Protein Blot Analysis Research Articles

Investigation of the Anti-Inflammatory Activity of Fusaproliferin Analogues Guided by Transcriptome Analysis.

Background: Excessive inflammation results in severe tissue damage as well as serious acute or chronic disorders, and extensive research has focused on finding new anti-inflammatory hit compounds with safety and efficacy profiles from natural products. As promising therapeutic entities for the treatment of inflammation-related diseases, fusaproliferin and its analogs have attracted great interest. However, the underlying anti-inflammatory mechanism is still poorly understood and deserves to be further investigated. Methods: For the estimation of the anti-inflammatory activity of fusaproliferin (1) and its analogs (2-4) in vitro and in vivo, lipopolysaccharide (LPS)-induced RAW264.7 macrophages and zebrafish embryos were employed. Then, transcriptome analysis was applied to guide subsequent western blot analysis of critical proteins in related signaling pathways. Surface plasmon resonance assays (SPR) combined with molecular docking analyses were finally applied to evaluate the affinity interactions between 1-4 and TLR4 and provide a possible interpretation of the downregulation of related signaling pathways. Results: 1-4 significantly attenuated the production of inflammatory messengers, including nitric oxide (NO), reactive oxygen species (ROS), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), as well as nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in LPS-induced RAW264.7 macrophages. Transcriptome analyses based on RNA-seq indicated the ability of compound 1 to reverse LPS stimulation and the nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPKs) signaling pathways contribute to the anti-inflammatory process. Experimental verification at the protein level revealed that 1 can inhibit the activation of inhibitor of NF-κB kinase (IKK), degradation of inhibitor of NF-κB (IκB), and phosphorylation of NF-κB and reduce nuclear translocation of NF-κB. 1 also decreased the phosphorylation of MAPKs, including p38, extracellular regulated protein kinases (ERK), and c-Jun N-terminal kinase (JNK). SPR assays and molecular docking results indicated that 1-4 exhibited affinity for the TLR4 protein with KD values of 23.5-29.3μM. Conclusion: Fusaproliferin and its analogs can be hit compounds for the treatment of inflammation-associated diseases.

Electrohydrodynamic Jet-Printed Ultrathin Polycaprolactone Scaffolds Mimicking Bruch's Membrane for Retinal Pigment Epithelial Tissue Engineering.

Age-related macular degeneration (AMD) is the leading cause of visual loss and affects millions of people worldwide. Dysfunction of the retinal pigment epithelium (RPE) is associated with the pathogenesis of AMD. The purpose of this work is to build and evaluate the performance of ultrathin scaffolds with an electrohydrodynamic jet (EHDJ) printing method for RPE cell culture. We printed two types of ultrathin (around 7 μm) polycaprolactone scaffolds with 20 μm and 50 μm pores, which possess mechanical properties resembling that of native human Bruch’s membrane and are biodegradable. Light microscopy and cell proliferation assay showed that adult human retinal pigment epithelial (ARPE-19) cells adhered and proliferated to form a monolayer on the scaffolds. The progress of culture matured on the scaffolds was demonstrated by immunofluorescence (actin, ZO-1, and Na+/K+-ATPase) and Western blot analysis of the respective proteins. The RPE cells cultured on EHDJ-printed scaffolds with 20 μm pores presented higher permeability, higher transepithelial potential difference, and higher expression level of Na+/K+-ATPase than those cultured on Transwell inserts. These findings suggest that the EHDJ printing can fabricate scaffolds that mimic Bruch’s membrane by promoting maturation of RPE cells to form a polarized and functional monolayered epithelium with potential as an in vitro model for studying retinal diseases and treatment methods.

Receptor activity\u2010modifying protein 1 regulates mouse skin fibroblast proliferation via the G\u03b1i3-PKA-CREB-YAP axis

BackgroundSkin innervation is crucial for normal wound healing. However, the relationship between nerve receptors and wound healing and the intrinsic mechanism remains to be further identified. In this study, we investigated the role of a calcitonin gene-related peptide (CGRP) receptor component, receptor activity‐modifying protein 1 (RAMP1), in mouse skin fibroblast (MSF) proliferation.MethodsIn vivo, Western blotting and immunohistochemical (IHC) staining of mouse skin wounds tissue was used to detect changes in RAMP1 expression. In vitro, RAMP1 was overexpressed in MSF cell lines by infection with Tet-On-Flag-RAMP1 lentivirus and doxycycline (DOX) induction. An IncuCyte S3 Live-Cell Analysis System was used to assess and compare the proliferation rate differences between different treatment groups. Total protein and subcellular extraction Western blot analysis, quantitative real-time-polymerase chain reaction (qPCR) analysis, and immunofluorescence (IF) staining analysis were conducted to detect signalling molecule expression and/or distribution. The CUT & RUN assay and dual-luciferase reporter assay were applied to measure protein-DNA interactions.ResultsRAMP1 expression levels were altered during skin wound healing in mice. RAMP1 overexpression promoted MSF proliferation. Mechanistically, total Yes-associated protein (YAP) and nuclear YAP protein expression was increased in RAMP1-overexpressing MSFs. RAMP1 overexpression increased inhibitory guanine nucleotide-binding protein (G protein) α subunit 3 (Gαi3) expression and activated downstream protein kinase A (PKA), and both elevated the expression of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) and activated it, promoting the transcription of YAP, elevating the total YAP level and promoting MSF proliferation.ConclusionsBased on these data, we report, for the first time, that changes in the total RAMP1 levels during wound healing and RAMP1 overexpression alone can promote MSF proliferation via the Gαi3-PKA-CREB-YAP axis, a finding critical for understanding RAMP1 function, suggesting that this pathway is an attractive and accurate nerve target for skin wound treatment.9cQ-_n7Xy-hvoWjx8xDqRQVideo

205 Targeting Homologous Repair to Overcome Genotoxic Therapy Resistance in Pancreatic Cancer

OBJECTIVES/GOALS: Pancreatic ductal adenocarcinoma (PDAC) is a relatively radioresistant disease, and inhibition of DNA homologous recombination (HR) repair in combination with radiation therapy (RT) is a potentially attractive strategy to overcome radioresistance. We have found that the expression of the HR protein RAD18 is upregulated in PDAC cells. METHODS/STUDY POPULATION: Standard clonogenic assays, γH2aX foci staining, HR-GFP reporter assay, and western blot analysis of DNA damage response proteins were performed in MIA-PaCa2 (MP2) and PANC-1 cells following knockdown of RAD18 in cells via short hairpin RNA (shRNA). Drug targeting of RAD18 was achieved through the use of a USP-7 inhibitor, P5091. Cells with or without stable knockdown of RAD18 were implanted orthotopically in the pancreas of athymic nude mice and treated with sham radiation or radiation to a dose of 20 Gy in 5 daily fractions once tumors reached 100-150 mm3. RESULTS/ANTICIPATED RESULTS: Stable knockdown of RAD18 in MP2 and PANC-1 resulted in decreased radiation clonogenic survival in vitro (dose enhancement factor (DEF)=1.52 and 1.51, respectively), decreased DNA repair after radiation as measured by the increased number of γH2aX nuclear foci assay at 6, 12, and 24 hours (all p<0.05), decreased HR activation following DNA damage via an HR-GFP reporter assay (p=0.039), and increased tumor growth delay following radiation in vivo (p<0.001). P5091 treatment of both MP2 and PANC-1 resulted in efficient knockdown of RAD18, which was confirmed through western blotting, qRT-PCR, and luciferase reporter assays. P5091 increased radiosensitization, yH2aX nuclear foci remained elevated at 12 and 24 hours (p<0.05), and HR repair was also reduced (p=0.014). DISCUSSION/SIGNIFICANCE: Herein, we show the HR repair protein RAD18, and that modulation of RAD18 expression correlates with in vitro and in vivo radiosensitization through altered HR-mediated DNA repair. USP7 inhibition successfully reduced RAD18 expression and resulted in enhanced radiosensitization.

The potential mechanism of Fructus Ligustri Lucidi promoting osteogenetic differentiation of bone marrow mesenchymal stem cells based on network pharmacology, molecular docking and experimental identification

ABSTRACT Recent studies have shown that the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteogenic lineages can promotes bone formation and maintains bone homeostasis, which has become a promising therapeutic strategy for skeletal diseases such as osteoporosis. Fructus Ligustri Lucidi (FLL) has been widely used for the treatment of osteoporosis and other orthopedic diseases for thousands of years. However, whether FLL plays an anti-osteoporosis role in promoting the osteogenic differentiation of BMSCs, as well as its active components, targets, and specific molecular mechanisms, has not been fully elucidated. First, we obtained 13 active ingredients of FLL from the Traditional Chinese Medicine Systems Pharmacology (TCSMP) database, and four active ingredients without any target were excluded. Subsequently, 102 common drug-disease targets were subjected to protein-protein interaction (PPI) analysis, Gene Oncology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The results of the three analyses were highly consistent, indicating that FLL promoted the osteogenic differentiation of BMSCs by activating the PI3K/AKT signaling pathway. Finally, we validated previous predictions using in vitro experiments, such as alkaline phosphatase (ALP) staining, alizarin red staining (ARS), and western blot analysis of osteogenic-related proteins. The organic combination of network pharmacological predictions with in vitro experimental validation comprehensively confirmed the reliability of FLL in promoting osteogenic differentiation of BMSCs. This study provides a strong theoretical support for the specific molecular mechanism and clinical application of FLL in the treatment of bone formation deficiency.

Asthma Susceptibility Gene ORMDL3 Promotes Autophagy in Human Bronchial Epithelium.

The genome-wide association study (GWAS)-identified asthma susceptibility risk alleles on chromosome 17q21 increase the expression of ORMDL3 (ORMDL sphingolipid biosynthesis regulator 3) in lung tissue. Given the importance of epithelial integrity in asthma, we hypothesized that ORMDL3 directly impacted bronchial epithelial function. To determine whether and how ORMDL3 expression impacts the bronchial epithelium, in studies using both primary human bronchial epithelial cells and human bronchial epithelial cell line, 16HBE (16HBE14o-), we assessed the impact of ORMDL3 on autophagy. Studies included: autophagosome detection by electron microscopy, RFP-GFP-LC3B to assess autophagic activity, and Western blot analysis of autophagy-related proteins. Mechanistic assessments included immunoprecipitation assays, intracellular calcium mobilization assessments, and cell viability assays. Coexpression of ORMDL3 and autophagy-related genes was measured in primary human bronchial epithelial cells derived from 44 subjects. Overexpressing ORMDL3 demonstrated increased numbers of autophagosomes and increased levels of autophagy-related proteins LC3B, ATG3, ATG7, and ATG16L1. ORMDL3 overexpression promotes autophagy and subsequent cell death by impairing intracellular calcium mobilization through interacting with SERCA2. Strong correlation was observed between expression of ORMDL3 and autophagy-related genes in patient-derived bronchial epithelial cells. Increased ORMDL3 expression induces autophagy, possibly through interacting with SERCA2, thereby inhibiting intracellular calcium influx, and induces cell death, impairing bronchial epithelial function in asthma.

Reduced Expression of Prion Protein With Increased Interferon-β Fail to Limit Creutzfeldt-Jakob Disease Agent Replication in Differentiating Neuronal Cells.

Immortalized uninfected septal (SEP) neurons proliferate but after physiological mitotic arrest they express differentiated neuronal characteristics including enhanced cell-to-cell membrane contacts and ≥ 8 fold increases in host prion protein (PrP). We compared proliferating uninfected and Creutzfeldt-Jakob Disease (CJD) agent infected cells with their arrested counterparts over 33 days by quantitative mRNA and protein blot analyses. Surprisingly, uninfected arrested cells increased interferon-β (IFN-β) mRNA by 2.5–8 fold; IFN-β mRNA elevations were not previously associated with neuronal differentiation. SEP cells with high CJD infectivity titers produced a much larger 40–68-fold increase in IFN-β mRNA, a classic host anti-viral response that is virucidal for RNA but not DNA viruses. High titers of CJD agent also induced dramatic decreases in host PrP, a protein needed for productive agent replication. Uninfected arrested cells produced large sustained 20–30-fold increases in PrP mRNA and protein, whereas CJD arrested cells showed only transient small 5-fold increases in PrP. A > 10-fold increase in infectivity, but not PrP misfolding, induced host PrP reductions that can limit CJD agent replication. In contrast to neuronal lineage cells, functionally distinct migratory microglia with high titers of CJD agent do not induce an IFN-β mRNA response. Because they have 1/50th of PrP of an average brain cell, microglia would be unable to produce the many new infectious particles needed to induce a large IFN-β response by host cells. Instead, microglia and related cells can be persistent reservoirs of infection and spread. Phase separations of agent-associated molecules in neurons, microglia and other cell types can yield new insights into the molecular structure, persistent, and evasive behavior of CJD-type agents.

Proteomic analysis of the mitochondrial glucocorticoid receptor interacting proteins reveals pyruvate dehydrogenase and mitochondrial 60 kDa heat shock protein as potent binding partners

Glucocorticoids are steroid hormones that regulate plethora biological actions such as growth and metabolism, immune response, and apoptosis. Glucocorticoids actions are mediated via glucocorticoid receptors which act mainly as transcription factors, but it is also found to be localized in mitochondria. Mitochondrial localization of the receptor indicates novel functions of the receptor. Characterization of the mitochondrial glucocorticoid receptor (mtGR) interacting proteins will shed light on these actions and the biochemical mechanisms that underlie mitochondrial glucocorticoid receptor import and functions. In this study, applying immunoprecipitation, mass spectrometry and Western blot analysis of the GR interacting proteins in total or mitochondrial extracts of HepG2 cells and of HepG2 cells overexpressing a mitochondrial targeted GR we found pyruvate dehydrogenase (PDH), chaperones such as and heat shock protein (HSP) −60, −70, −75 and −90, and 78 kDa glucose-regulated protein, mitochondrial transcription factors and enzymes involved in the regulation of the mitochondrial protein biosynthesis, lipid metabolism, ATP production and apoptosis as glucocorticoid receptor interacting proteins. Our results uncover potential novel mitochondrial partners of the receptor, suggesting possible new regulatory roles of mtGR in the control of mitochondrial-associated functions of the cell. SignificanceIn this study the mitochondrial GR interacting proteins were characterized highlighting novel regulatory roles of the receptor in mitochondria. Detection of the mtGR/PDH and mtGR/HSP60 interaction in almost all the analyses performed uncovered PDH and HSP60 proteins as potent mtGR binding partners. The interesting finding of the PDH/mtGR interaction possibly indicates involvement of mtGR in the regulation of the balance between glycolytic and oxidative phosphorylation energy production. Characterization of the mitochondrial heat shock −60, −70, −75 and 78 proteins as mtGR binding partners contribute to the characterization of the biochemical mechanisms of the mitochondrial import of the receptor. Moreover, identification of mitochondrial heat shock proteins, metabolic enzymes, transcription factors, OXPHOS, and regulatory molecules in mitochondrial protein biosynthesis as mtGR binding partners indicates possible new regulatory roles of mtGR in the glucocorticoids-induced regulation and orchestration of nuclear and mitochondrial functions, the exact biochemical mechanism of which remain to be established. The study discloses potential new regulatory roles of the receptor in mitochondria, pointing out its importance as a promising target molecule for the control of the mitochondria-associated pathophysiology of the cell.

Resveratrol attenuates chronic pulmonary embolism-related endothelial cell injury by modulating oxidative stress, inflammation, and autophagy

ObjectivesDue to Pulmonary Artery Endothelial Cell (PAEC) dysfunction, Pulmonary Hypertension (PH) persists even after the Pulmonary Embolism (PE) has been relieved. However, the mechanism behind this remains unclear. MethodHere, the authors incubated Human PAECs (HPAECs) with thrombin to simulate the process of arterial thrombosis. ResultsCCK8 results showed a decrease in the viability of HPAECs after thrombin incubation. In addition, the expression of Tissue Factor (TF), Monocyte Chemoattractant Protein 1 (MCP-1), VCAM-1, ICAM-1, cleaved caspase 3, cleaved caspase 9, and Bax protein were all increased after thrombin incubation, while Bcl-2 was decreased. The effects of 3-MA treatment further suggested that autophagy might mediate the partial protective effects of Resveratrol on HPAECs. To observe the effects of Resveratrol in vivo, the authors established a Chronic Thromboembolic Pulmonary Hypertension (CTEPH) model by repeatedly injecting autologous blood clots into a rat's left jugular vein. The results exhibited that Mean Pulmonary Arterial Pressure (mPAP) and vessel Wall Area/Total Area (WA/TA) ratio were both decreased after Resveratrol treatment. Moreover, Resveratrol could reduce the concentration and activity of TF, vWF, P-selectin, and promote these Superoxide Dismutase (SOD) in plasma. Western blot analysis of inflammation, platelet activation, autophagy, and apoptosis-associated proteins in pulmonary artery tissue validated the results in PHAECs. ConclusionsThese findings suggested that reduced autophagy, increased oxidative stress, increased platelet activation, and increased inflammation were involved in CTEPH-induced HPAEC dysfunction and the development of PH, while Resveratrol could improve PAEC dysfunction and PH.

Chondrogenic potential of manganese-loaded composite scaffold combined with chondrocytes for articular cartilage defect

Cartilage is an alymphatic, avascular and non-innervated tissue. Lack of potential regenerative capacity to reconstruct chondral defect has accelerated investigation and development of new strategy for cartilage repair. We prepared a manganese ion-incorporated natupolymer-based scaffold with chitosan-gelatin by freeze-drying procedure. The scaffold was characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy, energy dispersive spectroscopy, compressive testing, and analysis of porosity and flexibility. Live/dead assay confirmed the good cytocompatibility of prepared scaffold on rat articular chondrocytes after 10 days and 4 weeks of culture. The manganese-loaded composite scaffold upregulated the expression of chondrogenic-related markers (Sox9, integrin, and Col II) in chondrocytes. Western blot analysis of proteins extracted from chondrocytes grown on scaffolds indicated the signaling pathways of p-Akt and p-ERK1/2 played a key role. Histological analysis following implantation of current composite scaffold loaded with chondrocytes into a rat articular cartilage defect model showed that the scaffolds promoted the formation of collagen II and cartilage repair. These findings suggested the potential of manganese-loaded scaffold to promote new cartilage formation and a promising strategy for articular cartilage engineering application.

Rhizoctonia bataticola lectin induces apoptosis and inhibits metastasis in ovarian cancer cells by interacting with CA 125 antigen differentially expressed on ovarian cells.

A N-glycan specific lectin from Rhizoctonia bataticola [RBL] was shown to induce growth inhibitory and apoptotic effect in human ovarian, colon and leukemic cells but mitogenic effect on normal PBMCs as reported earlier, revealing its clinical potential. RBL has unique specificity for high mannose tri and tetra antennary N-glycans, expressed in ovarian cancer and also recognizes glycans which are part of CA 125 antigen, a well known ovarian cancer marker. Hence, in the present study diagnostic and therapeutic potential of RBL was investigated using human ovarian epithelial cancer SKOV3 and OVCAR3 cells known for differentially expressing CA 125. RBL binds differentially to human ovarian normal, cyst and cancer tissues. Flow cytometry, western blot analysis of membrane proteins showed the competitive binding of RBL and CA 125 antibody for the same binding sites on SKOV3 and OVCAR3 cells. RBL has strong binding to both SKOV3 and OVCAR3 cells with MFI of 173 and 155 respectively. RBL shows dose and time dependent growth inhibitory effect with IC50 of 2.5 and 8μg/mL respectively for SKOV3 and OVCAR3 cells. RBL induces reproductive cell death, morphological changes, nuclear degradation and increased release of ROS in SKOV3 and OVCAR3 cells leading to cell death. This is also supported by increase in hypodiploid population, altered MMP leading to apoptosis possibly involving intrinsic pathway. Adhesion, wound healing, invasion and migration assays demonstrated anti-metastasis effect of RBL apart from its growth inhibitory effect. These results show the promising potential of RBL both as a diagnostic and therapeutic agent.

Bone marrow mesenchymal stem cell-derived exosomes inhibit chondrocyte apoptosis and the expression of MMPs by regulating Drp1-mediated mitophagy

Osteoarthritis (OA) is a joint degenerative disease commonly seen in the elderly. Bone marrow mesenchymal stem cell-exosomes (BMSC-exosomes) are closely associated with the progression of OA. Here, we investigated whether BMSC-exosomes can affect OA development by regulating mitophagy. Primary rat chondrocytes were treated with advanced glycation end products (AGEs) to induce cell damage. The results of flow cytometry showed that AGEs treatment significantly promoted apoptosis of chondrocytes. AGEs treatment also enhanced the expression of matrix metalloproteinases (MMPs), MMP-3 and MMP-13, and dynamin-related protein 1 (Drp1) in chondrocytes. To investigate the impact of BMSC-exosomes on chondrocytes, chondrocytes were treated with BMSC-exosomes. AGEs-mediated increase of apoptosis and up-regulation of MMP-3, MMP-13, and Drp1 in chondrocytes were abrogated by BMSC-exosomes. Western blot analysis of autophagy-related proteins and Mito-Keima assay revealed that BMSC-exosome treatment elevated the expression of autophagy-related proteins, LC3-II/LC3-I and Beclin-1, and promoted mitophagy in the AGEs-treated chondrocytes. Moreover, Drp1 overexpression repressed the expression of LC3-II/LC3-I and Beclin-1, and enhanced apoptosis and the expression of MMP-3 and MMP-13 in AGEs-treated chondrocytes. BMSC-exosomes reversed the impact of Drp1 overexpression on AGEs-treated chondrocytes. In conclusion, this work demonstrates that BMSC-exosomes inhibit chondrocyte apoptosis and the expression of MMPs, which attributes to regulate Drp1-mediated mitophagy. Thus, BMSC-exosomes may be a potential treatment for OA.

Electroacupuncture in promoting neural repair after spinal cord injury: Inhibiting the Notch signaling pathway and regulating downstream proteins expression.

Spinal cord injury (SCI) is one of the serious central nervous system injuries and the incidence of SCI continues to increase. Previous studies have indicated that electroacupuncture (EA) is beneficial for promoting recovery after SCI. In the present study, we attempted to evaluate how EA can promote the neural repair in SCI model rats by observing changes in the Notch signaling pathway. Experimental rats were randomly divided into four groups. Each group had its own intervention period: 1 day, 7 days, 14 days, and 28 days, and five randomized subgroups: blank control (B) group, blank electroacupuncture (BE) group, sham operation (S) group, model control (M) group and EA group. Animals in the EA group and the BE group were treated with EA at Dazhui (GV14) and Mingmen (GV4) acupoints for 20 min. After the intervention period, the Basso-Beattie-Bresnahan (BBB) score was used to evaluate the neurological function. We found that BBB score increased in EA-treated groups. Hematoxylin and eosin staining was used to observe pathological changes in the injured spinal cord and the results showed that EA therapy could promote the repair of injured spinal cord tissue. Immunohistochemistry and Western blot methods were used to detect the expression of proteins Delta1, Presenilin1, Hes1, and Hes5 in the injured spinal cord. The results showed that the expression levels of Delta1, Presenilin1, Hes1, and Hes5 increased significantly after SCI and decreased after EA treatment. Our study suggested that the possible mechanism by which EA could benefit the recovery after SCI in rats may include inhibiting the Notch signaling pathway and regulating the downstream proteins expression. In addition, our study can provide reference for selecting acupoints and treatment cycle in the treatment of SCI.

Characterization and phytochemical constituents of Periploca hydaspidis Falc crude extract and its anticancer activities.

The current study aims to investigate the anticancer potential of Periploca hydaspidis extracts against HCCLM3 and MDA-MB 231 cell lines with invasive properties and to identify molecular targets underlying its action mechanism. Cytotoxic screening of plant extracts was done via MTT assay against liver and breast cancer cell lines and GC/MS of the best cytotoxic fraction was performed to identify its chemical composition. Flow cytometry detected apoptosis and cell-cycle changes after drug treatment. The specified cells were studied for migration and invasion potential along with performing western blot analysis of proteins involved in apoptosis, cell-cycle, metastasis, and MAPK (Mitogen-activated protein kinase) cell-signaling pathway. The results revealed the crude methanol (PHM) fraction of P. hydaspidis shown dose and time dependent cell-proliferative inhibition response. GC/MS analysis detected 54 compounds of which fatty acids (29.8%), benzenoids (15.7%), and esters (14.3%) constituted the bulk. The inhibitory effect against cancer cells was linked with cell-cycle arrest at G0/G1 phase, induction of apoptosis, reduced migration and invasion capabilities post treatment. PHM induced apoptosis via downregulation of anti-apoptotic (survivin, B-cell lymphoma Extra-large; BCL-XL, X-linked inhibitor of apoptosis protein; XIAP, Myelocytomatosis; C-myc), metastatic (Matrix metallopeptidases 9/2; MMP9/2), and cell-cycle regulatory (cyclin D1 and E) proteins, whereas upregulation of pro-apoptotic proteins (Bcl-2 homologous antagonist/killer; BAK, Bcl-2-Associate X protein; BAX, cleaved caspases; 3,7,8,9, and PARP) and activation of MAPK (Jun amino-terminal kinase; JNK and P38) pathway. P38 was needed for PHM-induced apoptosis, where the inhibition of P38 by pharmacological inhibitor (SB239063) diminished the apoptotic effects. Overall, our results conclude that PHM can inhibit cell-proliferation and induce apoptotic effects by activation of P38 MAPK cell-signaling pathway. This suggests the methanol fraction of P. hydaspidis (PHM) to have anticancer compounds, potentially useful for treating liver and breast cancer. In future, one-step advance studies of PHM regarding its role in metastatic inhibition, immune response modulation for reducing tumor, and inducing apoptosis in suitable animal models would be an interesting and promising research area.

Two γ-zeins induce the unfolded protein response.

The rapid, massive synthesis of storage proteins that occurs during seed development stresses endoplasmic reticulum (ER) homeostasis, which activates the ER unfolded protein response (UPR). However, how different storage proteins contribute to UPR is not clear. We analyzed vegetative tissues of transgenic Arabidopsis (Arabidopsis thaliana) plants constitutively expressing the common bean (Phaseolus vulgaris) soluble vacuolar storage protein PHASEOLIN (PHSL) or maize (Zea mays) prolamins (27-kDa γ-zein or 16-kDa γ-zein) that participate in forming insoluble protein bodies in the ER. We show that 16-kDa γ-zein significantly activates the INOSITOL REQUIRING ENZYME1/BASIC LEUCINE ZIPPER 60 (bZIP60) UPR branch-but not the bZIP28 branch or autophagy-leading to induction of major UPR-controlled genes that encode folding helpers that function inside the ER. Protein blot analysis of IMMUNOGLOBULIN-BINDING PROTEIN (BIP) 1 and 2, BIP3, GLUCOSE REGULATED PROTEIN 94 (GRP94), and ER-localized DNAJ family 3A (ERDJ3A) polypeptides confirmed their higher accumulation in the plant expressing 16-kDa γ-zein. Expression of 27-kDa γ-zein significantly induced only BIP3 and ERDJ3A transcription even though an increase in GRP94 and BIP1/2 polypeptides also occurred in this plant. These results indicate a significant but weaker effect of 27-kDa γ-zein compared to 16-kDa γ-zein, which corresponds with the higher availability of 16-kDa γ-zein for BIP binding, and indicates subtle protein-specific modulations of plant UPR. None of the analyzed genes was significantly induced by PHSL or by a mutated, soluble form of 27-kDa γ-zein that traffics along the secretory pathway. Such variability in UPR induction may have influenced the evolution of storage proteins with different tissue and subcellular localization.

NIR Laser Photobiomodulation Induces Neuroprotection in an In Vitro Model of Cerebral Hypoxia/Ischemia

Brain photobiomodulation (PBM) is an innovative treatment for a variety of neurological conditions, including cerebral ischemia. However, the capability of PBM for ischemic stroke needs to be further explored and its mechanisms of action remain currently unclear. The aim of the present research was to identify a treatment protocol capable of inducing neuroprotection and to investigate the molecular mechanisms activated by a dual-wavelength near infrared (NIR) laser source in an organotypic hippocampal slice model of hypoxia/ischemia. Hippocampal slices were exposed to oxygen and glucose deprivation (OGD) for 30 min followed by NIR laser light (fluence 3.71, 7.42, or 14.84 J/cm2; wavelengths 808 nm and 905 nm) delivered immediately or 30 min or 60 min after OGD, in order to establish a therapeutic window. Neuronal injury was assessed by propidium iodide fluorescence 24 h later. Our results show that NIR laser irradiation attenuates OGD neurotoxicity once applied immediately or 30 min after OGD. Western blot analysis of proteins involved in neuroinflammation (iNOS, COX-2, NFkB subunit p65, and Bcl-2) and in glutamatergic-mediated synaptic activity (vGluT1, EAAT2, GluN1, and PSD95) showed that the protein modifications induced by OGD were reverted by NIR laser application. Moreover, CA1 confocal microscopy revealed that the profound morphological changes induced by OGD were reverted by NIR laser radiation. In conclusion, NIR laser radiation attenuates OGD neurotoxicity in organotypic hippocampal slices through attenuation of inflammatory mechanisms. These findings shed light on molecular definition of NIR neuroprotective mechanisms, thus underlining the potential benefit of this technique for the treatment of cerebral ischemia.

RAMS11 promotes CRC through mTOR-dependent inhibition of autophagy, suppression of apoptosis, and promotion of epithelial-mesenchymal transition

BackgroundLong non-coding RNAs (lncRNAs), a class of non-coding RNAs (ncRNAs) associated with diverse biological processes of cells. Over the past decades, cumulating research evidences revealed that abnormal expressions of lncRNAs are associated with colorectal cancer (CRC) initiation, progression, metastasis, and resistance to therapies. Moreover, their usefulness as candidate biomarkers for CRC diagnosis and prognosis are well evident throughout previous literature. In the current study, we examined the role and molecular mechanisms of newly identified lncRNA named RNA associated with metastasis-11 (RAMS11) in CRC development.MethodsThe expression of RAMS11 in CRC cell lines DLD-1, HT-29, HCT-116, and SW480 and colon normal cells CCD-112-CoN were evaluated by quantitative RT-qPCR. The results showed that the RAMS11 is significantly upregulated in CRC cell lines compared to the normal cells. The CCK-8 proliferation assay, colony formation assay, and migration assay were performed to evaluate the biological and physiological functions of RAMS11 in vitro. To decipher the molecular mechanisms of RAMS11 medicated CRC progression, we further performed western blot analysis of the key pathway proteins (e.g., AMPK, AKT, and mTOR).ResultsOur results revealed that higher expression of RAMS11 is associated with increased CRC proliferation, migration, and development of metastasis. Knockdown of RAMS11 induced autophagy, apoptosis along with reduction of epithelial-mesenchymal transition (EMT) suggesting that RAMS11 is involved in CRC progression. The molecular mechanisms of RAMS11 indicated that knockdown of RAMS11 significantly inhibited CRC carcinogenesis through mTOR-dependent autophagy induction.ConclusionsIn sum, our results suggested that RAMS11 is an important oncogene in CRC pathogenesis. Targeting RAMS11 could be a potential therapeutic strategy for CRC management.

Altered Grooming Syntax and Amphetamine-Induced Dopamine Release in EAAT3 Overexpressing Mice.

The excitatory amino acid transporter EAAT3 plays an important role in the neuronal uptake of glutamate regulating the activation of glutamate receptors. Polymorphisms in the gene-encoding EAAT3 have been associated with obsessive–compulsive disorder (OCD), although the mechanisms underlying this relationship are still unknown. We recently reported that mice with increased EAAT3 expression in forebrain neurons (EAAT3glo/CMKII) display behavioral and synaptic features relevant to OCD, including increased grooming, higher anxiety-like behavior and altered cortico-striatal synaptic function. The dopamine neurotransmitter system is implicated in ritualistic behaviors. Indeed, dopaminergic neurons express EAAT3, and mice lacking EAAT3 exhibit decreased dopamine release and decreased expression of the dopamine D1 receptor. Moreover, EAAT3 plays a role on the effect of the psychostimulant amphetamine. As such, we sought to determine if the OCD-like behavior in EAAT3glo/CMKII mice is accompanied by altered nigro-striatal dopaminergic transmission. The aim of this study was to analyze dopamine transmission both in basal conditions and after an acute challenge of amphetamine, using behavioral, neurochemical, molecular, and cellular approaches. We found that in basal conditions, EAAT3glo/CMKII mice performed more grooming events and that they remained in phase 1 of the grooming chain syntax compared with control littermates. Administration of amphetamine increased the number of grooming events in control mice, while EAAT3glo/CMKII mice remain unaffected. Interestingly, the grooming syntax of amphetamine-control mice resembled that of EAAT3glo/CMKII mice in basal conditions. Using in vivo microdialysis, we found decreased basal dopamine levels in EAAT3glo/CMKII compared with control mice. Unexpectedly, we found that after acute amphetamine, EAAT3glo/CMKII mice had a higher release of dopamine compared with that of control mice, suggesting that EAAT3 overexpression leads to increased dopamine releasability. To determine postsynaptic effect of EAAT3 overexpression over dopamine transmission, we performed Western blot analysis of dopaminergic proteins and found that EAAT3glo/CMKII mice have higher expression of D2 receptors, suggesting a higher inhibition of the indirect striatal pathway. Together, the data indicate that EAAT3 overexpression impacts on dopamine transmission, making dopamine neurons more sensitive to the effect of amphetamine and leading to a disbalance between the direct and indirect striatal pathways that favors the performance of repetitive behaviors.

Knockdown of Lingo-1 by short hairpin RNA promotes cognitive function recovery in a status convulsion model

The purpose of this study was to determine the dynamic changes of the Nogo-66 receptor 1 (NgR1) pathway during epileptogenesis and the potential beneficial of leucine-rich repeat and Ig-like domain-containing Nogo receptor interacting protein 1 (Lingo-1) inhibition on epilepsy rats. The hippocampal changes of the NgR1 pathway during epileptogenesis were determined by western blot analysis of multiple proteins, including neurite outgrowth inhibitor protein A (NogoA), myelin-associated glycoprotein (MAG), oligodendrocyte-myelin glycoprotein (OMgp), Lingo-1, ras homolog family member A (RhoA) and phosphorylated RhoA (p-RhoA). Lentivirus-mediated short hairpin RNA (shRNA) was used to knockdown the hippocampal expression of Lingo-1. Novel object recognition (NOR) test and Morris Water Maze (MWM) test were employed to determine the cognitive functions of rats. Hematoxylin and eosin (H&E) staining, protein expressions of RhoA, p-RhoA, and myelin basic protein (MBP), as well as convulsion susceptibility test were additionally performed. Our results showed that the NgR1 pathway was activated during epileptogenesis, characterized by up-regulation of NogoA, MAG, OMgp, and Lingo-1, which was especially significant at the chronic phase of epilepsy. The cognitive function, convulsion susceptibility and hippocampal neuronal survival of rats were impaired at the chronic phase of epileptogenesis but all improved by Lingo-1 inhibition; besides, the hippocampal protein expressions of p-RhoA and MBP were significantly decreased at the chronic phase of SC rats but increased after Lingo-1 inhibition. Our results demonstrated that Lingo-1 shRNA can improve epilepsy-induced cognitive impairment, which may be related with the pro-myelination and neuroprotection effects of Lingo-1 inhibition.

Apoptosis in platelets from adult patients with chronic idiopathic thrombocytopenic purpura.

Adult chronic idiopathic thrombocytopenic purpura (cITP) is a chronic and usually life-long haemorrhagic disorder in which enhanced platelet destruction and weakened platelet production lead to thrombocytopenia. Platelets were isolated from blood samples collected from 40 adult patients with cITP and 40 healthy volunteers. Mitochondrial membrane potential (ΔΨm) and plasma membrane phosphatidylserine externalization were determined by flow cytometry, and activation of caspase-3 and expressions of Bax, Bak and Bcl-xL were analysed by western blotting. Flow cytometry showed increased mitochondrial depolarization and lower ΔΨm in platelets from adult patients with cITP. In addition, plasma membrane phosphatidylserine externalization was observed on platelets from adult patients with cITP, but rarely from healthy volunteers. Western blot analysis of platelet proteins revealed that, in adult cITP patients, caspase-3 was activated, which cleaved gelsolin and to release a 47-kDa fragment. Moreover, the expressions of Bax and Bak were elevated, and Bcl-xL was decreased markedly in platelets from adult patients with cITP. Our findings reveal, based on loss of mitochondrial membrane potential (Δψm), phosphatidylserine exposure, caspase-3 activation, enhanced expression of Bax and Bak, and attenuated expression of Bcl-xL, that platelet death in the pathogenesis of thrombocytopenia in chronic ITP in adults is apoptotic.