Intron-based genomic editing: a highly efficient method for generating knockin zebrafish

The TALEN and CRISPR/Cas9 nuclease systems have been extensively utilized in genomic engineering of model organisms. In zebrafish, the nuclease systems have been successfully applied in generating loss-of–function knockout lines. However, genome-specific knockin techniques in zebrafish are still at the beginning. In this perspective, we briefly summarize the recent progresses on knockin approaches in zebrafish with a special focus on the newly developed intron-based knockin method.     

RET mutation p.S891A in a Chinese family with familial medullary thyroid carcinoma and associated cutaneous amyloidosis binding OSMR variant p.G513D

There are no reports on the relationship between familial medullary thyroid carcinoma (FMTC) associated with cutaneous amyloidosis (CA) and RET or OSMR/IL31RA gene mutations. In this study, we investigated a Chinese family with FMTC/CA and found a recurrent RET c.2671T>G (p.S891A) mutation in six of 17 family members. Three of the six p.S891A mutation carriers presented with medullary thyroid carcinoma (MTC). Of them, three (two with and one without MTC) were diagnosed as having combined lichen/macular biphasic CA. We also identified a novel RET variant, c.1573C>T (p.R525W) in five members. Of them, three carriers had no evidence of thyroid/skin or basal serum/stimulated calcitonin abnormalities. In vitro cell proliferation assay indicated that oncogenic activity of RET p.S891A was slightly enhanced by p.R525W, whereas p.R525W alone had no effect on cell proliferation. Meanwhile, we identified a novel OSMR variant, c.1538G>A (p.G513D) in seven members. We noticed that three OSMR p.G513D carriers presenting with CA also had the RET p.S891A mutation. Our investigation indicated that the RET p.S891A mutation combined with OSMR p.G513D may underlie a novel phenotype manifesting as FMTC and CA.     

Ergosterol purified from medicinal mushroom Amauroderma rude inhibits cancer growth in vitro and in vivo by up-regulating multiple tumor suppressors

We have previously screened thirteen medicinal mushrooms for their potential anti-cancer activities in eleven different cell lines and found that the extract of Amauroderma rude exerted the highest capacity in inducing cancer cell death. The current study aimed to purify molecules mediating the anti-cancer cell activity. The extract of Amauroderma rude was subject to fractionation, silica gel chromatography, and HPLC. We purified a compound and identified it as ergosterol by EI-MS and NMR, which was expressed at the highest level in Amauroderma rude compared with other medicinal mushrooms tested. We found that ergosterol induced cancer cell death, which was time and concentration dependent. In the in vivo experiment, normal mice were injected with murine cancer cell line B16 that is very aggressive and caused mouse death severely. We found that treatment with ergosterol prolonged mouse survival. We found that ergosterol-mediated suppression of breast cancer cell viability occurred through apoptosis and that ergosterol up-regulated expression of the tumor suppressor Foxo3. In addition, the Foxo3 down-stream signaling molecules Fas, FasL, BimL, and BimS were up-regulated leading to apoptosis in human breast cancer cells MDA-MB-231. Our results suggest that ergosterol is the main anti-cancer ingredient in Amauroderma rude, which activated the apoptotic signal pathway. Ergosterol may serve as a potential lead for cancer therapy.     

Macrolide analog F806 suppresses esophageal squamous cell carcinoma (ESCC) by blocking β1 integrin activation

The paucity of new drugs for the treatment of esophageal squamous cell carcinoma (ESCC) limits the treatment options. This study characterized the therapeutic efficacy and action mechanism of a novel natural macrolide compound F806 in human ESCC xenograft models and cell lines. F806 inhibited growth of ESCC, most importantly, it displayed fewer undesirable side effects on normal tissues in two human ESCC xenograft models. F806 inhibited proliferation of six ESCC cells lines, with the half maximal inhibitory concentration (IC₅₀) ranging from 9.31 to 16.43 μM. Furthermore, F806 induced apoptosis of ESCC cells, contributing to its growth-inhibitory effect. Also, F806 inhibited cell adhesion resulting in anoikis. Mechanistic studies revealed that F806 inhibited the activation of β1 integrin in part by binding to a novel site Arg610 of β1 integrin, suppressed focal adhesion formation, decreased cell adhesion to extracellular matrix and eventually triggered apoptosis. We concluded that F806 would potentially be a well-tolerated anticancer drug by targeting β1 integrin, resulting in anoikis in ESCC cells.     

Hepatitis B virus core antigen as a carrier for Chlamydia trachomatis MOMP multi-epitope peptide enhances protection against genital chlamydial infection

Chlamydia trachomatis (Ct) is the leading cause of sexually transmitted diseases worldwide. There is no safe and effective vaccine to control the spread of Ct. In development of Ct vaccine, selection of appropriate candidate antigens and an effective delivery system may be the main challenges. Multi-epitope of major outer membrane protein (MOMPm) is the most suitable candidate for a Ct vaccine, while hepatitis B virus core antigen (HBcAg) has unique advantages as vaccine delivery system. Therefore, in this study, we evaluated the immunogenicity and protective immune response of a novel candidate vaccine in a murine model of chlamydial genital infection. This candidate vaccine comprises MOMPm peptide delivered with HBcAg. Our results of Ct-specific serum IgG and secretory IgA assay, cytokine assay, and cytotoxic T-lymphocyte assay revealed that immunogenicity of the candidate vaccine was much better than that of the corresponding synthetic MOMPm peptide. Furthermore, the protective effect of the candidate vaccine was also shown much better than that of the synthetic peptide by calculating the isolation of Chlamydia from vaginal swabs and histopathological analysis. Taken together, our results indicate that HBcAg carrying Ct MOMPm could be an effective immune prophylactic for chlamydial infection.     

大鼠骨髓基质细胞体外培养向神经干细胞的诱导分化

目的:已证实骨髓基质细胞可分化为中胚层组织细胞,实验予进一步探讨体外分离培养的骨髓基质细胞向神经干细胞分化的可能性,以及是否能继续定向分化为神经细胞及神经胶质细胞,为神经系统疾病细胞移植治疗提供种子细胞.方法:实验于2007-02/09在泸州医学院神经生物学研究室进行.①动物:选择5只普通级SD大鼠,由泸州医学院实验动物中心提供,实验过程中对动物的处胃符合动物伦理学标准.②实验方法:大鼠戊巴比妥钠腹腔麻醉,取双侧胫骨和股骨,磷酸盐缓冲液冲洗骨髓腔.采用密度梯度离心法从大鼠骨髓中分离培养骨髓基质细胞,胰蛋白酶与EDTA联合消化,传至第4代,用含20 μg/L碱性成纤维细胞生长因子、20 μ g/L表皮生长因子、N2辅助因子的DMEM/F12培养液向神经干细胞诱导分化.③实验评估:观察原代、传代培养及诱导分化后的骨髓基质细胞生长情况和形态变化,采用SABC法进行免疫细胞化学榆测神经细胞特异性标志的表达.结果:①骨髓基质细胞形态观察:原代细胞接种l d后开始贴壁增殖,3 d后多数贴壁,贴壁细胞呈梭形或扁平形;10 d后90%细胞融合,以长梭形为主,突起粗大,形成网状、片状;传代后细胞贴壁速度加快,增殖能力更强,7 d左右达到融合.②诱导分化后细胞生长情况和形态变化:第4代骨髓基质细胞向神经干细胞诱导分化7 d,成球的细胞脱离瓶底,悬浮在细胞液中.将细胞离心弃上清,换成血清分化液后,细胞球逐渐贴壁,球周围很快发出突起,分化为星形胶质细胞样细胞、神经元样细胞及少突胶质细胞样细胞.③神经细胞特异性标志的表达:骨髓源性细胞球表达巢蛋白,呈棕黄色,为神经干细胞:从细胞球分化的细胞抗胶质纤维酸性蛋白、微管相关蛋白2及半乳糖脑苷脂均呈阳性.结论:骨髓基质细胞能在体外诱导分化出神经干细胞,且骨髓源性神经干细胞可进一步定向分化为神经细胞及神经胶质细胞.     

TSLP regulates intestinal immunity and inflammation in mouse models of helminth infection and colitis

Intestinal epithelial cells (IECs) produce thymic stromal lymphopoietin (TSLP); however, the in vivo influence of TSLP–TSLP receptor (TSLPR) interactions on immunity and inflammation in the intestine remains unclear. We show that TSLP–TSLPR interactions are critical for immunity to the intestinal pathogen Trichuris. Monoclonal antibody–mediated neutralization of TSLP or deletion of the TSLPR in normally resistant mice resulted in defective expression of Th2 cytokines and persistent infection. Susceptibility was accompanied by elevated expression of interleukin (IL) 12/23p40, interferon (IFN) γ, and IL-17A, and development of severe intestinal inflammation. Critically, neutralization of IFN-γ in Trichuris-infected TSLPR^−/− mice restored Th2 cytokine responses and resulted in worm expulsion, providing the first demonstration of TSLPR-independent pathways for Th2 cytokine production. Additionally, TSLPR^−/− mice displayed elevated production of IL-12/23p40 and IFN-γ, and developed heightened intestinal inflammation upon exposure to dextran sodium sulfate, demonstrating a previously unrecognized immunoregulatory role for TSLP in a mouse model of inflammatory bowel disease.Intestinal epithelial cells (IECs) are a critical cell population that maintains intestinal immune homeostasis through both barrier function and the ability to actively modulate intestinal immune responses (1–3). One IEC-derived cytokine with immunomodulatory properties is thymic stromal lymphopoietin (TSLP) (4). TSLP is a four-helix bundle cytokine that is expressed both in humans and mice. Despite poor sequence homology, human and mouse TSLP exhibit similar biological functions (4). Expression of TSLP is regulated by NF-κB and can be induced by exposure to viral, bacterial, and parasitic pathogens, inflammatory cytokines, and the Th2 cell–associated cytokines IL-4 and IL-13 (3, 5–8). TSLP binds to its high affinity receptor, a heterodimer composed of a unique TSLPRα chain and the IL-7Rα chain, that is expressed on hematopoietic cell lineages, including B cells, T cells, mast cells, and DCs (4, 5, 9–12).In vitro studies demonstrated that TSLP-conditioned human DCs can promote Th2 cell responses (11, 13–15). Mechanistically, TSLP treatment of DCs induces Th2 cell differentiation by inhibiting IL-12 production while simultaneously inducing OX40L expression (14–16). The in vivo functions of TSLP have been most extensively studied in the skin and the lung (11, 13, 17, 18). Transgenic overexpression of TSLP in cutaneous or pulmonary epithelial cells results in the onset of Th2 cytokine–mediated inflammation resembling atopic dermatitis or asthma, respectively (17, 18). Based on these studies, it has been proposed that TSLP is both necessary and sufficient for the initiation of Th2 cytokine–driven inflammation (4, 19, 20). We recently showed that TSLP responsiveness is an important component of early immunity to the intestinal nematode pathogen Trichuris (2). However, the mechanisms and absolute requirements for TSLP–TSLPR interactions in the regulation of intestinal immunity and inflammation in vivo remain undefined.In this study, we identify constitutive TSLP expression in IECs throughout the lower gastrointestinal (GI) tract, with the highest level of expression in the proximal large intestine. When challenged with Trichuris, genetically resistant WT mice in which TSLP was neutralized or TSLPR^−/− mice failed to express protective Th2 cell–associated cytokines and maintained persistent parasites beyond day 34 after infection. Disruption of the TSLP–TSLPR pathway additionally resulted in increased expression of IL-12/23p40, IFN-γ, and IL-17A, and the development of severe infection-induced inflammation. Recombinant TSLP inhibited production of IL-12/23p40 in DCs in vitro, and DCs isolated from infected TSLPR^−/− mice exhibited dysregulated production of IL-12/23p40 ex vivo. Significantly, blockade of IFN-γ in Trichuris-infected TSLPR^−/− mice restored expression of Th2 cytokines and host protective immunity. Additionally, TSLPR^−/− mice exhibited elevated expression of proinflammatory cytokines and early onset of intestinal inflammation in a mouse model of inflammatory bowel disease (IBD). Collectively, these data suggest that within the intestinal microenvironment, one function of TSLP–TSLPR interactions may be to limit proinflammatory cytokine production and inflammation.     

Akt/Nrf2 Activated Upregulation of Heme Oxygenase-1 Involves in the Role of Rg1 Against Ferrous Iron-Induced Neurotoxicity in SK-N-SH Cells

Iron accumulation is considered to be involved in the pathogenesis of Parkinson’s disease (PD). Our previous studies have observed that Rg1, a major pharmacologically active ingredient from Ginseng, could protect dopaminergic neurons by reducing nigral iron levels through regulating the expression of iron transporters in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mice. The aim of this study is to investigate other mechanism involved in the cytoprotection of Rg1 against iron-induced neurotoxicity in human neuroblastoma SK-N-SH cells. Significant rescue of Rg1 on cell viability against 100 μM ferrous iron-induced neurotoxicity was observed. Upregulation of heme oxygenase-1 (HO-1) and Cu–Zn superoxide dismutase (Cu/Zn SOD) were observed in Rg1 pretreated group. Moreover, Rg1 pretreatment induces Nrf2 nuclear translocation, which is upstream of HO-1 expression, and activated PI3K/Akt pathway was also observed in Rg1 pretreated group. This could antagonize iron-induced increase in intracellular reactive oxygen species and decrease in mitochondrial transmembrane potential. These results suggest that the neuroprotective effects of Rg1 against iron toxicity are attributed to the anti-oxidative properties by activating Akt/Nrf2 pathway and increasing Nrf2-induced expression of HO-1 and Cu/Zn SOD.     

Surgical treatment of high grade dural arteriovenous fistulae

Dural arteriovenous fistulae (dAVF) with direct cortical venous drainage (CVD, Borden Type III) have a high risk of hemorrhage, particularly when symptomatic. Stereotactic radiosurgery is therefore not recommended, and endovascular treatment can be limited by access, incomplete obliteration, and recanalization. Of 70 cerebral dAVF seen at our institution over the past 8 years, 35 were Borden Type III (50%). Twenty-four were treated via microsurgery (69%). Presentation included hemorrhage in nine patients (38%), nonhemorrhagic neurologic deficits in five (21%), asymptomatic in five (21%), headache in three (13%), and seizure in two patients (8%). Only eight of 19 patients with symptomatic dAVF were independent (modified Rankin Scale [mRS] 0–2) preoperatively (42%). The dAVF location was tentorial in six patients (25%), petrosal in six (25%), superior sagittal sinus in four (17%), torcular in two (7%), floor of the anterior fossa in two (7%), and sphenoid ridge, transverse-sigmoid, inferior sagittal sinus and jugular in one patient each (4%). Four patients had failed endovascular therapy (17%). The angiographic obliteration rate was 96%. The combined permanent morbidity and mortality rate was 17%. After a mean follow-up of 2.1 years, 13 patients improved (54%), seven were the same, (29%) and four were worse (17%). Thirteen patients were asymptomatic (mRS 0, 54%), and 18 were independent (mRS 0–2, 75%). Our results reinforce that surgical treatment of dAVF with direct CVD is associated with a high angiographic cure rate with acceptable morbidity and mortality, particularly in light of the lesions’ natural history.