V(D)J recombination is initiated by a specialized transposase consisting of the subunits RAG-1 and RAG-2. The susceptibility of gene segments to DNA cleavage by the V(D)J recombinase is correlated with epigenetic modifications characteristic of active chromatin, including trimethylation of histone H3 on lysine 4 (H3K4me3). Engagement of H3K4me3 by a plant homeodomain (PHD) in RAG-2 promotes recombination in vivo and stimulates DNA cleavage by RAG in vitro. We now show that H3K4me3 acts allosterically at the PHD finger to relieve autoinhibition imposed by a separate domain within RAG-2. Disruption of this autoinhibitory domain was associated with constitutive increases in recombination frequency, DNA cleavage activity, substrate binding affinity and catalytic rate, thus mimicking the stimulatory effects of H3K4me3. Our observations support a model in which allosteric control of RAG is enforced by an autoinhibitory domain whose action is relieved by engagement of active chromatin.
In patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe discovery of non-peptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used for discovery of other classes of antigen surrogates.
Chronic Lymphocytic Leukemia; Combinatorial Chemistry; B Cell Receptor; Antigen Surrogate; Antibody
N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation. Ac-SDKP plasma concentration is increased 5-fold after angiotensin-converting enzyme inhibition. Here we studied the effect of Ac-SDKP on monocyte/macrophage infiltration, fibroblast proliferation, and collagen deposition in the rat heart in renovascular hypertension.
Methods and Results
We investigated whether long-term Ac-SDKP administration would prevent left ventricular (LV) hypertrophy and interstitial collagen deposition in rats with 2-kidney, 1-clip (2K-1C) hypertension. Ac-SDKP (400 μg · kg−1 · d−1) did not affect development of hypertension. Mean blood pressure was similar in rats with 2K-1C hypertension whether they were given vehicle or Ac-SDKP and was higher than in controls. Both LV weight and cardiomyocyte size were significantly increased in rats with 2K-1C hypertension compared with controls and were unaffected by Ac-SDKP. Proliferating cell nuclear antigen- and monocyte/macrophage-positive cells were increased in the LV of 2K-1C hypertensive rats; this increase was significantly blunted by Ac-SDKP (P<0.001). LV interstitial collagen fraction was also increased in 2K-1C hypertensive rats given vehicle (10.1±0.8%) compared with sham (5.3±0.1%, P<0.0001), and this increase was prevented by Ac-SDKP (5.4±0.4%, P<0.001).
Ac-SDKP inhibited monocyte/macrophage infiltration, cell proliferation, and collagen deposition in the LV of hypertensive rats without affecting blood pressure or cardiac hypertrophy, suggesting that it may be partly responsible for the cardioprotective effect of angiotensin-converting enzyme inhibitors.
angiotensin; hypertension, renal; blood pressure; inhibitors; collagen; myocardium
Primary Sjögren’s syndrome (pSS) is one of the most common chronic systemic autoimmune diseases, and thrombocytopenia is one of the hematological manifestations of pSS. When platelet and endothelial cells are activated, P-selectin is expressed on the cell surface. This study aimed to investigate the role of P-selectin autoantibodies in the pathogenesis of thrombocytopenia in pSS.
P-selectin autoantibodies were measured by enzyme-linked immunosorbent assay (ELISA) in 38 pSS patients without thrombocytopenia and 32 pSS patients with thrombocytopenia, 32 idiopathic thrombocytopenic purpura (ITP) patients, and 35 healthy controls.
The plasma P-selectin autoantibodies (A490) in ITP patients and pSS patients with/without thrombocytopenia were significantly higher than those in healthy controls, but there were no significant differences between ITP patients and pSS patients with thrombocytopenia. The positive rate of P-selectin autoantibodies in pSS patients with thrombocytopenia was significantly higher than that in ITP patients. The platelet count was lower in P-selectin autoantibodies-positive patients, while among pSS patients with thrombocytopenia, the platelet count was lower in P-selectin autoantibodies-positive patients than in P-selectin autoantibodies-negative patients. In ITP patients and pSS patients with thrombocytopenia, the platelet count was lower in P-selectin autoantibodies-positive patients.
Elevated plasma P-selectin autoantibodies may play a role in the pathogenesis of thrombocytopenia in pSS patients.
Autoantibodies, P-Selectin; Endothelial Cells; Purpura, Thrombocytopenic, Idiopathic; Sjögren’s Syndrome, Primary; Thrombocytopenia
Innate immune responses are regulated in the intestine to prevent excessive inflammation. Here we show that a subset of mouse colonic macrophages constitutively produce the anti-inflammatory cytokine IL-10. In mice infected with Citrobacter rodentium, a model for enteropathogenic Escherichia coli infection in humans, these macrophages are required to prevent intestinal pathology. IL-23 is significantly increased in infected mice with a myeloid cell-specific deletion of IL-10, and the addition of IL-10 reduces IL-23 production by intestinal macrophages. Furthermore, blockade of IL-23 leads to reduced mortality in the context of macrophage IL-10 deficiency. Transcriptome and other analyses indicate that IL-10-expressing macrophages receive an autocrine IL-10 signal. Interestingly, only transfer of the IL-10 positive macrophages could rescue IL-10 deficient infected mice. Therefore, these data indicate a pivotal role for intestinal macrophages that constitutively produce IL-10, in controlling excessive innate immune activation and preventing tissue damage after an acute bacterial infection.
To explore the protective effect of dexmedetomidine (Dex) on rats with renal ischemia-reperfusion injury and the influence of Dex on the expression of tight junction protein in kidney. Grouped 40 SPF male rats into 4 groups, sham operation group (group S), ischemia-reperfusion group (group I/R), pretreatment with Dex group (group Pre/Dex), post-treatment with Dex group (group Post/Dex), randomly, 10 rats each group. Rats in group S were anaesthetized and set up with removal of right kidney; rats in group I/R were set up with removal of right kidney and left renal artery clamping for 45 min followed by 60 min reperfusion; rats in group Pre/Dex were intravenous injected with Dex (1 μg/kg) for 30 min after indwelling catheter via femoral vein puncture; rats in group Post/Dex were intravenous injected with Dex (1 μg/kg) for 30 min after left renal reperfusion. The kidneys in each group were made out pathologic slices after 6 h I/R, stained with HE; blood samples were taken with separation plasma, creatinine (Scr) and urea nitrogen (BUN) were detected by automatic biochemical analyzer; IL-1β and TNF-α were detected by Enzyme-linked Immunosorbent Assay (ELISA); the expression level of tight junction protein ZO-1 and protein occludin in kidney were detected by Western-blot. The results of HE staining showed that, comparing to group S, the tissue of kidney in group I/R were damaged heavily with tubules dilatation and inflammation obviously, while lightened in group Pre/Dex and group Post/Dex. The results of detection of renal function and inflammatory factors showed that, comparing to group S, Scr, BUN, IL-1β and TNF-α were all enhanced in group I/R, group Pre/Dex and group Post/Dex, significantly (P < 0.05), while the inflammatory factors in group Pre/Dex and group Post/Dex were lower than in group I/R, significantly (P < 0.05). The results of Western-blot showed that the expression of protein ZO-1 and occludin in group Pre/Dex and group Post/Dex were higher than in group I/R, significantly (P < 0.05). Dex could reduce renal dysfunction induced by I/R, inhibit inflammatory response, up-regulate the expression of protein ZO-1 and occludin and protect renal.
Dexmedetomidine (Dex); renal; ischemia-reperfusion; renal function; tight junction protein
Immune-mediated liver injury is widely seen during hepatitis B virus (HBV) infection. Unsuccessful immune clearance of HBV results in chronic hepatitis and increases the risk of liver cirrhosis and hepatocellular carcinoma. HBV-related liver fibrosis (HBVLF), occurring as a result of HBV-induced chronic hepatitis, is a reversible, intermediate stage of chronic hepatitis B (CHB) and liver cirrhosis. Therefore, defining the pathogenesis of HBVLF is of practical significance for achieving better clinical outcomes. Recently, the homeostasis of CD4+ T cells was considered to be pivotal in the process of HBVLF. To better uncover the underlying mechanisms, in this review, we systematically retrospect the impacts of different CD4+ T-cell subsets on CHB and HBVLF. We emphasize CD4+ T-cell homeostasis and the important balance between regulatory T (Treg) and T helper 17 (Th17) cells. We discuss some cytokines associated with Treg and Th17 cells such as interleukin (IL)-17, IL-22, IL-21, IL-23, IL-10, IL-35 and IL-33, as well as surface molecules such as programmed cell death protein 1, cytotoxic T lymphocyte-associated antigen 4, T cell immunoglobulin domain and mucin domain-containing molecule 3 and cannabinoid receptor 2 that have potential therapeutic implications for the homeostasis of CD4+ T cells in CHB and HBVLF.
Homeostasis; Regulatory T cells; T helper 17 cells; CD4+ T cells; Liver fibrosis; Chronic hepatitis B; Pathogenesis; Therapy
Background and Objectives
Darapladib is a lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibitor. This study evaluated the pharmacokinetics, pharmacodynamics and safety of darapladib in healthy Chinese subjects.
Twenty-four subjects received darapladib 160 mg orally, approximately 1 hour after a standard breakfast, as a single dose and once daily for 28 days. Non-compartmental methods were used to determine the single and multiple dose pharmacokinetics of darapladib and its metabolite SB-553253. Repeat dose Lp-PLA2 activity and safety were evaluated.
Systemic exposure (AUC(0-T), Cmax geometric mean (CVb%)) of darapladib was higher after multiple-dosing (519 ng.h/mL (33.3%), 34.4 ng/mL (49.9%)) compared to single-dose administration (153 ng.h/mL (69.0%), 17.9 ng/mL (55.2%). The steady-state accumulation ratio was less than unity (Rs = 0.80), indicating time-dependent pharmacokinetics of darapladib. Darapladib steady-state was reached by Day 14 of once daily dosing. Systemic exposure to SB-553253 was lower than darapladib with median (SB-553253: darapladib) ratios for AUC(0-τ) of 0.0786 for single dose and 0.0532 for multiple dose administration. On Day 28, pre-dose and maximum inhibition of Lp-PLA2 activity was approximately 70% and 75% relative to the baseline value, respectively and was dependent of darapladib concentration. The most common adverse events (≥ 21% subjects) were abnormal faeces, abnormal urine odour, diarrhoea and nasopharyngitis.
Darapladib 160 mg single and repeat doses were profiled in healthy Chinese subjects. Single dose systemic exposure to darapladib in healthy Chinese subjects was consistent with that observed previously in Western subjects whereas steady-state systemic exposure was approximately 65% higher in Chinese than Western subjects. The Lp-PLA2 activity and adverse event profile were similar in healthy Chinese and previous reports in Western subjects. Ethnic-specific dose adjustment of darapladib is not considered necessary for the Chinese population.
Kinins exert cardioprotective effects via 2 G-protein-coupled receptors, B1 and B2. Using B1 kinin receptor gene knockout mice (B1
−/−), we tested the hypotheses that the B1 receptor plays an important role in preservation of cardiac function, whereas lack of B1 may accelerate cardiac remodeling and dysfunction after myocardial infarction, and that B2 receptors may compensate for lack of B1, whereas blockade of B2 receptors in B1
−/− mice may cause further deterioration of cardiac function and remodeling. Female B1
−/− mice and wild-type controls (C57BL/6J, B1
+/+) underwent sham surgery or myocardial infarction and were treated with either vehicle or B2-antagonist (icatibant, 500 μg/kg per day, subcutaneous) for 8 weeks. We found that in sham myocardial infarction, B1
−/− mice had a larger left ventricular diastolic chamber dimension both initially and at 4 to 8 weeks compared with B1
+/+. Left ventricular mass and myocyte size were also larger in B1
−/− with sham operation than in B1
+/+, although cardiac function did not differ between strains. After myocardial infarction, cardiac remodeling and function were similar in both strains, although B1
−/− mice tended to have lower blood pressure. Blockade of B2 receptors tended to worsen cardiac remodeling and dysfunction in B1
−/− but not in B1
+/+. These results may suggest that B2 receptors play an important role in compensating for lack of B1 receptors in mice with myocardial infarction. Dual blockade of both B1 and B2 eliminates this compensation, leading to further deterioration of cardiac dysfunction and remodeling after myocardial infarction.
kinins; myocardial infarction; mice
Emerging evidence suggests that synaptic dysfunction occurs prior to neuronal loss in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS). Therefore, monitoring synaptic activity during early stages of neurodegeneration may provide valuable information for the development of diagnostic and/or therapeutic strategies. Here, we describe an electrophysiological method routinely applied in our laboratory for investigating synaptic activity of the neuromuscular junction (NMJ), the synaptic connection between motoneurons and skeletal muscles. Using conventional intracellular sharp electrodes, both spontaneous synaptic activity (miniature end-plate potentials) and evoked synaptic activity (end-plate potentials) can be readily recorded in acutely isolated nerve–muscle preparations. This method can also be adapted to various simulation protocols for studying short-term plasticity of neuromuscular synapses.
Amyotrophic lateral sclerosis; Miniature end-plate potential; End-plate potential; Intracellular recording; Neuromuscular junction; Neurodegeneration
Shikonin is an anthraquinone derivative extracted from the root of lithospermum. Shikonin is traditionally used in the treatment of inflammatory and infectious diseases such as hepatitis. Shikonin also inhibits proliferation and induces apoptosis in various tumors. However, the effect of shikonin on gliomas has not been fully elucidated. In the present study, we aimed to investigate the effects of shikonin on the migration and invasion of human glioblastoma cells as well as the underlying mechanisms. U87 and U251 human glioblastoma cells were treated with shikonin at 2.5, 5, and 7.5 μmol/L and cell viability, migration and invasiveness were assessed with CCK8, scratch wound healing, in vitro Transwell migration, and invasion assays. The expression and activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) and the expression of phosphorylated β-catenin (p-β-catenin) and phosphorylated PI3K/Akt were also checked. Results showed that shikonin significantly inhibited the cell proliferation, migration, invasion, and expression of MMP-2 and MMP-9 in U87 and U251 cells. The expression of p-β-catenin showed contrary trends in two cell lines. It was significantly inhibited in U87 cells and promoted in U251 cells. Results in this work indicated that shikonin displayed an inhibitory effect on the migration and invasion of glioma cells by inhibiting the expression and activity of MMP-2 and -9. In addition, shikonin also inhibited the expression of p-PI3K and p-Akt to attenuate cell migration and invasion and MMP-2 and MMP-9 expression in both cell lines, which could be reversed by the PI3K/Akt pathway agonist, insulin-like growth factor-1 (IGF-1).
shikonin; glioma; migration; invasion; β-catenin; phosphorylated PI3K/Akt
Mechanistic (or mammalian) target of rapamycin (mTOR) plays important roles in cell growth and proliferation. In esophageal squamous cell carcinoma (SCC), high expression of phosphorylated (activated) mTOR (p-mTOR) has been reported as an adverse prognostic factor in some but not all studies. The signals of mTOR pathway and mitogen-activated protein kinase (MAPK) pathway converge on 4E-binding protein 1 (4EBP1), which drives the downstream proliferative signals. We previously found that high expression of phosphorylated 4EBP1 (p-4EBP1) is an adverse prognostic factor in esophageal SCC. Podoplanin is a type-1 transmembrane glycoprotein expressed in various normal human tissues, including lymphatic endothelium. Our previous study showed that high podoplanin expression correlates with clinical nodal metastasis, which is associated with short survival in esophageal SCC. In current study, we investigated p-mTOR expression by immunohistochemistry in 75 cases of surgically resected esophageal SCC. The result was correlated with p-4EBP1 expression, podoplanin expression, clinicopathologic features and patient survival. We found that high p-mTOR expression was significantly associated with high podoplanin expression (P = 0.0030) and high tumor grade (P = 0.0014). No correlation with p-4EBP1 expression, patient survival or other clinicopathologic features was found. Recently, podoplanin expression in astrocytic brain tumors was found to be regulated by the phosphatidylinositol 3-kinase (PI3K)/AKT/activator protein-1 (AP-1) pathway. Similarly, mTOR is activated by a PI3K/AKT/mTOR pathway. The association of p-mTOR and podoplanin expression in our study could be due to a common upstream pathway. Since both mTOR and podoplanin are potential therapeutic targets, the possible benefit of combined targeted therapy warrants further investigation.
Mechanistic target of rapamycin (mTOR); podoplanin; 4E-binding protein 1 (4EBP1); tumor grade; esophagus; squamous cell carcinoma
NADPH is a key reductant carrier that maintains internal redox and antioxidant status, and that links biosynthetic, catabolic and signalling pathways. Plants have a mitochondrial external NADPH oxidation pathway, which depends on Ca2+ and pH in vitro, but concentrations of Ca2+ needed are not known. We have determined the K0.5(Ca2+) of the external NADPH dehydrogenase from Solanum tuberosum mitochondria and membranes of E. coli expressing Arabidopsis thaliana NDB1 over the physiological pH range using O2 and decylubiquinone as electron acceptors. The K0.5(Ca2+) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K0.5(Ca2+) for NADPH oxidation was high (≈100 μM), yet 20-fold lower K0.5(Ca2+) values were determined at pH 6.8. Lower K0.5(Ca2+) values were observed with decylubiquinone than with O2 as terminal electron acceptor. NADPH oxidation responded to changes in Ca2+ concentrations more rapidly than NADH oxidation did. Thus, cytosolic acidification is an important activator of external NADPH oxidation, by decreasing the Ca2+-requirements for NDB1. The results are discussed in relation to the present knowledge on how whole cell NADPH redox homeostasis is affected in plants modified for the NDB1 gene.
The development of renewable biofuels is a global priority, but success will require novel technologies that greatly improve our understanding of microbial systems biology. An approach with great promise in enabling functional characterization of microbes is activity-based protein profiling (ABPP), which employs chemical probes to directly measure enzyme function in discrete enzyme classes in vivo and/or in vitro, thereby facilitating the rapid discovery of new biocatalysts and enabling much improved biofuel production platforms. We review general design strategies in ABPP, and highlight recent advances that are or could be pivotal to biofuels processes including applications of ABPP to cellulosic bioethanol, biodiesel, and phototrophic production of hydrocarbons. We also examine the key challenges and opportunities of ABPP in renewable biofuels research. The integration of ABPP with molecular and systems biology approaches will shed new insight on the catalytic and regulatory mechanisms of functional enzymes and their synergistic effects in the field of biofuels production.
Activity-based protein profiling (ABPP); Cellulosic bioethanol; Biodiesel; Protein redox; Proteomics
The long non-coding RNA Colorectal neoplasia differentially expressed (CRNDE) is a novel gene that activated early in colorectal neoplasia, but it is also up-regulated in many other solid tumors. Herein, the function and underlying mechanism of CRNDE in regulating glioma stem cells (GSCs) were investigated. We found that CRNDE expression was up-regulated while miR-186 expression was down-regulated in GSCs. Overexpression of CRNDE could promote the cellular proliferation, migration, invasion and inhibit the apoptosis in GSCs. Overexpression of miR-186 exerted functions of inhibiting the proliferation, migration and invasion of GSCs and promoting apoptosis. And CRNDE decreased the expression levels of XIAP and PAK7 by binding to miR-186 and negatively regulating it. In addition, miR-186 binded to XIAP and PAK7 3′UTR region, and decrease the expression of them, thus regulating the expression levels of downstream target proteins such as caspase 3, BAD, cyclin D1 and MARK2. The in vivo effect of CRNDE and miR-186 showed that the tumor formation rate was minimum in tumor-bearing nude mice with the knockdown of CRNDE and the overexpression of miR-186. In conclusion, CRNDE played an oncogenic role of GSCs through the negative regulation of miR-186. Both CRNDE and miR-186 could be regarded as potential targets in the glioma therapy.
long non-coding RNAs; CRNDE; microRNAs; miR-186; glioma stem cells
Hemophilia is a hereditary disease with impaired blood coagulation due to a genetic deficiency of blood coagulation factors. Hemophilia often causes spontaneous life-threatening bleeding, so patients with hemophilia are often not suitable for any surgery that may cause iatrogenic bleeding and threaten the life of the patient. Therefore, surgery in lung cancer patients with hemophilia is extremely rare. The present study reported the case of a lung cancer patient with hemophilia who presented with a persistent cough. A mass was revealed by computed tomography and the patient underwent a successful thoracoscopic right lower lobectomy. The study discusses the patient's diagnosis and treatment options for hemophilia A and lung cancer, including indications for thoracoscopic lobectomy, pre-operative preparation and post-operative care, and other treatment options are discussed. The literature is also reviewed on this subject.
lung cancer; hemophilia; thoracoscopy; coagulation factor VIII
Influenza virus vaccine (IVV) is a promising research domain that is closely related to global health matters, which has been acknowledged not only by scientists and technology developers, but also by policy-makers. Meanwhile, patents encompass valuable technological information and reflect the latest technological inventions as well as the innovative capability of a nation. However, little research has examined this up-and-coming research field using patent bibliometric method. Thus, this paper (a) designs the technology classification system and search strategy for the identification of IVV; and (b) presents a longitudinal analysis of the global IVV development based on the European Patent Office (EPO) patents. Bibliometric analysis is used to rank countries, institutions, inventors and technology subfields contributing to IVV technical progress. The results show that the global trends of IVV are a multi-developing feature of variety but an uneven technical resource distribution. Although the synthetic peptide vaccine is a comparatively young field, it already demonstrates the powerful vitality and the enormous development space. With the worldwide competition increasing, all nations especially China should be looking to increase devotion, enhance capability and regard effectiveness of technological innovation.
Gefitinib resistance has been shown to complicate cancer therapy. Lovastatin is a proteasome inhibitor that enhances gefitinib-induced antiproliferation in non-small cell lung cancer. The objective of this study is to investigate the mechanism of lovastatin-induced antiproliferation in gefitinib-resistant human cholangiocarcinoma.
Two gefitinib-resistant cholangiocarcinoma cell lines, SSP-25 and HuH-28, were used in this study to determine how to compensate gefitinib resistance. The combined effect of these two drugs was examined using the MTT assay, qPCR, immunoblotting, flow cytometry, and in vivo xenograft. Results indicated that lovastatin enhanced TNF-α-induced cell death in vitro. In addition, the combination of lovastatin with gefitinib enhanced accumulation of TNF-α. Furthermore, the treatment induced a synergistic cytotoxic effect and antiproliferation through apoptosis in SSP-25 cells and cell cycle arrest in HuH-28 cells. Reproductive results were also observed in in vivo xenografts. These observations suggest that the combination of gefitinib and lovastatin might have additive antiproliferative effects against gefitinib-resistant cholangiocarcinoma cells. Based on these observations, we concluded that the combination of gefitinib and lovastatin could be used to overcome gefitinib resistance in cholangiocarcinoma cells.
cholangiocarcinomas; combination therapy; lovastatin; gefitinib
An endo-β-1,4-glucanase gene, cel7A, was cloned from the thermophilic cellulase-producing fungus Neosartorya fischeri P1 and expressed in Pichia pastoris. The 1,410-bp full-length gene encodes a polypeptide of 469 amino acids consisting of a putative signal peptide at residues 1–20, a catalytic domain of glycoside hydrolase family 7 (GH7), a short Thr/Ser-rich linker and a family 1 carbohydrate-binding module (CBM 1). The purified recombinant Cel7A had pH and temperature optima of pH 5.0 and 60°C, respectively, and showed broad pH adaptability (pH 3.0–6.0) and excellent stability at pH3.0–8.0 and 60°C. Belonging to the group of nonspecific endoglucanases, Cel7A exhibited the highest activity on barley β-glucan (2020 ± 9 U mg–1), moderate on lichenan and CMC-Na, and weak on laminarin, locust bean galactomannan, Avicel, and filter paper. Under simulated mashing conditions, addition of Cel7A (99 μg) reduced the mash viscosity by 9.1% and filtration time by 24.6%. These favorable enzymatic properties make Cel7A as a good candidate for applications in the brewing industry.
Glioma is the most common and aggressive primary adult brain tumor. Long non-coding RNAs (lncRNAs) have important roles in a variety of biological properties of cancers. Here, we elucidated the function and the possible molecular mechanisms of lncRNA HOTAIR in human glioma U87 and U251 cell lines. Quantitative RT-PCR demonstrated that HOTAIR expression was up-regulated in glioma tissues and cell lines. Knockdown of HOTAIR exerted tumor-suppressive function in glioma cells. Further, HOTAIR was confirmed to be the target of miR-326 and miR-326 mediated the tumor-suppressive effects of HOTAIR knockdown on glioma cell lines. Moreover, over-expressed miR-326 reduced the FGF1 expression which played an oncogenic role in glioma by activating PI3K/AKT and MEK 1/2 pathways. In addition, the in vivo studies also supported the above findings. Taken together, knockdown of HOTAIR up-regulated miR-326 expression, and further inducing the decreased expression of FGF1, these results provided a comprehensive analysis of HOTAIR-miR-326-FGF1 axis in human glioma and provided a new potential therapeutic strategy for glioma treatment.
lncRNA; HOTAIR; miR-326; glioma; FGF1
The aim of this study was 2-fold: first, to assess the prognostic significance on overall survival (OS) of the 3-point tumor regression grade (TRG) in patients with esophageal squamous cell carcinoma (ESCC) who received neoadjuvant chemoradiotherapy (nCRT); second, to investigate the associations of TRG with the clinicopathological characteristics of the study patients.
A total of 357 ESCC patients were retrospectively enrolled. The 3-point TRG was determined by assessing the percentage of viable residual tumor cells (VRTC) in the resected specimens as follows: TRG 1, 0% VRTC; TRG 2, 1% to 50% VRTC; and TRG 3, >50% VRTC.
A TRG of 1, 2, and 3 was found in 32.2%, 38.9%, and 28.9% of the specimens, respectively. High TRG values were significantly associated with advanced pretreatment clinical stage, longer tumor length, and higher posttreatment tumor depth of invasion (yT), the presence of lymph node metastases (LNM), and lymphovascular invasion. We observed a stepwise decrease in 5-year OS rates with increasing TRG, as follows: 51% for patients with a TRG of 1, 28% for patients with a TRG of 2, and 22% for patients with a TRG of 3 (P < 0.001). TRG and LNM were independent predictors of OS in multivariate analysis. Notably, the prognostic impact of TRG on OS was greater in patients without LNM (P < 0.001) and ypT3 disease (P = 0.021).
TRG is independently associated with OS in ESCC patients treated with nCRT. The interrelationships between TRG, LNM, and depth of tumor invasion may improve the prognostic stratification in esophageal cancer.
Epoxyeicosatrienoic acids (EETs), cytochrome P450-derived metabolites of arachidonic acid, have been reported to increase intracellular calcium concentration in aortic vascular smooth muscle cells (SMCs). As EETs are labile, we synthesized a new stable urea EET analog with agonist and soluble epoxide hydrolase (sEH) inhibitor properties. We refer to this analog, 12-(3-hexylureido)dodec-8-enoic acid, as 8-HUDE. Measuring tension of vascular rings, intracellular calcium signaling by confocal laser scanning microscopy and gene expression by reverse-transcription-PCR and western blots, we examined the effects of 8-HUDE on pulmonary vascular tone and calcium signaling in rat pulmonary artery (PA) SMCs (PASMCs). 8-HUDE increased the tension of rat PAs to 145% baseline, whereas it had no effect on the tension of mesenteric arteries (MAs). The 8-HUDE-induced increase in vascular tone was abolished by removal of extracellular Ca2+ or by pretreatment with either La3+ or SKF96365, which are inhibitors of canonical transient receptor potential channels (TRPCs). Furthermore, 8-HUDE-evoked increases in [Ca2+]i in PASMCs could be blunted by inhibition of TRPC with SKF96365, removal of extracellular calcium or depletion of intracellular calcium stores with caffeine, cyclopiazonic acid or 2-aminoethoxydiphenyl borate, but not by the voltage-activated calcium channel blocker nifedipine. In addition to immediate effects on calcium signaling, 8-HUDE upregulated the expression of TRPC1 and TRPC6 at both mRNA and protein levels in rat PASMCs, whereas it suppressed the expression of sEH. Our observations suggest that 8-HUDE increases PA vascular tone through increased release of calcium from intracellular stores, enhanced [Ca2+]i influx in PASMCs through store-operated Ca2+ channels and modulated the expression of TRPC and sEH proteins in a proconstrictive manner.
canonical transient receptor potential channel (TRPC); 8-HUDE; epoxyeicosatrienoic acids (EETs); pulmonary arteries; store-operated Ca2+ channels (SOCCs); 12-(3-hexylureido)dodec-8-enoic acid
Classical genetic approaches to examine the requirements of genes for T cell differentiation during infection are time-consuming. Here we developed a pooled approach to screen 30–100+ genes individually in separate antigen-specific T cells during infection using short hairpin RNAs in a microRNA context (shRNAmir). Independent screens using T cell receptor (TCR)-transgenic CD4+ and CD8+ T cells responding to lymphocytic choriomeningitis virus (LCMV) identified multiple genes that regulated development of follicular helper (Tfh) and T helper-1 (Th1) cells, and short-lived effector and memory precursor cytotoxic T lymphocytes (CTL). Both screens revealed roles for the positive transcription elongation factor (P-TEFb) component Cyclin T1 (Ccnt1). Inhibiting expression of Cyclin T1, or its catalytic partner Cdk9, impaired development of Th1 cells and protective short-lived effector CTL, and enhanced Tfh and memory precursor CTL formation in vivo. This pooled shRNA screening approach should have utility in numerous immunological studies.
This study aims to explore the effects of arachidonic acid (ARA) on learning and memory dysfunction in rats exposed to repeated isoflurane anesthesia and the underlying mechanisms. Fifty rats were randomly divided into five groups: sham control group, isoflurane group, low dose ARA + isoflurane group, moderate dose ARA + isoflurane group, high dose ARA + isoflurane group. The Morris water maze test was performed to assess learning and memory function and the hippocampus tissues were obtained for biochemical analysis. The results showed that administration of ARA improved learning and memory deficit induced by repeated isofluane anesthesia in Morris water maze test and in a dose-dependent manner. Additionally, ARA increased the activities of choline acetyl transferase (ChAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the levels of acetycholine (Ach) and γ-amino-butyric acid (GABA), whereas decreased the activity of acetylcholine esterase (AchE), the content of glutamate (Glu) and malondialdehyde (MDA), and the radio of Glu/GABA. Meanwhile, ARA elevated the ratio of Bcl-2/Bax and inhibited the activity of caspase-3. In conclusion, ARA has potential therapeutic value in alleviating isoflurane-induced learning and memory impairment. The mechanism might be involved in regulating the cholinergic and Glu/GABA regulatory system, decreasing oxidative damage and inhibiting cell apoptosis.
ARA; isoflurane anesthesia; learning and memory dysfunction; cholinergic system; Glu/GABA regulatory system