The impact of weight loss on circulating cytokines in Beagle dogs

Chronic low-grade inflammation in obesity is characterized by an increased production of pro-inflammatory and chemotactic cytokines that are contributing to insulin resistance and related co-morbidities. Cytokines act in networks and exhibit pleiotropic effects so we investigated the circulating levels of a wide array of cytokines (pro and anti-inflammatory, chemotactic and growth factors) in a canine model of weight loss. The dogs served as their own control in order to study the impact of weight loss independent of potential confounding factors, such as history of excess weight or gender. While low-grade inflammation had been previously investigated in obese dogs by measuring changes in adipokines, acute phase proteins and key pro-inflammatory cytokines, to the best of our knowledge this is the first study to evaluate how weight loss impacts a wide array of circulating cytokines.Eighteen overweight Beagle dogs were recruited (six spayed females and 12 neutered males), and none of them were grossly obese according to the body condition score (BCS). All the dogs reached an ideal weight by the end of the program. Parameters were assessed before (baseline), at mid-point (month 3) and at end-point (month 6). Plasma GM-CSF, IL-2, Il-4, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, IFNγ, IP-10, TNFα, monocyte chemotactic protein 1 (MCP-1), keratinocyte chemokine (KC) were measured with canine multiplex immunoassays. Fat mass was assessed by dual energy X-ray absorption (DEXA).Several cytokines decreased throughout the weight loss program (p < 0.01) and were correlated with the percentage of fat measured by DEXA (p < 0.05): chemotactic (MCP-1), growth factors (GM-CSF, IL-7 and IL-2), and pro-inflammatory (KC and IL-18). We could not show trends for several cytokines, possibly because their level may be lower than the assay sensitivity: anti-inflammatory (IL-4 and IL-10), and pro-inflammatory (IL-6 and TNFα).In conclusion, while our findings for several pro-inflammatory and chemotactic cytokines are in accordance with human and rodent studies, we may have identified additional cytokines, such as growth factors, related to obesity-induced low-grade inflammation. Considering the weight loss was enabled by an adjusted diet, the role of this association of cytokines in insulin resistance and related co-morbidities needs to be clarified. Our results could help better understand the cytokine biology in dogs, and as such are relevant for further elucidating the relationship between immune function and metabolism/nutrition.     

Effect of miR-181b on cell proliferation and expression of Bcl-2 and SP1 in HepG2 cells

AIM: To investigate the role of miR-181b in the expression of Bcl-2 and SP1 at mRNA and protein levels in the human hepatoma G2 cells (HepG2), and to explore the effect of miR-181b on the regulation of HepG2 cell proliferation. METHODS: The synthetic double-strand complementary DNA based on the sequence of miR-181b was inserted into the vector of miRNASelect^TM pEGP-miR. The microRNA high-expression plasmid was cloned, and the sequences were identified. The miR-181b plasmid was transfected into HepG2 cells with liposomes. The stable cell line was screened by puromycin. The mRNA and protein levels of Bcl-2 and SP1 were measured by RT-PCR and Western blotting, respectively. Methyl thiazolyl tetrazolium (MTT) method was used to analyze the proliferation of HepG2 cells. RESULTS: The Western blotting results showed that miR-181b inhibited the protein expression of Bcl-2 and SP1. The result of RT-PCR also indicated that the mRNA expression of Bcl-2 and SP1 was suppressed. Compared with the control, the growth rate of HepG2 with high expression of miR-181b was significantly decreased.CONCLUSION: miR-181b inhibits the proliferation of HepG2, which may be related to the down-regulation of Bcl-2 and SP1.     

香蕉枯萎病菌myosin-1基因的功能分析及外施dsRNA介导的抗性评估

 香蕉枯萎病是由尖孢镰刀菌古巴专化型(Fusarium oxysporum f. sp. cubense,Foc)引起的香蕉毁灭性土传病害,其中 4号生理小种(Foc4)能感染几乎所有的香蕉品系,危害最严重。SMART在线软件分析myosin-1基因具有肌球蛋白马达蛋白(myosin motor domain, MMD),肌动蛋白尾结构TH1(myosin tail)和 Src家族同源结构域SH3(src homology domain 3),与禾谷镰刀菌中氰烯菌酯靶标基因myosin-5具高度的蛋白同源性,相似性高达83%。利用Split-marker基因重组技术获得Foc4的myosin-1基因敲除突变体,Δmyosin-1突变株丧失了对氰烯菌酯的敏感性,菌丝生长缓慢,产孢量减少且孢子畸形,对香蕉致病力严重下降,证实myosin-1是氰烯菌酯在Foc4中的作用靶标基因。外施靶向myosin-1体外转录的dsRNA,能抑制菌丝的生长,降低菌丝活性;菌丝膨胀扭曲分枝增多,出现典型的球状结构,与氰烯菌酯处理后的表型一致。在盆栽活体人工接种实验中,体外施用dsRNA可以明显抑制枯萎病外部症状的发展,推迟发病时间,赋予寄主抗性,结果说明体外施用dsRNA可以作为新型杀菌剂防治香蕉枯萎病。综上,myosin-1基因作为氰烯菌酯在Foc4中的靶标基因具有高度的序列保守,在调控菌丝生长发育,产孢以及致病力等方面发挥重要作用,而外施dsRNA具有防治香蕉枯萎菌的巨大潜力。     

NA-1株鹅源副黏病毒表面结构蛋白HN的真核表达

应用特异性引物,从鹅源副黏病毒NA-1株中扩增出HN蛋白基因,PCR产物纯化后克隆入pGEM-T载体,得到重组质粒pTHN。用EcoRⅠ和NotⅠ双酶切pTHN,回收目的基因HN片段并将其定向克隆到pPICZαA中,构建重组质粒pPICZαAHN。用SacⅠ酶切pPICZαAHN使其线性化,并电击转化至感受态毕赤酵母细胞GS115。PCR法鉴定阳性重组子,用1%甲醇诱导表达后,进行SDS-PAGE及Western-blot分析。结果在酵母菌培养基上清中检测到相对分子质量为63000的重组蛋白,且该重组蛋白可与NA-1株病毒多克隆抗体发生特异性血清学反应,表明NA-1的HN蛋白片段在Pichiapastoris中获得成功表达。     

Serum Biomarkers of Clinical and Cytologic Response in Dogs with Idiopathic Immune‐Mediated Polyarthropathy

Background

Immune‐mediated polyarthopathy (IMPA) is common in dogs, and is monitored by serial arthrocenteses.

Hypothesis/Objectives

Plasma C‐reactive protein (CRP), interleukin‐6 (IL‐6), and CXCL8 (interleukin‐8) would serve as noninvasive markers of joint inflammation in IMPA.

Animals

Nine client‐owned dogs with idiopathic IMPA; 6 healthy controls.

Methods

Prospective study. Plasma CRP, IL‐6, and CXCL8 were measured by ELISA at baseline, 2, and 4 weeks during treatment with prednisone at 50 mg/m²/day. Arthrocenteses, the canine brief pain inventory (CBPI), and accelerometry collars were used to assess joint inflammation, lameness, and mobility at all 3 time points.

Results

C‐reactive protein concentrations were higher in IMPA dogs (median 91.1 μg/mL, range 76.7–195.0) compared with controls (median <6.3 μg/mL, <6.3–13.7; P = .0035), and were significantly lower at week 2 (10.6 μg/mL, <6.3–48.8) and week 4 (<6.3 μg/mL, <6.3–24.4; P < .001).C‐reactive protein was correlated with median CBPI scores (r = 0.68; P = .0004), joint cellularity (r = 0.49, P = .011), and mobility by accelerometry (r = −0.42, P = .048). Plasma IL‐6 concentrations were also higher in IMPA dogs (median 45.9 pg/mL), compared with controls (median <15.7 pg/mL; P = .0008). IL‐6 was lower in IMPA dogs by week 4 (<15.7 pg/mL; P = .0099), and was modestly correlated with CBPI scores (r = 0.47, P = .023). CXCL8 did not differ significantly between IMPA and healthy dogs.

Conclusions

Plasma CRP and IL‐6 might be useful surrogate markers of synovial inflammation and disease activity in dogs with IMPA.     

茄子新品种湘杂早红的选育

以南县紫圆茄的自交系92147为母本，陕西汉中紫茄的自交系96267为父本配制成一代杂种湘杂早红。从定植到始收约35d（天），果实紫红色，卵圆形，果肉白色，肉质细嫩，单果质量250g左右。对青枯病和绵疫病的抗性比湘早茄强。每667m^2产量3000—4500kg，适合春栽。     

黑甜糯玉米新品种黑甜糯639的选育

黑甜糯639是以糯玉米自交系HNF为母本,以甜玉米自交系D91为父本配制而成的黑甜糯玉米一代杂种。生育期为91 d(天),株高263 cm,穗位151 cm,雄穗主轴与分枝角度中等,一级分枝18～23个,果穗筒型,籽粒黑紫色,甜糯籽粒比例为1∶3,籽粒可溶性糖含量为12.5%,支链淀粉占总淀粉含量的98.1%,口感佳。高抗丝黑穗病、茎腐病、大斑病,抗穗腐病和矮花叶病,平均每667 m~2鲜穗产量900 kg左右。适宜在山西省鲜食糯玉米主产区种植。     

非典型犬瘟热病毒核衣壳蛋白基因序列分析及其在大肠杆菌中的表达

为分析当地非典型犬瘟热病毒(CDV)核衣壳蛋白(N)基因的序列特征及其表达产物的抗原性,根据已发表CDV的N基因序列设计引物,用RT-PCR方法从引起非典型症状的CDV细胞培养物中扩增N基因,进行克隆和序列分析,结果表明:该非典型CDV的N基因与已发表的12个CDV强毒株的核苷酸序列和氨基酸序列同源性分别在96.6%～99.2%和97.9%～99.4%之间,与已发表的4个CDV疫苗弱毒株的同源性分别在93.2%～93.6%和96.4%～97.5%之间;在N基因系统发育进化树上,非典型CDV与12个强毒株处在同一亚群,而且与9个中国分离毒株的亲缘关系近于3个国外毒株。N基因在大肠杆菌中表达的重组N蛋白的分子量为62 ku,主要以包涵体的形式存在;用western blot分析,重组N蛋白可与CDV阳性血清发生特异性反应;以纯化的重组N蛋白为抗原建立的CDV抗体间接ELISA检测方法具有良好的特异性。     

金雀异黄素对雌性大鼠体内促性腺激素及胰岛素样生长因子表达的影响

本试验旨在研究金雀异黄素(genistein,GEN)对雌性大鼠体内促性腺激素及胰岛素样生长因子表达的影响。选取40只SD雌性大鼠[体重(200±20)g],随机分为5组,分别为阴性对照(NC)组、GEN低(L)、中(M)、高剂量(H)组及阳性对照(PC)组,每组8只,NC组灌胃花生油(其他组灌胃试剂以此为溶剂);L、M、H组分别灌胃15、30、60 mg/(kg BW·d)GEN,PC组灌胃己烯雌酚0.5 mg/(kg BW·d)。试验期30 d。采用酶联免疫吸附试验(ELISA)法检测血清中卵泡刺激素(FSH)、黄体生成素(LH)、胰岛素样生长因子-1(IGF-1)、胰岛素样生长因子结合蛋白-1(IGFBP-1)含量;实时定量PCR法检测卵巢IGF-1、IGFBP-1 mRNA表达水平。结果表明:与NC组比较,试验组血清中FSH、LH含量有升高趋势,但差异不显著(P0.05),作用效果与PC组一致;试验组血清IGF-1含量略有降低,但差异不显著(P0.05),PC组显著降低(P0.05);试验组血清IGFBP-1含量显著或极显著升高(P0.05或P0.01),PC组显著升高(P0.05);试验组卵巢组织中IGF-1、IGFBP-1 mRNA表达水平均升高,其中M、H组显著升高(P0.05),与PC组变化一致。由此可见,GEN能够提高雌性大鼠血清FSH、LH含量、降低血清IGF-1含量、提高血清IGFBP-1含量,同时提高卵巢中IG FBP-1、IG F-1 mRNA的表达水平,这些指标协同作用于卵巢,能够促进卵泡的成熟,调节卵巢功能。