CircSEMA4B targets miR-431 modulating IL-1β-induced degradative changes in nucleus pulposus cells in intervertebral disc degeneration via Wnt pathway

Intervertebral disc (IVD) degeneration (IDD), characterized by elevated levels of proinflammatory mediators, increased Aggrecan and collagen degradation, and increased degradation of extracellular matrix (ECM), has been widely regarded as a significant contributor to low back pain. Genetics are significant factors contribute to IDD. Based on previous data, circular RNA SEMA4B (circSEMA4B) is down-regulated in IDD specimens; herein, we demonstrated circSEMA4B overexpression could attenuate the effect of IL-1β on nucleus pulposus cell (NPC) proliferation, senescence, and ECM and Aggrecan degradation in IDD via Wnt signaling. Moreover, miR-431, a direct target of circSEMA4B, could bind to the 3′UTR of SFRP1 or GSK-3β, two inhibitory regulators of Wnt signaling, to inhibit their expression thus playing a role similar to the activator of Wnt signaling in NPCs. The effect of circSEMA4B knockdown on NPCs was partially reversed by miR-431 inhibition; circSEMA4B serves as a miR-431 sponge to compete with SFRP1 or GSK-3β for miR-431 binding, thus inhibiting IL-1β-induced degenerative process in NPCs through Wnt signaling. Rescuing circSEMA4B expression in NPCs in IDD might present a potential strategy for IDD improvement.     

Zika virus non-structural protein 4B interacts with DHCR7 to facilitate viral infection

Zika virus (ZIKV) evolves non-structural proteins to evade immune response and ensure efficient replication in the host cells. Cholesterol metabolic enzyme 7-dehydrocholesterol reductase (DHCR7) was recently reported to impact innate immune responses in ZIKV infection. However, the vital non-structural protein and mechanisms involved in DHCR7-mediated viral evasion are not well elucidated. In this study, we demonstrated that ZIKV infection facilitated DHCR7 expression. Notably, the upregulated DHCR7 in turn facilitated ZIKV infection and blocking DHCR7 suppressed ZIKV infection. Mechanically, ZIKV non-structural protein 4B (NS4B) interacted with DHCR7 to induce DHCR7 expression. Moreover, DHCR7 inhibited TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) phosphorylation, which resulted in the reduction of interferon-beta (IFN-β) and interferon-stimulated genes (ISGs) productions. Therefore, we propose that ZIKV NS4B binds to DHCR7 to repress TBK1 and IRF3 activation, which in turn inhibits IFN-β and ISGs, and thereby facilitating ZIKV evasion. This study broadens the insights on how viral non-structural proteins antagonize innate immunity to facilitate viral infection via cholesterol metabolic enzymes and intermediates.     

Vitamin B6-deficient diet plus 4-deoxypyridoxine (4-DPD) reduces the inflammatory response induced by T. spiralis in diaphragm,masseter and heart muscle tissue of mice

Animals fed diets deficient in vitamin B6 develop microcytic anemia, alterations of growth, and other pathologies. 4-deoxypirydoxine is a potent antagonist of vitamin B6 coenzyme which depresses IL-1, TNF and IL-6 and has anti-inflammatory properties. The aim of this study was to show the anti-infl ammatory effects of 4-DPD on chronic inflammation caused by the nematode parasite T. spiralis, specifically on the recruitment and the activation of inflammatory cells. Two groups of mice, 6 weeks of age, were used: one was maintained on a vitamin B6-deficient synthetic pellet diet for 15 days before injection of the nematode, and administered an intraperitoneal injection (i.p.) of 4-DPD (250 g/mouse) for 15 days (the first, 5 days before infection), and the second group was maintained on a normal diet for the total duration of the experiment. These two groups were then injected with 150 larvae (L1-T. spiralis) per os.Chronic inflammation was caused by infection of treated or untreated mice with T. spiralis parasite. After 14 days post-infection all mice developed a chronic inflammatory response. Mice fed with a B6-deficient diet showed a significant decrease in the number of cysts found in the diaphragm when compared to mice treated with normal diet. In addition, in all mice treated with vitamin B6-deficient diet plus 4-DPD the average body weight was significantly lower, compared to the mice on normal diet in all weeks examined. Moreover, in sections of the diaphragm, masseter and miocardium muscles, the infiltration of inflammatory cells, such as macrophages, lymphocytes, and eosinophils were more intense in untreated mice compared to those fed a vitamin B6-deficient diet.These results show that BALB/c mice infected with T. spiralis and fed a vitamin B6-deficient diet plus the vitamin B6 antagonist, 4-DPD, prolong the time of invasion of the larvae in the muscle cells, influence the recruitment of inflammatory cells and the intensity of the inflammatory reaction compared to infected untreated mice (control)     

Inhibitory effect of glycoprotein isolated from Opuntia ficus-indica var. saboten MAKINO on activities of allergy-mediators in compound 48/80-stimulated mast cells

The present study was performed to investigate the anti-allergy potentials of glycoprotein (90 kDa) isolated from Opuntia ficus-indica var. saboten MAKINO (OFI glycoprotein) in vivo (ICR mice) and in vitro (RBL-2H3 cells). At first, to know whether the OFI glycoprotein has an inhibitory ability for allergy in vivo, we evaluated the activities of allergy-related factors such as histamine and β-hexosaminidase release, lactate dehydrogenase (LDH), and interleukin 4 (IL-4) in compound 48/80 (8 ml/kg BW)-treated ICR mice. After that, we studied to found the effect for anti-allergy in vitro such as nuclear factor kappa B (NF-κB) and inducible nitric oxide synthase (iNOS), extracellular signal-regulated kinase (ERK) 1/2, arachidonic acid, and cyclooxygenase-2 (COX-2) in compound 48/80 (5 μg/ml)-treated RBL-2H3 cells. Our results showed that the OFI glycoprotein (5 mg/kg) inhibited histamine and β-hexosaminidase release, lactate dehydrogenase (LDH), and interleukin 4 (IL-4) in mice serum. Also OFI glycoprotein (25 μg/ml) has suppressive effects on the expression of MAPK (ERK1/2), and on protein expression of anti-allergic proteins (iNOS and COX-2). Thus, we speculate that the OFI glycoprotein is an example of natural compound that blocks anti-allergic signal transduction pathways.     

A stimulatory role for the La-related protein 4B in translation

La-related proteins (LARPs) belong to an evolutionarily conserved family of factors with predicted roles in RNA metabolism. Here, we have analyzed the cellular interactions and function of LARP4B, a thus far uncharacterized member of the LARP family. We show that LARP4B is a cytosolic protein that accumulates upon arsenite treatment in cellular stress granules. Biochemical experiments further uncovered an interaction of LARP4B with the cytosolic poly(A) binding protein 1 (PABPC1) and the receptor for activated C Kinase (RACK1), a component of the 40S ribosomal subunit. Under physiological conditions, LARP4B co-sedimented with polysomes in cellular extracts, suggesting a role in translation. In agreement with this notion, overexpression of LARP4B stimulated protein synthesis, whereas knockdown of the factor by RNA interference impaired translation of a large number of cellular mRNAs. In sum, we identified LARP4B as a stimulatory factor of translation. We speculate that LARP4B exerts its function by bridging mRNA factors of the 3′ end with initiating ribosomes.     

Shikonin improve sepsis-induced lung injury via regulation of miRNA-140-5p/TLR4—a vitro and vivo study

Shikonin is an anti-inflammatory agent extracted from natural herbs. The aim of this study is to explain the treatment effects and mechanism of Shikonin in acute lung injury induced by sepsis. In this study, first, we evaluate different Shikonin concentrations for the anti-inflammation of acute lung injury induced by sepsis in an in vivo study. On the basis of the results, we confirm that 50.0 mg/kg was the best therapeutic Shikonin concentration. As a second step, we discuss the mechanism of Shikonin by a vitro cell experiment. Finaly, we validate that Shikonin has effective treatment effects on acute lung injury via regulation of microRNA-140-5p/toll-like receptor 4 (miRNA-140-5p/TLR4) in the in vivo study. The results of vitro and vivo study showed that Shikonin could improve acute lung injury induced by sepsis. The mechanism might be correlation miRNA-140-5p expression increasing, and regulated targeted gene TLR4, with TLR4 expression depressing, the downstream myeloid differentiation protein 88 and nuclear factor κB proteins expression were suppressed. In conclusion, Shikonin improved sepsis induced lung injury by regulation miRNA-140-5p/TLR4.     

丙型肝炎病毒非结构蛋白NS4B诱导细胞非折叠蛋白反应

用RT-PCR和免疫印迹的方法检测稳定表达NS4B的HeLa细胞中的XBP1;通过RT-PCR的方法在表达NS4B的HeLa和Huh-7细胞中检测ATF6,Grp78和caspase-12的转录,并且通过报告基因的方法分析XBP1和Grp78启动子活性。实验结果表明:在表达NS4B的HeLa细胞中检测到XBP1的两种形式(剪接和未剪接),此外,在细胞中ATF6、Grp78的转录水平和XBP1、Grp78启动子的荧光素酶活性较没有表达NS4B的HeLa和Huh-7细胞中的量有所增加;通过染色质免疫沉淀实验(ChIP)分析,这些增加可能是由于XBP1结合到了这些基因的启动子上引起的。总之,实验结果可提示HCVNS4B通过ATF6或XBP1途径引起内质网压力,导致UPR反应。NS4B可能在HCV的致病性中起着重要的作用,特别是在慢性肝炎,甚至肝细胞癌中。     

Inflammation-dependent downregulation of miR-194-5p contributes to human intervertebral disc degeneration by targeting CUL4A and CUL4B

Inflammation is one of the major causes of intervertebral disc degeneration (IDD). Emerging evidence has revealed that increase in the levels of pro-inflammatory cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α), can activate a variety of signaling pathways, eventually resulting in IDD. Here, we show that the two cullin family genes, CUL4A and CUL4B, but not other cullins, are specifically overexpressed in IDD samples compared with healthy controls, and the CUL4A and CUL4B levels are positively correlated with the severity of IDD. In vitro analyses in human osteoblast cells (hFOB1.19), nucleus pulposus cells (hNPCs), and annulus fibrosus cells (hAFCs) indicated that treatment with IL-6 and TNF-α can increase CUL4A and CUL4B levels. By performing a microRNA-based microarray analysis, we found a set of microRNAs (miRNAs) that were differentially expressed in IDD samples compared with samples from healthy controls. Of these miRNAs, miR-194-5p, was significantly downregulated in IDD samples and could bind to the three prime untranslated regions (3′-UTRs) of both CUL4A and CUL4B, thereby downregulating their expression. The in vitro overexpression or downregulation of miR-194-5p, with a miR-194-5p-mimic or with anti-miR-194-5p, can cause the repression or induction of both CUL4A and CUL4B, respectively. Interestingly, treatment with IL-6 and TNF-α inhibitors in primary hNPCs and hAFCs that were isolated from patients with IDD led to the downregulation of CUL4A and CUL4B. Together, these findings provide insight into how the inflammation-dependent downregulation of miR-194-5p contributes to the pathogenesis of IDD, which may aid in the development of new therapeutic approaches for IDD by directly targeting miR-194-5p or CUL4A and CUL4B.     

食管癌LAPTM4B mRNA表达及其启动子区甲基化

研究溶酶体相关4次跨膜蛋白B（lysosome associated protein transmembrane 4 beta,LAPTM4B）基因在食管癌中的表达,及其启动子区甲基化状态,为进一步揭示LAPTM4B在不同肿瘤中表达高低机理提供参考.采用半定量RT-PCR法,确定42对食管癌中LAPTM4B mRNA表达.采用5对肝癌中LAPTM4B mRNA表达做内对照（利用灰度值比较）,分析该基因在食管癌中的表达强度.选取其中3对食管癌组织样品（癌组织和癌旁正常组织）,提取基因组DNA,采用亚硫酸氢钠修饰法,联合基因测序法分析LAPTM4B启动子区是否有甲基化修饰位点存在.结果发现,在42对食管癌组织中,癌组织和癌旁正常组织LAPTM4B mRNA表达存在差异：癌组织中LAPTM4B mRNA表达阳性为37/42（88.1%）,癌旁正常组织中LAPTM4B mRNA表达阳性为26/42（61.9%）.经基因测序法分析3对食管癌组织经通用引物PCR扩增的片段,发现1例癌旁正常组织样品中有3个CpG位点.以上结果表明,LAPTM4B基因与肝癌比较在食管癌中低表达,其启动子区1例癌旁正常组织在靠近转录起始点上游-418、-416和-398位置,存在3个CpG位点,而其他2例癌旁正常组织和3例癌组织中,没有发现CpG位点.这提示,LAPTM4B基因启动子区甲基化是其表达调节的重要方式.     

Design and synthesis of spirocyclic compounds as HCV replication inhibitors by targeting viral NS4B protein

Two novel series of spirocyclic piperidine analogs appended to a pyrazolo[1,5-a]pyridine core were designed, synthesized and evaluated for their anti-HCV activity. A series of piperidine ketals afforded dispiro 6p which showed excellent in vitro anti-HCV activities (EC₅₀ of 1.5 nM and 1.2 nM against genotype 1a and 1b replicons, respectively). A series of piperidine oxazolidinones afforded 27c which showed EC₅₀’s of 10.9 nM and 6.1 nM against 1a and 1b replicons, respectively. Both compounds 6p and 27c bound directly to non-structural NS4B protein in vitro (IC₅₀’s = 10.2 and 30.4 nM, respectively) and exhibited reduced potency in replicons containing resistance mutations encoding changes in the NS4B protein.     

Reduced toxicity of amphotericin B methyl ester (AME) vs. amphotericin B and fungizone in tissue culture

Summary The comparative toxicities of amphotericin B methyl ester (AME), the parent antibiotic amphotericin B (AB), and the deoxycholate solubilized complex of AB, Fungizone² (FZ), toward five cell lines has been determined as measured by early membrane damage (⁵¹Cr release), 24 hr survival, 72 hr viability, and growth rate. Cells used were of turtle (TH-1), marsupial (PT K2), human MA 160), rabbit (RK-13) and hamster (BHK-21) origin. AME: (a) caused less membrane damage at 1 hr than AB or FZ; (b) was less toxic than AB or FZ as indicated by 24 hr cell survival and 72 hr cell viability; and (c) was required in higher levels than AB or FZ to reduce the growth rate of all five cell lines. Spectrophotometric analysis of residual polyene levels indicated that AME had good stability in tissue culture medium. Previous studies have indicated that AME has the same in vitro antifungal activity as the parent antibiotic AB (1, 2). These findings suggest that AME may prove to be superior to AB and FZ for use as an antifungal agent in tissue culture systems. Fungizone^R. Trade mark. E. R. Squibb and Sons. This investigation was supported in part by Contract NIH 69-2161, NIH Grant No. AI-02095 and NIH Training Grant No. GM 507 from the National Institute of General Medical Sciences.     

巨细胞病毒肝炎患儿gB基因型分析及细胞因子水平测定

探讨新生儿肝炎患儿CMVgB基因型分布,研究不同gB基因型致病性的差异。采用巢式聚合酶链反应(nested PCR),限制性片段长度多态性(RFLP)分析技术等测定45例新生儿肝炎患儿尿中巨细胞病毒gB基因型,并分析gB基因型与其临床症状的关系。并用ELISA方法测定患儿血清TNF-α和IFN-γ水平。结果显示,新生儿肝炎在gB1~4型中均可发生,以gB1型为主。病理性黄疸为惟一症状的患儿只在gB1型和gB3型中出现,且预后较好。新生儿肝炎患儿血清TNF-α和IFN-γ皆高于对照组,可能与CMV肝炎的病理变化和病损程度有密切的关系。     

Minocycline improves the functional recovery after traumatic brain injury via inhibition of aquaporin-4

Traumatic brain injury (TBI) is one of the main concerns worldwide as there is still no comprehensive therapeutic intervention. Astrocytic water channel aquaporin-4 (AQP-4) system is closely related to the brain edema, water transport at blood-brain barrier (BBB) and astrocyte function in the central nervous system (CNS). Minocycline, a broad-spectrum semisynthetic tetracycline antibiotic, has shown anti-inflammation, anti-apoptotic, vascular protection and neuroprotective effects on TBI models. Here, we tried to further explore the underlying mechanism of minocycline treatment for TBI, especially the relationship of minocycline and AQP4 during TBI treatment. In present study, we observed that minocycline efficaciously reduces the elevation of AQP4 in TBI mice. Furthermore, minocycline significantly reduced neuronal apoptosis, ameliorated brain edema and BBB disruption after TBI. In addition, the expressions of tight junction protein and astrocyte morphology alteration were optimized by minocycline administration. Similar results were found after treating with TGN-020 (an inhibitor of AQP4) in TBI mice. Moreover, these effects were reversed by cyanamide (CYA) treatment, which notably upregulated AQP4 expression level in vivo. In primary cultured astrocytes, small-interfering RNA (siRNA) AQP4 treatment prevented glutamate-induced astrocyte swelling. To sum up, our study suggests that minocycline improves the functional recovery of TBI through reducing AQP4 level to optimize BBB integrity and astrocyte function, and highlights that the AQP4 may be an important therapeutic target during minocycline treating for TBI.     

VEGF-A signaling through Flk-1 is a critical facilitator of early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis

Vascular endothelial growth factor-A (VEGF-A) signaling directs both vasculogenesis and angiogenesis. However, the role of VEGF-A ligand signaling in the regulation of epithelial-mesenchymal interactions during early mouse lung morphogenesis remains incompletely characterized. Fetal liver kinase-1 (Flk-1) is a VEGF cognate receptor (VEGF-R2) expressed in the embryonic lung mesenchyme. VEGF-A, expressed in the epithelium, is a high affinity ligand for Flk-1. We have used both gain and loss of function approaches to investigate the role of this VEGF-A signaling pathway during lung morphogenesis. Herein, we demonstrate that exogenous VEGF 164, one of the 3 isoforms generated by alternative splicing of the Vegf-A gene, stimulates mouse embryonic lung branching morphogenesis in culture and increases the index of proliferation in both epithelium and mesenchyme. In addition, it induces differential gene and protein expression among several key lung morphogenetic genes, including up-regulation of BMP-4 and Sp-c expression as well as an increase in Flk-1-positive mesenchymal cells. Conversely, embryonic lung culture with an antisense oligodeoxynucleotide (ODN) to the Flk-1 receptor led to reduced epithelial branching, decreased epithelial and mesenchymal proliferation index as well as downregulating BMP-4 expression. These results demonstrate that the VEGF pathway is involved in driving epithelial to endothelial crosstalk in embryonic mouse lung morphogenesis.     

Kruppel样因子4对内毒素所致IL-6基因表达的调控及机制研究

探讨Kruppel样因子4(KLF4)对内毒素所致白介素(IL-6)的基因表达以及释放的影响,并对其调控机制做了初步研究．使用RT-PCR和Western blot检测KLF4 mRNA和蛋白质的表达．采用KLF4过表达的RAW264.7巨噬细胞株或反义寡核苷酸技术抑制内源性KLF4的表达,用RT-PCR和ELISA检测内毒素(LPS)刺激后IL-6 mRNA和蛋白质的表达．采用荧光素酶报告基因检测RAW264.7细胞中KLF4过表达对IL-6基因启动子报告基因转录活性的影响．使用EMSA法检测细胞中KLF4与IL-6基因启动子区KLF4元件的结合．结果表明：LPS可以诱导RAW264.7巨噬细胞KLF4的表达以及IL-6蛋白表达．KLF4过表达明显抑制IL-6的mRNA和蛋白质的表达,而KLF4缺失使这种作用消失．荧光素酶报告基因的结果显示,KLF4可以抑制LPS所致的IL-6基因启动子的转录活性．EMSA显示KLF4不能与IL-6启动子区的KLF4结合元件直接结合．结果表明,LPS可以促进RAW264.7小鼠巨噬细胞KLF4的表达和IL-6的释放．KLF4能抑制LPS诱导的IL-6表达和释放,其机制是抑制IL-6启动子的转录活性,但KLF4的抑制作用不是通过直接与IL-6基因的启动子区相结合而实现的．     

Cleavage of the Babuvirus Movement Protein B4 into Functional Peptides Capable of Host Factor Conjugation is Required for Virulence

Banana bunchy top virus(BBTV) poses a serious danger to banana crops worldwide. BBTV-encoded protein B4 is a determinant of pathogenicity. However, the relevant molecular mechanisms underlying its effects remain unknown. In this study, we found that a functional peptide could be liberated from protein B4, likely via proteolytic processing. Site-directed mutagenesis indicated that the functional processing of protein B4 is required for its pathogenic effects, including dwarfism and sterility, in plants. The released protein fragment targets host proteins, such as the large subunit of RuBisCO(RbcL)and elongation factor 2(EF2), involved in protein synthesis. Therefore, the peptide released from B4(also a precursor) may act as a non-canonical modifier to influence host–pathogen interactions involving BBTV and plants.