesis and development of HCC. This study aimed to establish the mechanism underlying circ-HOMER1 involvement in HCC. To this end, we identified a binding site for miR-1322 via bioinformatics, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and dual-luciferase reporter assays providing evidence of a direct link between circ-HOMER1 and miR-1322. Similarly, the target gene of miR-1322 was investigated. Moreover, we determined the specific function of circ-HOMER1 in HCC with the aid of qRT-PCR based on patient clinical records, Cell Counting Kit-8, acridine orange/ethidium bromide double fluorescence staining, flow cytometry, and wound-healing and transwell assays. Notably, circ-HOMER1 was upregulated in both HCC cells and tissues. This aberrant expression pattern was closely correlated with larger tumor size, higher tumor-node-metastasis stage, and poorer prognosis for the patients with HCC. Moreover, silenced circ-HOMER1 inhibited cell proliferation, migration, and invasion concomitant with the promotion of apoptosis in HCC cells, and vice versa. Mechanistically, circ-HOMER1 enhanced the inhibition of miR-1322 on CXCL6 in HCC. Furthermore, we found that circ-HOMER1 promoted HCC cell growth and aggressiveness by miR-1322/CXCL6 axis. This study may provide a potential prognostic indicator and therapeutic target for patients with HCC.

Gene Regulatory Effect of Pyruvate Kinase M2 is Involved in Renal Inflammation in Type 2 Diabetic Nephropathy

The inflammation of glomerular endothelial cells induces and promotes the activation of macrophages and contributes to the development of diabetic nephropathy. Thus, this study aimed to investigate the gene regulatory effect and potential role of pyruvate kinase M2 (PKM2) in inflammatory response in diabetic nephropathy. The plasma PKM2 levels of patients with diabetes were evaluated. Eight-week-old mice were divided into three groups (WT, db/db mice, and db/db mice treated with TEPP-46) and raised for 12 weeks. Blood and kidney samples were collected at the end of the experiment. Endothelial cells were stimulated with high glucose with or without TEPP-46. The expression of intercellular adhesion molecule 1 (ICAM-1), interleukin 6 (IL-6), interleukin 1 beta (IL-1β), phospho-PKM2, PKM2, phospho-STAT3(signal transducer and activator of transcription), STAT3, nuclear factor kappa B (NF-kB), and phospho-NF-kB in vivo and in vitro were determined using Western blot. The activation of macrophages (CD68+CD86+) in the glomeruli was assessed via fluorescent double staining. Moreover, immune endothelial adhesion experiments were performed. The plasma PKM2 levels of patients with type 2 diabetes increased. P-PKM2 was up-regulated in vivo and in vitro. TEPP-46 decreased inflammatory cell infiltration and ICAM-1 expression in vivo and in vitro and inhibited the differentiation of macrophages to M1 cells in db/db mice with diabetic nephropathy. PKM2 regulated the phosphorylation of STAT3 and NF-kB. Furthermore, high glucose levels induced the transition from tetramer to dimer and the nuclear translocation of PKM2. The gene regulatory effect of PKM2 is involved in renal inflammation in type 2 diabetic nephropathy by promoting the phosphorylation of STAT3 and NF-kB and the expression of intercellular adhesion molecule 1. Thus, the down-regulation of phosphorylated PKM2 may have protective effects against diabetic nephropathy by inhibiting renal inflammation.

Cyclocarya paliurus extract attenuates hepatic lipid deposition in HepG2 cells by the lipophagy pathway

Context Cyclocarya paliurus (CP) (Batal.) Iljinsk (Cyclocaryaceae), a plant native to China, is the sole species in the genus Cyclocarya. Its leaves have been widely used as a remedy for hyperlipidaemia in traditional folk medicine. However, the mechanism underlying CP-induced lipolysis, especially in the liver, has not been entirely elucidated. Objective This study investigates the effect of CP ethanol extract (CPE) on hepatic steatosis and the underlying molecular mechanisms involved. Materials and methods The effect of CPE at concentrations of 0, 6.25, 12.5, 25, 50, and 100 μg/mL on the viability of HepG2 cells was examined using the cell counting kit-8 (CCK-8) assay after incubation for 24 h. CPE-induced changes in intracellular lipid content were assessed by measuring the absorbance of oil red O staining at 520 nm, and the possible underlying mechanisms were further studied using quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis, western blotting, immunofluorescence studies and transmission electron microscopy. Results The half-maximal inhibitory concentration (IC50) of CPE in HepG2 cells was 97.27 μg/mL. Treatment with 50 μg/mL CPE increased lipid clearance, which was associated with increased autophagy in HepG2 cells. CPE-induced autophagy involved downregulation of phosphorylation level of mammalian target of rapamycin (0.87 ± 0.08 vs. 1.31 ± 0.10). Fluorescent double staining and electron microscopy images showed lipid deposits within autolysosomes, thereby confirming the abovementioned findings. Discussion and conclusions CPE can induce hepatic fat clearance through the autophagy-lysosome pathway known as lipophagy. CPE has potential as a functional food.

Kidney and liver are the main organs of expression of a key metabolic enzyme alanine:glyoxylate aminotransferase 2 in humans

BackgroundThe metabolic syndrome is a cluster of cardiovascular risk factors and is highly predictive for development of cardiovascular diseases. An association between elevated plasma levels of the endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA) and risk of cardiovascular diseases has been demonstrated in numerous epidemiological studies. ADMA can be catabolized by dimethylarginine dimethylaminohydrolase (DDAH) or metabolized through a much less understood alternative pathway by alanine:glyoxylate aminotransferase 2 (AGXT2) with the formation of α-keto-δ-(N,N-dimethylguanidino)valeric acid (ADGV). Previous RT-PCR and Western Blot studies suggested that Agxt2 is expressed in the mouse kidney and liver at comparable levels, while Northern Blot and in-situ RNA-hybridisation experiments demonstrated that the kidney is the main organ of Agxt2 expression in rats. Given this discrepancy, the goal of the current study was to analyse the expression of AGXT2 in human tissues. Material and methodsWe analyzed AGXT2 expression in human tissues from a normal tissue bank by RT-PCR and further validated the results by Western Blot. We also performed immunohistochemical staining for AGXT2 and double fluorescent staining with an anti-AGXT2 antibody and a monoclonal anti-mitochondrial antibody. ResultsWe saw the strongest expression of AGXT2 in the kidney and liver and confirmed this results on protein level. By IHC staining we were able to show that AGXT2 is present in the convoluted tubule in the kidney and in the liver hepatocytes. The double fluorescent staining revealed mitochondrial localization of AGXT2. ConclusionsOur current data suggest that both hepatocytes and kidney tubular epithelial cells are the major sources of AGXT2 in humans. We also demonstrated the mitochondrial localization of human AGXT2 enzyme.

Up-regulated LINC00261 predicts a poor prognosis and promotes a metastasis by EMT process in cholangiocarcinoma

ObjectiveLINC00261 plays a vital role in tumorigenesis and metastasis of digestive system cancer. However, an influence of LINC00261 on cholangiocarcinoma has a little research. There, we investigated clinical role and molecular mechanisms of LINC00261 in cholangiocarcinoma. MethodsThe qRT-PCR was performed for the detection of LINC00261 level in 50 paired specimens from CCA patients and six cell lines. Cell proliferation were explored by CCK-8 and colony formation assays in QBC939 and RBE cells after transfected with si-LINC00261 or si-NC. Then, AO/EB double fluorescence staining and flow cytometric assays were performed to assess cell apoptosis. Transwell and wound healing assays were selected to evaluate migratory and invasive property of cells. Protein levels, such as PCNA, Bax, Bcl-2, and several epithelial-to-mesenchymal transition markers, including E-cadherin, N-cadherin and Vimentin, were detected by western blot assays. Furthermore, we use a R2 platform to evaluate the correlation between LINC00261 and EMT makers and predict the overall survival and relapse-free survival for CCA patients by the expression of LINC00261/ EMT makers. ResultsLINC00261 was overexpressed in cancerous tissues and CCA cell lines compared with adjacent tissues and HIBEC, respectively. Up-regulation of LINC00261 was related to larger tumor size (p = 0.009), positive lymph node metastasis (p = 0.021), advanced TNM stages (p = 0.017) and higher postoperative recurrence (p = 0.009) for CCA patients. Additionally, univariate and multivariate analysis displayed that LINC00261 an independent prognostic factor in CCA patients. Knockdown of LINC00261 expression in RBE and QBC939 cell lines inhibited cell proliferation, migration and invasion property and increased cell apoptosis and the EMT progression. Moreover, there was a strong correlation between LINC00261 and E-cadherin (CDH1) (p < 0.05), and low expression of E-cadherin (CDH1) has a poor overall survival and relapse-free survival in CCA patients (p < 0.05). ConclusionOverall, high level of LINC00261 in CCA predicts a poor prognosis, and promotes a metastasis via EMT process. Thus, LINC00261 could be a promising biomarker and therapeutic target for CCA, and in the high level of LINC00261 in CCA, E-cadherin or CDH1 might be an effective factor for tumor metastasis or poor prognosis.

Circular RNA circ_0074026 indicates unfavorable prognosis for patients with glioma and facilitates oncogenesis of tumor cells by targeting miR-1304 to modulate ERBB4 expression.

Glioma (GM) is a common malignancy all over the world. A novel circular RNA (circRNA), circ_0074026, has been documented to be upregulated in GM tissues than that of normal counterparts, as confirmedby circRNA microarray. However, the biological mechanism of circ_0074026 is still unreported in GM. The expression of circ_0074026 in GM specimens or cells was detected by quantitative real-time polymerase chain reaction. Fisher's exact test was utilized to evaluate the clinical relevance of circ_0074026 in patients with GM. Kaplan-Meier and Cox regression analysis were used to estimate the prognostic value of circ_0074026. Cell counting kit-8 (CCK-8), acridine orange/ethidium bromide double fluorescence staining, flow cytometry, wound healing, and Transwell assays were used to detect the malignant behaviors of GM cells including cell growth, apoptosis, migration, and invasion. CCK-8 and flow cytometric experiments were utilized to evaluate whether circ_0074026 had a side effect on normal human astrocyte cells. The interaction between miR-1304 and circ_0074026 or ERBB4 3'-untranslated region (3'-UTR) was predicted withcircular RNA Interactome and TargetScan, respectively, and then confirmed by the dual-luciferase reporter test. The levels of circ_0074026 were both apparently increased in GM samples and cells. The elevated expression of circ_0074026 was linked to patients' tumor size, WHO grade, disease-free survival, and overall survival. The depletion of circ_0074026 can block cell growth, migration, invasion, and impel cell apoptosis in the LN229 cell line. However, ectopically expressed circ_0074026 caused the opposite effect in the U251 cell line. The following dual-luciferase reporter assay demonstrated that miR-1304 interacted with circ_0074026 and ERBB4 3'-UTR. Furthermore, the rescue assay indicated that circ_0074026 modulated ERBB4 to promote tumor progression by regulating miR-1304. Thus, anovel regulatory pathway may provide a new therapeutic target for patients with GM.

The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells.

Slit1 is one of the known signaling factors of the slit family and can promote neurite growth by binding to its receptor, Robo2. Upregulation of Slit1 expression in dorsal root ganglia (DRG) after peripheral nerve injury plays an important role in nerve regeneration. Each sensory neuronal soma in the DRG is encapsulated by several surrounding satellite glial cells (SGCs) to form a neural structural unit. However, the temporal and spatial patterns of Slit1 upregulation in SGCs in DRG and its molecular mechanisms are not well understood. This study examined the spatial and temporal patterns of Slit1 expression in DRG after sciatic nerve crush by immunohistochemistry and western blotting. The effect of neuronal damage signaling on the expression of Slit1 in SGCs was studied in vivo by fluorescent gold retrograde tracing and double immunofluorescence staining. The relationship between the expression of Slit1 in SGCs and neuronal somas was also observed by culturing DRG cells and double immunofluorescence labeling. The molecular mechanism of Slit1 was further explored by immunohistochemistry and western blotting after intraperitoneal injection of Bright Blue G (BBG, P2X7R inhibitor). The results showed that after peripheral nerve injury, the expression of Slit1 in the neurons and SGCs of DRG increased. The expression of Slit1 was presented with a time lag in SGCs than in neurons. The expression of Slit1 in SGCs was induced by contact with surrounding neuronal somas. Through injured cell localization, it was found that the expression of Slit1 was stronger in SGCs surrounding injured neurons than in SGCs surrounding non-injured neurons. The expression of vesicular nucleotide transporter (VNUT) in DRG neurons was increased by injury signaling. After the inhibition of P2X7R, the expression of Slit1 in SGCs was downregulated, and the expression of VNUT in DRG neurons was upregulated. These results indicate that the ATP-P2X7R pathway is involved in signal transduction from peripheral nerve injury to SGCs, leading to the upregulation of Slit1 expression.

In vitro apoptosis induction ability of methanolic extract of Paramignya trimera root (Xao tam phan) in breast cancer stem cells

Objective: Cancer has been considered as one of the world's leading causes of death. Recently, the Paramignya trimera plant, locally called ``Xao Tam Phan'', has become a popular Vietnamese medicinal herb that is used as alternative medicine for cancer treatment support with minimal side effects. In this study, we aimed to demonstrate the cytotoxicity of methanolic extract of Paramignya trimera on a Vietnamese breast cancer stem cell line (VNBRCA1) in vitro.&#x0D; Methods: We used the MTT assay to determine the cytotoxicity of the extract on VNBRCA1 cells and human fibroblast (HF) cell line was used as a control for the plant extract treatment. Clinically used anticancer drug, doxorubicin, was used as a control drug (for relative comparison to the plant extract) to evaluate the selective cytotoxicity of the plant extract on VNBRCA1 and HF cells. We examined the apoptosis induction by the plant extract on VNBRCA1by Annexin V/7AAD staining and flow cytometry analysis. In addition, the morphology of apoptotic nuclei of treated cells was observed by fluorescent microscopy using double fluorescent staining: Hoechst 33342 and propidium iodide (PI).&#x0D; Results: In comparison between the cytotoxicity of the plant extract and Doxorubicin on both cell lines (VBRCA1 and HF), we observed that plant extract was selectively cytotoxic against VNBRCA1 with an IC50 value of 10610 μg/mL, while Doxorubicin was discriminatorily cytotoxic against HF with an IC50 value of 0.135+/-0.09 μg/mL. We also found that the plant extract induced apoptosis VNBRCA1 in a dose-dependent manner. In addition, fluorescent microscopy revealed disintegrated nuclei of plant extract-treated cells, representing a hallmark of apoptosis.&#x0D; Conclusions: These results showed that Paramignya trimera methanolic extract selectively killed VNBRCA1 cell lines, indicating that Paramignya trimera methanolic extract may represent a potential agent for cancer treatment.

Mitotic activity, modulation of DNA processing, and purinergic signalling in the adult rat auditory brainstem following sensory deafferentation.

A complex scenario of cellular network reorganization is caused by unilateral sensory deafferentation (USD) in the adult rat central auditory system. We asked whether this plasticity response involves mitosis. Immunohistochemistry was applied to brainstem sections for the detection and localization of mitotic markers Ki67 and PCNA, the growth-associated protein Gap43 and purine receptor P2X4. Fluorescent double staining was done for Ki67:PCNA and for both of them with HuC/HuD (neurons), S100 (astrocytes), Iba1 (microglia) and P2X4. Inquiring 1-7days after USD, we found Ki67 expression to be changed in cellular profiles of cochlear nucleus (CN) with a significant increase in number by 1-3days, followed by reset to control level within 1week. USD-induced mitosis exclusively occurred in microglia and was absent elsewhere in the auditory brainstem. PCNA staining of small cellular profiles increased similarly but remained elevated. PCNA staining intensity also changed in CN, superior olive and inferior colliculus in neuronal nuclei, suggesting shifts in DNA processing. No apoptotic cell death was detected in any region of the adult auditory brainstem after USD. A comparison of anterograde and retrograde effects of nerve damage revealed proliferating microglia expressing P2X4 receptors in CN upon USD, but not in the facial nucleus after facial nerve transection. In conclusion, the deafferentation model studied here permits insight into the capacity of the adult mammalian brain to invoke mitosis among glia cells, adjustment of gene processing in neurons and purinergic signalling between them, jointly accounting for a multilayered neuro- and glioplastic response.

Semaphorin 3A gets involved in the establishment of mouse tootheruptive pathway.

The accurately establishment of the eruptive pathway is of vital importance. The mechanisms governing tooth eruption pathway remain little known. This study is to elucidate the roles of Semaphorin 3A (Sema 3A) in mouse tooth eruptive pathway. C57BL/6 mice (11-13 and 15-17days after birth) were chosen to observe eruptive pathway of mouse lower first molar. Expressions of Sema 3A and its receptor neuropilin 1 and plexin A1 were detected. Osteoclasts were identified by TRAP staining. Co-localization of Sema 3A and osteoclast maker CD68 was detected by double immunofluorescence staining. Picrosirius red staining was applied to observe collagen fibers during mucosal penetration phase. In vitro, Bone marrow-derived macrophages (BMMs) were prepared from 4week C57BL/6 mice to observe the effect of Sema 3A on the differentiation of BMMs into osteoclasts by TRAP staining. Expressions of Sema 3A was observed by immunofluorescence and western blotting. At osseous eruption phase, many TRAP-positive multi-nucleated cells were distributed around occlusal alveolar bone. The positive expressions of Sema 3A were observed in the multi-nucleated cells. Fluorescence double staining showed that Sema 3A and CD68 were co-expressed in osteoclasts. Its receptor neuropilin 1 and plexin A1 were also found in osteoclasts. In vitro, Sema3A negatively regulated osteoclast differentiation. At mucosal penetration, occlusal alveolar bone had been completely resorbed and collagen fires were gradually degraded for eruptive pathway. Similar positive expressions of Sema 3A and its receptor neuropilin 1 and plexin A1 were also found in the mucosalpenetration pathway. Sema 3A gets involved in the establishment of mouse tootheruptive pathway by modulating osteoclast activity. Sema3A should be considered as a novel nervous agent or a potential biomarker for mouse tootheruptive pathway.

Antiapoptotic Effect of Granulocyte-Colony Stimulating Factor After Peripheral Nerve Trauma

Granulocyte-colony stimulating factor (G-CSF) has been observed to have direct protective effects on neurons after stroke in experimental models and in humans. In the present study, the antiapoptotic effects of G-CSF on spinal α-motoneurons after inducement of peripheral sciatic nerve lesions were evaluated in a rat model. Of 48 rats, 24 were treated with G-CSF and 24 were treated with glucose 5% solution (control group). The spinal cord of 6 rats in each group were removed at days 1, 4, 7, and 14. The α-motoneurons of spinal cord section L4-L6 were counted and investigated for the expression of choline acetyltransferase (ChAT), G-CSF receptor (G-CSFR), and Bcl-2 and Bax proteins. Additionally, α-motoneuron fluorescence double staining was performed for ChAT/Bcl-2, ChAT/Bax, and ChAT/G-CSFR. Without G-CSF treatment, the number of ChAT-positive α-motoneurons on the lesion side was significantly decreased (P < 0.001). The number of α-motoneurons with Bcl-2 and G-CSFR positivity on the lesion side was significantly decreased (P < 0.05). In contrast, the number of α-motoneurons with Bax positivity was significantly greater (P < 0.05). After G-CSF treatment, the differences in the number of α-motoneurons on the 2 sides were not statistically significant. Fluorescence double staining of α-motoneurons was positive for ChAT/Bcl-2, ChAT/Bax, and ChAT/G-CSFR. The results indicated that G-CSF has neuroprotective properties in spinal α-motoneurons and contributes to antiapoptotic effects after peripheral nerve lesions. The relevance of G-CSF, its precise mode of action, and the effect of these findings in clinical situations remains to be elucidated and require examination in further studies.

Cyclopamine Suppresses Human Esophageal Carcinoma Cell Growth by Inhibiting Glioma-Associated Oncogene Protein-1, a Marker of Human Esophageal Carcinoma Progression.

BackgroundEsophageal carcinoma is a common gastrointestinal tumor in humans. Cyclopamine, a Hedgehog (Hh)-pathway-specific inhibitor, is an effective chemotherapeutic drug for suppressing tumor cell differentiation, with unclear mechanisms. We investigated glioma-associated oncogene protein-1 (Gli-1) expression in human esophageal carcinoma tissue and the inhibition of cyclopamine on EC9706 esophageal carcinoma cell growth.Material/MethodsGli-1 in tumor tissue was measured by immunohistochemistry (IHC). EC9706 cells were treated with different concentrations of cyclopamine and incubated for different times. MTT method, flow cytometry, and Acridine orange/ethidium bromide (AO/EB) double-fluorescence staining were applied to detect cell proliferation and apoptosis. Western blot (WB) analysis was performed to assess Gli-1 expression.ResultsGli-1 was associated with patient age, gender, lymphatic metastasis, tumor recurrence, and stage, with significantly (P<0.05) positive correlations with age, lymphatic metastasis, tumor recurrence, and stage. At 12 h (F=214.57), 24 h (F=76.832), 48 h (F=236.90), and 72 h (F=164.55), the higher the concentration of cyclopamine, the higher the inhibition rate of suppressing EC9706 proliferation, and this effect was significant (P<0.05). The number of early-apoptosis cells increased as the concentration of cyclopamine increased. Morphology of EC9706 cells appeared as round with rough edges, karyopyknosis, and karyorrhexis. After 48 h, apoptosis rates of EC9706 cells treated with different concentrations of cyclopamine were (7.73±1.25)% at 2.5 μM, (13.37±1.42)% at 5.0 μM, (22.3±2.92)% at 10.0 μM, and (33.57±1.75)% at 20.0 μM, and the effect was dose-dependent. Gli-1 was obviously reduced after cyclopamine treatment and the effect was dose-dependent.ConclusionsGli-1 is highly expressed in human esophageal carcinoma, and could be a marker for use in assessing tumor stage and the deciding on treatment target.

Identification and anti-cancer activity in 2D and 3D cell culture evaluation of an Iranian isolated marine microalgae Picochlorum sp. RCC486.

PurposeCancer disease is the second cause of death in the world. Now a days, high percentage of drugs, which are involved in treatment of cancers, have natural origin. Introduction of microalgae strains as anti-cancer drugs origin is a valuable approach for cancer therapy.MethodsIn the present study we describe the isolation, characterization, and anti-proliferative activity of a new microalga strain (Picochlorum sp. RCC486) from Iran. The cytotoxic activity of four different algal extracts including methanol, ethyl acetate, chloroform, and hexane were evaluated against MDA-MB-231, MCF-7, Hep-G2, and A-549 cell liens. Cell viability was determined using MTT assay in both monolayer and spheroids 3D cultures. The apoptosis was confirmed by different methods such as AO/EB and Annexin V-FITC/PI double staining, caspase-3 colorimetric assay, ROS and MMP assay.ResultsThe results of MTT assay and fluorescent double staining confirmed that methanol and ethyl acetate extracts showed the best cytotoxic activity against the cancer cell lines. The production of ROS, caspase-3 activity and depolarized MMP were quite significant in MDA-MB-231 cell line treated with methanol and ethyl acetate extracts.ConclusionIn this research we revealed that cytotoxicity and apoptotic effects of the methanol and ethyl acetate extracts in human cancer cells make them good candidates for further pharmacological studies to discover effective drugs for cancer therapy. Graphical abstractThe present study describes the isolation, characterization, and anti-proliferative activity of different extracts of a new microalga strain (Picochlorum sp. RCC486) from Iran. The antiproliferative and apoptosis inducing activity of ethyl acetate and methanol extracts with high content of phenol and carotenoid make them as good candidates for further pharmacological studies to discover effective drugs for cancer therapy. Electronic supplementary materialThe online version of this article (10.1007/s40199-018-0213-5) contains supplementary material, which is available to authorized users.

Sulbactam Protects Hippocampal Neurons Against Oxygen-Glucose Deprivation by Up-Regulating Astrocytic GLT-1 via p38 MAPK Signal Pathway.

Sulbactam is an atypical β-lactam medication and reported to be neuroprotective by up-regulating glial glutamate transporter-1 (GLT-1) in rats. The present study was undertaken to study the role of p38 MAPK signal pathway in sulbactam induced up-regulation of GLT-1 expression in astrocytes and anti-ischemic effect. Neuron-astrocyte co-cultures and astrocyte cultures from neonatal Wistar rats were used. Cerebral ischemia was mimicked by oxygen-glucose deprivation (OGD). Hoechst (HO)/propidium iodide (PI) double fluorescence staining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay were used to evaluate neuronal death and cell viability, respectively. Immunocytochemistry and Western blot were used to detect protein expressions. Sulbactam pre-incubation significantly and dose-dependently prevented neuronal death and decline in cell viability induced by OGD in neuron-astrocyte co-cultures, and upregulated GLT-1 expression in astrocyte cultures endured OGD, which suggested that sulbactam might protect neurons against OGD by up-regulating astrocytic GLT-1 expression. It was further shown that the phosphorylated-p38 MAPK expression in astrocytes was up-regulated after the sulbactam pre-incubation and this up-regulation was moderate in amplitude. Especially, the time course of the up-regulation of phosphorylated-p38 MAPK was obviously earlier than that of GLT-1, which suggested possibility that p38 MAPK might be an upstream signal for GLT-1 up-regulation induced by sulbactam. We further found that SB203580, the specific inhibitor of p38 MAPK, dose-dependently inhibited the GLT-1 up-regulation induced by sulbactam either in non- or OGD-treated astrocytes and the protective effect of sulbactam on co-cultured neurons against OGD. Taken together, it might be concluded that sulbactam protects cerebral neurons against OGD by up-regulating astrocytic GLT-1 expression via p38 MAPK signal pathway.

Impact of COX2 Inhibitor for Regulation of PD-L1 Expression in Non-small Cell Lung Cancer.

There is no clear evidence in the literature regarding the regulation of programmed cell death-ligand 1 (PD-L1) expression by cyclo-oxygenase-2 (COX2). In this study, whether PD-L1 expression was regulated by COX2 activity was examined in vitro. Resected lung cancer specimens were analyzed for PD-L1 and COX2 expression by immunohistochemical analysis. Next, co-localization of PD-L1 and COX2 expression was analyzed by double-fluorescence staining. Lastly, the effect of COX2 inhibition on the expression of PD-L1 was examined using lung cancer cell lines. PD-L1 expression was significantly correlated with COX2 expression in the resected specimens. The majority of cancer cells that expressed PD-L1 also co-expressed COX2. However, treatment of lung cancer cell lines with a COX2 inhibitor had no impact on PD-L1 expression. Our results suggest that COX2 inhibition might have no effect on the usage of immune checkpoint inhibitors in lung cancer treatment.

Application of the Fluorescent Dye BODIPY in the Study of Lipid Dynamics of the Rice Blast Fungus Magnaporthe oryzae.

Rice blast is one of the most serious diseases affecting rice yield which is caused by Magnaporthe oryzae, a model organism for studies on plant pathogenic fungi. Lipids stored in M. oryzae cells have been shown to be crucial for the development of appressorium turgor and the ability of the pathogen to cause infection. Nile red staining is a common method to study lipid dynamics in phytopathogenic fungi. However, the disadvantages of this dye include its wide spectrum, poor water solubility, and susceptibility to quenching. Boron dipyrromethene (BODIPY) is a new type of fluorescent dye that has a different emission wavelength to that of Nile red as well as many desirable spectral and chemical properties. In this study, we used BODIPY to stain the lipids in M. oryzae cells to seek a possible substitute to Nile red in the study of lipid dynamics in plant pathogenic fungi. Our data showed that through simple and routine procedures, BODIPY was able to distinctly label lipids in the cells of mycelia and conidia. The positions of lipids labeled by BODIPY were essentially identical to those labeled by Nile red, but with more clear fluorescence labelling, lower background, and higher specificity. The use of BODIPY to stain germinating M. oryzae conidia allowed the lipid dynamics to be clearly tracked during this process. We also achieved double and multiple fluorescent staining conidia by combining BODIPY with the red fluorescent protein mCherry and other fluorescent dyes, such as Calcofluor white and DAPI, in conidia, mycelia, and sexual structures of M. oryzae. These results indicate that BODIPY is an ideal fluorescent dye for staining fungal lipids and provide a method for the study of the lipid dynamics and lipid metabolism in plant pathogenic fungi.

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of its labeling was affected by various factors, here, we provide a refined protocol for the application of high molecular weight BDA for studying optimal neural labeling in the central nervous system. After stereotactic injection of BDA into the ventral posteromedial nucleus (VPM) of the thalamus in the rat through a delicate glass pipette, BDA was stained with fluorescent streptavidin-Alexa (AF) 594 and counterstained with fluorescent Nissl stain AF500/525. On the background of green Nissl staining, the red BDA labeling, including neuronal cell bodies and axonal terminals, was more distinctly demonstrated in the somatosensory cortex. Furthermore, double fluorescent staining for BDA and the calcium-binding protein parvalbumin (PV) was carried out to observe the correlation of BDA labeling and PV-positive interneurons in the cortical target, providing the opportunity to study the local neural circuits and their chemical characteristics. Thus, this refined method is not only suitable for visualizing high quality neural labeling with the high molecular weight BDA through reciprocal neural pathways between the thalamus and cerebral cortex, but also will permit the simultaneous demonstration of other neural markers with fluorescent histochemistry or immunochemistry.

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat.

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of its labeling was affected by various factors, here, we provide a refined protocol for the application of high molecular weight BDA for studying optimal neural labeling in the central nervous system. After stereotactic injection of BDA into the ventral posteromedial nucleus (VPM) of the thalamus in the rat through a delicate glass pipette, BDA was stained with fluorescent streptavidin-Alexa (AF) 594 and counterstained with fluorescent Nissl stain AF500/525. On the background of green Nissl staining, the red BDA labeling, including neuronal cell bodies and axonal terminals, was more distinctly demonstrated in the somatosensory cortex. Furthermore, double fluorescent staining for BDA and the calcium-binding protein parvalbumin (PV) was carried out to observe the correlation of BDA labeling and PV-positive interneurons in the cortical target, providing the opportunity to study the local neural circuits and their chemical characteristics. Thus, this refined method is not only suitable for visualizing high quality neural labeling with the high molecular weight BDA through reciprocal neural pathways between the thalamus and cerebral cortex, but also will permit the simultaneous demonstration of other neural markers with fluorescent histochemistry or immunochemistry.

Inhibition of Yiqi Jiedu formula on proliferation of nasopharyngeal carcinoma cells by MAPK/ERK signaling pathway

To study the effect of aqueous extracts of Yiqi Jiedu formula (YQ) on the proliferation of CNE2 cells in human nasopharyngeal carcinoma, and investigate its mechanism to provide a new theoretical basis for the clinical application of YQ. CNE2 cells were treated with different concentrations (0.125, 0.25, 0.5, 0.25 g·L⁻¹) of YQ, positive control medicine (cisplatin 4.0 mg·L⁻¹), inhibitor PD98059 (50 μmol·L⁻¹), activator isoproterenol hydrochloride (20 μmol·L⁻¹), activator isoproterenol hydrochloride (ISO)+YQ 0.5 g·L⁻¹. Then cell labeling by using real-time analyzer (RTCA) and CCK 8 method were used to detect cell proliferation activity, and the half inhibitory concentration (IC₅₀) was calculated. The cell cycle distribution was detected by fluorescence double dye flow cytometry PI staining, and Western blot method was used to detect the expression levels of related protein and MAPK/ERK signaling pathway. The results of RTCA and CCK-8 test showed that as compared with the control group, YQ group could effectively inhibit the proliferation of CNE2 cells (P<0.01), with a dose and time dependence, and 48 h IC₅₀ value was 0.5 g·L⁻¹. The results of cell cycle showed that after 48 h of water extract treatment, the cell cycle was significantly changed, the proportion of G₀/G₁ was reduced, the ratio of G₂/M increased, and the cell cycle was in G₂/M period (P<0.01). Western blot results showed that after 48 h treatment with different concentrations of aqueous extract, cell cycle-related proteins cyclinD1, cyclinD3 and CDK2 expression levels were down-regulated; MAPK/ERK signaling pathway related protein p-c-Raf, p-MEK, p-ERK1/2 expression level significantly lower as compared with the control group (P<0.05). After adding activator and inhibitor in MAPK/ERK signaling pathway on this basis, the results showed that after adding activator ISO, cell proliferation was significantly higher than that in the Control group; the cycle related proteins cyclinD1, cyclinD3, and CDK2 expression levels were increased; at the same time, key protein p-c-Raf, p-MEK, p-ERK1/2 expression levels in the signal pathways were relatively increased. While after the addition of inhibitor PD98059, the cell proliferation was significantly lower than that in the Control group, and the expression level of corresponding protein was decreased, which was significantly different from the Control group (P<0.05). So YQ could block cell cycle and inhibit the proliferation of CNE2 cells mainly by reducing the expression of MAPK/ERK signaling pathway key protein p-c-Raf, p-MEK and p-ERK1/2.

202 Targeting Galactosyl-α-1,3-Galactose (αGal) Epitopes for Multi-Species Embryo Immunosurgery

Immunosurgical isolation of the inner cell mass (ICM) from blastocysts is based on complement-mediated lysis of antibody-coated trophectoderm (TE) cells. Conventionally, anti-species antisera, containing antibodies against multiple undefined TE cell epitopes, have been used as antibody source. We previously generated α-1,3-galactosyltransferase deficient (GTKO) pigs to prevent hyper-acute rejection of pig-to-primate xenotransplants. Because GTKO pigs lack galactosyl-α-1,3-galactose (αGal) but are exposed to this antigen (e.g. αGal on gut bacteria), they are expected to produce anti-αGal antibodies. In this study, we examined whether serum from GTKO pigs can be used as a novel antibody source for embryo immunosurgery. First, the presence of αGal epitopes in mouse (E3.5), rabbit (Day 4), pig (Day 6–7), and bovine (Day 7–8) blastocysts was examined by staining with fluorescein isothiocyanate (FITC)-conjugated BSI-B4 lectin (Sigma, St. Louis, MO, USA) that binds αGal. Expression of αGal epitopes on the surface of TE cells was detected in blastocysts of all examined species. Next, pig blastocysts were incubated with a medium containing GTKO pig serum. Swollen TE cells were observed in some of the blastocysts already after 2 min and, after 10 min, almost all TE cells of these blastocysts were completely destroyed. No lysis was recorded when the same experiment was done with wild-type pig serum, suggesting the presence of sufficient quantities of anti-αGal antibodies in GTKO serum to coat the TE cells and induce their complement-mediated lysis. Finally, GTKO serum was systematically tested for immunosurgery. Zona-free blastocysts of the species mentioned above were incubated with heat-inactivated GTKO pig serum for 1 h at 38°C. After washing, the blastocysts were labelled with Hoechst 33342 and TE was stained with FITC-conjugated concanavalin A (ConA) to distinguish the ICM from TE cells. Eventually, the blastocysts were individually incubated in complement solution for 30 to 40 min. Complement-mediated lysis of TE cells was efficiently induced in mouse, rabbit, pig, and bovine blastocysts (10/10, 7/7, 10/10, and 5/6, respectively), and intact ICM were successfully recovered from all species (100, 100, 60, and 80%, respectively). Double fluorescent staining with Hoechst 33342 and ConA clearly showed that the majority of isolated ICM was not contaminated with TE cells. Our study demonstrates that GTKO pig serum is a reliable source of antibodies targeting the αGal epitope of TE cells. Major advantages of using GTKO serum for embryo immunosurgery are (1) that it can be produced easily in large batches, thus reducing experimental variation; and (2) that it reacts with a large number of different species, except for humans, apes, and old world monkeys that lack αGal epitopes. Interesting applications include the preparation of TE and ICM for transcriptome profiling or chimeric embryo complementation experiments. This work is supported by the German Research Council (TR-CRC 127).