Clinical Profiles Associated with Influenza Disease in the Ferret Model

Influenza A viruses continue to pose a threat to human health; thus, various vaccines and prophylaxis continue to be developed. Testing of these products requires various animal models including mice, guinea pigs, and ferrets. However, because ferrets are naturally susceptible to infection with human influenza viruses and because the disease state resembles that of human influenza, these animals have been widely used as a model to study influenza virus pathogenesis. In this report, a statistical analysis was performed to evaluate data involving 269 ferrets infected with seasonal influenza, swine influenza, and highly pathogenic avian influenza (HPAI) from 16 different studies over a five year period. The aim of the analyses was to better qualify the ferret model by identifying relationships among important animal model parameters (endpoints) and variables of interest, which include survival, time-to-death, changes in body temperature and weight, and nasal wash samples containing virus, in addition to significant changes from baseline in selected hematology and clinical chemistry parameters. The results demonstrate that a disease clinical profile, consisting of various changes in the biological parameters tested, is associated with various influenza A infections in ferrets. Additionally, the analysis yielded correlates of protection associated with HPAI disease in ferrets. In all, the results from this study further validate the use of the ferret as a model to study influenza A pathology and to evaluate product efficacy.     

The Metastasis Suppressor,N-myc Downregulated Gene 1 (NDRG1), Is a Prognostic Biomarker for Human Colorectal Cancer

Metastasis remains to be one of the most prevalent causes leading to poor long-term survival of colorectal cancer (CRC) patients. The clinical significances of tumor metastatic suppressor, N-myc downregulated gene 1 (NDRG1), have been inconsistently reported in a variety of cancerous diseases. In this study with 240 CRC clinical specimens, we showed that NDRG1 expression was significantly decreased in most of CRC tissues compared to the paired non-tumor counterparts. Statistical analysis revealed a significant inverse correlation of NDRG1 expression with tumor stage, differentiation status and metastasis. Compared with NDRG1-negative group, NDRG1-positve group had better disease-free/overall survival (p = 0.000) over 5 years’ follow-up. Furthermore, NDRG1 was considered to be an independent prognostic factor for overall survival (p = 0.001) and recurrence (p = 0.003). Our study concludes that NDRG1 is a novel favorable predictor for the prognosis in CRC patients.     

Macrophage Depletion Impairs Corneal Wound Healing after Autologous Transplantation in Mice

Purpose

Macrophages have been shown to play a critical role in the wound healing process. In the present study, the role of macrophages in wound healing after autologous corneal transplantation was investigated by depleting local infiltrated macrophages.

Methods

Autologous corneal transplantation model was used to induce wound repair in Balb/c mice. Macrophages were depleted by sub-conjunctival injections of clodronate-containing liposomes (Cl₂MDP-LIP). The presence of CD11b⁺ F4/80⁺ macrophages, α-smooth muscle actin⁺ (α-SMA⁺) myofibroblasts, CD31⁺ vascular endothelial cells and NG₂ ⁺ pericytes was examined by immunohistochemical and corneal whole-mount staining 14 days after penetrating keratoplasty. Peritoneal macrophages were isolated from Balb/c mice and transfused into conjunctiva to examine the recovery role of macrophages depletion on wound healing after autologous corneal transplantation.

Results

Sub-conjunctival Cl₂MDP-LIP injection significantly depleted the corneal resident phagocytes and infiltrated macrophages into corneal stroma. Compared with the mice injected with PBS-liposome, the Cl₂MDP-LIP-injected mice showed few inflammatory cells, irregularly distributed extracellular matrix, ingrowth of corneal epithelium into stroma, and even the detachment of donor cornea from recipient. Moreover, the number of macrophages, myofibroblasts, endothelial cells and pericytes was also decreased in the junction area between the donor and recipient cornea in macrophage-depleted mice. Peritoneal macrophages transfusion recovered the defect of corneal wound healing caused by macrophages depletion.

Conclusions

Macrophage depletion significantly impairs wound healing after autologous corneal transplantation through at least partially impacting on angiogenesis and wound closure.     

Hepatic Parenchymal Changes following Transcatheter Embolization and Chemoembolization in a Rabbit Tumor Model

Objective

To compare the effects of transcatheter arterial chemoembolization (TACE) with transcatheter arterial embolization (TAE) on liver function, hepatic damage, and hepatic fibrogenesis in a rabbit tumor model.

Materials and Methods

Thirty-nine New Zealand white rabbits implanted with VX2 tumors in the left liver lobes were randomly divided into three groups: TAE, TACE, and control group. In the TAE group (n = 15), polyvinyl alcohol particles (PVAs) were used for left hepatic artery embolization. In the TACE group (n = 15), the tumors were treated with left hepatic arterial infusions of a suspension of 10-hydroxycamptothecin and lipiodol, followed by embolization with PVAs. In the control group (n = 9), the animals received sham treatment with distilled water. Serum and liver samples were collected at 6 hours, 3 days and 7 days after treatment. Liver damage was measured using a liver function test and histological analyses. Liver fibrogenesis and hepatic stellate cell (HSC) activation were evaluated using Sirius Red and anti-alpha-smooth muscle actin (α-SMA) immunohistochemical stains.

Results

TACE caused liver injury with greater increases in serum alanine aminotransferase and aspartate aminotransferase levels on day 3 (P<0.05). Histological analyses revealed increased hepatic necrosis in adjacent non-tumorous liver tissue from day 3 compared to the TAE group (Suzuki score of 2.33±1.29 versus 1.13±1.18, P = 0.001). HSC activation and proliferation were significantly increased in the TACE group compared to the control group at 3 and 7 days after treatment (0.074±0.014 vs. 0.010±0.006, and 0.088±0.023 vs. 0.017±0.009, P<0.05). Sirius Red staining demonstrated a statistically significant increase in collagen deposition in the livers in the TACE group 7 days after embolization compared to the control group (0.118±0.012 vs. 0.060±0.017, P = 0.05).

Conclusion

The results of this animal study revealed that TACE induced prominent hepatocellular damage and hepatic fibrogenesis, which compromised liver function and may be responsible for chronic liver decompensation.     

Ginsenoside Rd Attenuates Myocardial Ischemia/Reperfusion Injury via Akt/GSK-3β Signaling and Inhibition of the Mitochondria-Dependent Apoptotic Pathway

Evidence suggests Ginsenoside Rd (GSRd), a biologically active extract from the medical plant Panax Ginseng, exerts antioxidant effect, decreasing reactive oxygen species (ROS) formation. Current study determined the effect of GSRd on myocardial ischemia/reperfusion (MI/R) injury (a pathological condition where ROS production is significantly increased) and investigated the underlying mechanisms. The current study utilized an in vivo rat model of MI/R injury and an in vitro neonatal rat cardiomyocyte (NRC) model of simulated ischemia/reperfusion (SI/R) injury. Infarct size was measured by Evans blue/TTC double staining. NRC injury was determined by MTT and lactate dehydrogenase (LDH) leakage assay. ROS accumulation and apoptosis were assessed by flow cytometry. Mitochondrial membrane potential (MMP) was determined by 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetrathylbenzimidazol carbocyanine iodide (JC-1). Cytosolic translocation of mitochondrial cytochrome c and expression of caspase-9, caspase-3, Bcl-2 family proteins, and phosphorylated Akt and GSK-3β were determined by western blot. Pretreatment with GSRd (50 mg/kg) significantly augmented rat cardiac function, as evidenced by increased left ventricular ejection fraction (LVEF) and ±dP/dt. GSRd reduced myocardial infarct size, apoptotic cell death, and blood creatine kinase/lactate dehydrogenase levels after MI/R. In NRCs, GSRd (10 µM) inhibited SI/R-induced ROS generation (P<0.01), decreased cellular apoptosis, stabilized the mitochondrial membrane potential (MMP), and attenuated cytosolic translocation of mitochondrial cytochrome c. GSRd inhibited activation of caspase-9 and caspase-3, increased the phosphorylated Akt and GSK-3β, and increased the Bcl-2/Bax ratio. Together, these data demonstrate GSRd mediated cardioprotective effect against MI/R–induced apoptosis via a mitochondrial-dependent apoptotic pathway.     

侵染中华蜜蜂6日龄幼虫的蜜蜂球囊菌的微小RNA差异表达谱及调控网络

【目的】蜜蜂球囊菌(Ascosphaeraapis,简称球囊菌)是专性侵染蜜蜂幼虫的致死性真菌病原。MicroRNA(miRNA)作为一类重要的基因表达调控因子,能够广泛参与真菌及其宿主的相互作用过程。本研究通过比较分析球囊菌孢子(AaCK)和侵染中华蜜蜂(Apis cerana cerana,简称中蜂) 6日龄幼虫肠道内的球囊菌(AaT)的smallRNA(sRNA)组学数据对球囊菌的差异表达miRNA(differentiallyexpressed miRNA,DEmiRNA)、靶mRNA及二者间的调控网络进行全面解析,旨在揭示miRNA介导的球囊菌对中蜂幼虫的侵染机制。【方法】对于球囊菌侵染的中蜂6日龄幼虫肠道的small RNA-seq (sRNA-seq)数据,利用BLAST工具连续比对东方蜜蜂(Apiscerana)和球囊菌的参考基因组筛滤得到AaT的sRNA组学数据。分别将AaCK和AaT的sRNA组学数据比对miRBase数据库,对球囊菌侵染宿主前后miRNA的数量和结构特征进行分析。联用RNAhybrid+svm_light、Miranda和TargetScan软件预测AaCK vs AaT比较组中DEmiRNA的靶mRNA,进而利用相关生物信息学软件对上述靶mRNA进行GO分类和KEGG代谢通路富集分析。通过Cytoscape软件对DEmiRNA-mRNA调控网络进行可视化。利用Stem-loop RT-PCR、RT-qPCR和分子克隆验证测序结果的可靠性。【结果】在AaCK和AaT中分别鉴定到380和387个miRNA。结构特征分析结果显示,AaCK和AaT的mi RNA皆集中分布在18–25 nt,且首位碱基主要偏向于U。AaCKvsAaT比较组共有270个DEmiRNA,包含155个上调miRNA和115个下调miRNA,分别靶向结合6091和6145个mRNA。GO分类结果显示,上述靶mRNA主要涉及代谢进程、细胞进程、应激反应等15个生物学进程;细胞、细胞组分、细胞器等12个细胞组分;催化活性、结合、转运子活性等11个分子功能。KEGG代谢通路富集分析结果显示,上述靶mRNA富集在123条代谢通路,参与对氨基酸代谢、碳水化合物代谢以及核苷酸代谢等物质代谢,氧化磷酸化、硫代谢、氮代谢等能量代谢,以及MAPK和Hippo等信号通路的调控。球囊菌DEmiRNA与靶mRNA之间存在复杂的调控关系,其中miR-29-x、miR-250-x、miR-4968-y、miR-11200-x、novel-m0023-5p、novel-m0130-5p和novel-m0135-5p等DEmiRNA可靶向结合与球囊菌的半胱氨酸蛋白酶、DNA甲基化转移酶以及几丁质酶相关的mRNA;此外,miR-7-x、miR-9-z、miR-319-y和miR-5951-y等同时参与调控MAPK信号通路;进一步分析发现,miR-250-x同时参与对DNA甲基化转移酶、MAPK信号通路及其他酶类合成与代谢途径的调控,并可能参与球囊菌与中蜂6日龄幼虫之间的跨界调控。通过Stem-loopRT-PCR和RT-qPCR验证了4个DEmiRNA的差异表达,并利用分子克隆和Sanger测序证实miR-7-x的序列与测序结果一致。【结论】本研究解析了侵染中蜂6日龄幼虫的球囊菌的miRNA差异表达谱及DEmiRNA的调控网络,揭示了球囊菌DEmiRNA可能通过调控病原的物质和能量代谢、增殖、毒力、信号通路及相关mRNA参与对中蜂幼虫的侵染过程。miR-7-x、miR-250-x、novel-m0023-5p等关键DEmiRNA有望作为白垩病治疗的新型分子靶点。     

透射电子显微镜(TEM)技术在大孢子化石 超微结构研究中的应用

透射电子显微镜(TEM)具有优异的纳米尺度显微成像性能,因此被广泛应用于材料科学、物理学和生物学等学科的超微结构研究领域。文中根据应用TEM技术研究大孢子化石壁超微结构的实践体会,系统归纳和总结了大孢子化石TEM样品制备的前处理过程,主要步骤包括:材料选取、梯度脱水、包埋剂配比、梯度渗透、包埋、聚合、超薄切片和染色。运用上述TEM实验技术,文中以云南省曲靖市沾益区龙华山剖面中泥盆统吉维特阶上双河组分散拟网龙华山大孢(Longhuashanispora reticuloides Lu and Ouyang, 1978)的壁层超微结构为研究案例,揭示了这类孢子壁主要由三层薄壁组成,包括内部基底层、中部疏松层和外部致密层,其射线唇基底层下部具有近平行排列的多细纹带结构。此类大孢子的多细纹带结构与从加拿大新不伦瑞克省下泥盆统埃姆斯阶Campbellton组同孢植物化石Leclercqia complexa Banks et al., 1972中发现的原位小孢子射线唇基底层下部的多细纹带结构特征非常相似。此外,两者都具有相似的完全弓形脊和远极表面刺瘤状二型纹饰。因此综合外壁超微结构和纹饰形态特征,文中认为Longhuashanispora reticuloides的母体植物可能属于同孢植物向异孢植物演化过程中的过渡类型植物,并与Leclercqia complexa具有较近的亲缘关系。当前研究表明TEM技术在孢粉化石壁超微结构研究领域中具有独特优势,可为深入研究大孢子形态分类和揭示其与母体植物的亲缘关系提供新的线索,也可以被推广和应用到其他微体有机壁类化石的超微结构研究。