Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing

Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.     

Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.Complexities of natural biological systems make it difficult to understand and define precisely the roles of individual genes and their integrated functions. The use of model organisms with a relatively small number of genes enables the isolation of core biological processes from their complex regulatory networks for extensive characterization. However, even the simplest natural microbes contain many genes of unknown function, as well as genes that can be singly or simultaneously deleted without any noticeable effect on growth rate in a laboratory setting (Hutchison et al. 1999; Glass et al. 2006; Posfai et al. 2006). Ill-defined genes and those mediating functional redundancies both compound the challenge of understanding even the simplest life forms.Toward generating a minimal cell where every gene is essential for the axenic viability of the organism, we are pursuing strategies to reduce the 1-Mb genome of Mycoplasma mycoides JCVI-syn1.0 (Gibson et al. 2010). Because we can (1) introduce this genome into yeast and maintain it as a plasmid (Benders et al. 2010; Karas et al. 2013a); and (2) “transplant” the genome from yeast into mycoplasma recipient cells (Lartigue et al. 2009), genetic tools in yeast are available for reducing this bacterial genome. Several systems offer advanced tools for bacterial genome engineering. Here we further exploit distinctive features of yeast for this purpose.Methods for serially replacing genomic regions with selectable markers are limited by the number of available markers. One effective approach is to reuse the same marker after precise and scarless marker excision (Storici et al. 2001). We have previously used a self-excising marker (Noskov et al. 2010) six times in yeast to generate a JCVI-syn1.0 genome lacking all six restriction systems (JCVI-syn1.0 ∆1-6) (Karas et al. 2013a). Despite the advantages of scarless engineering, sequential procedures are time-consuming. When applied to poorly characterized genes with the potential to interact with other genes, some paths for multigene knockout may lead to dead ends that result from synergistic mutant phenotypes. When a dead end is reached, sequentially returning to a previous genome in an effort to find a detour to a viable higher-order multimutant may be prohibitively time-consuming.An alternative approach to multigene engineering, available in yeast, is to prepare a set of single mutants and combine the deletions into a single strain via cycles of mating and meiotic recombination (Fig. 1A; Pinel et al. 2011; Suzuki et al. 2011, 2012). With a green fluorescent protein (GFP) reporter gene inserted in each deletion locus, the enrichment of higher-order yeast deletion strains in the meiotic population can be accomplished using flow cytometry. Here we apply this method to the JCVI-syn1.0 ∆1-6 exogenous, bacterial genome harbored in yeast to nonsequentially assemble deletions for genes predicted to be individually deletable based on biological knowledge or transposon-mediated disruption data. The functional identification of simultaneously deletable regions is expected to accelerate the effort to construct a minimal genome.Open in a separate windowFigure 1.Progressive clustering of deleted genomic segments. (A) Scheme of the method. Light blue oval represents a bacterial cell. Black ring or horizontal line denotes a bacterial genome, with the orange box indicating the yeast vector used as a site for linearization and recircularization. Gray shape denotes a yeast cell. Green dot in the genome indicates a deletion replaced with a GFP marker. (B) Map of deleted regions. Orange box indicates the yeast vector sequence used for genome linearization and recircularization. Green boxes indicate regions deleted in multimutant mycoplasma strains. Blue boxes denote restriction modification (RM) systems that are also deleted in the strains. (C) Pulsed-gel electrophoresis result for deleted genomes. The starting strain was the JCVI-syn1.0 ∆1–6 strain (1062 kb). Two strains were analyzed for each design of simultaneous deletion (962 kb for eight-deletion or 974 kb for seven-deletion genome). Ladder is a set of yeast chromosomes (New England BioLabs). (D) GFP-RFP ratio sorting result. Standard sorting was compared with sorting based on a GFP-RFP ratio (Methods).     

江苏省沿海野生栝楼资源分布特点与评价

对江苏省沿海野生栝楼的形态、分布、生态环境与群落类型等进行了资料分析、踏查、样地调查。结果表明,栝楼在东台、射阳沿海滩涂较多分布于人工刺槐林、银杏林下和林缘,在村落周围和滩涂草甸也有分布,极少分布于农田和沟渠。对东台市沿海130.04 km2代表区域内天花粉蕴藏量估算值达731.663 7 t;射阳县沿海113.89 km2代表区域内天花粉的蕴藏量约为125.618 6 t。对江苏省沿海栝楼野生资源的生产发展提出了建议,为开展栝楼野生资源的可持续利用奠定基础。     

不同放疗靶区对食管癌疗效及安全性的影响

目的:分析不同临床靶区勾画对食管癌同步放化疗疗效及安全性的影响,探讨食管癌三维适形放疗的临床靶区范围。方法:2009年1月至2012年1月收治的60例食管癌患者随机分为非预防组和预防组,均接受同步放化疗。非预防组28例,CTV包括原发灶上下外扩3cm、周围外扩0.8-1.0cm及肿大淋巴结累及区;预防组32例,食管癌原发灶CTV外扩同非预防组,根据原发灶部位不同,给予区域淋巴结引流区的预防照射。结果:非预防组和预防组1、2年的生存率分别为67.9%、57.1%和68.8%、50.0%;1、2年局部控制率分别为71.4%、60.7%和71.9%、59.4%;野内淋巴结复发率分别为7.1%、6.3%。非预防组Ⅲ级以上放射性肺炎、放射性食管炎及骨髓抑制为3.6%、7.1%、14.3%;预防组Ⅲ级以上放射性肺炎、放射性食管炎及骨髓抑制发生率为6.3%、12.5%、15.6%。结论:预防组在提高生存率、局部控制率及降低野内淋巴结复发率方面未表现出明显优势,两组疗效相当(P>0.5)。预防组放射性肺炎、放射性食管炎及骨髓抑制发生率均高于非预防组,但两组比较无统计学意义(P>0.5)。预防组肺V10(%)、肺V20(%)、肺V30(%)三项指标均大于非预防组,两者差异有统计学意义(P<0.05)。     

1 585 例甲状腺癌的临床病理特点及总结分析

目的:研究甲状腺乳头状癌（pnpillary thyroid cancer,PTC ）的发病趋势及临床病理特点。方法:回顾性分析湖北省人民医院2001年1 月至2013年7 月甲状腺疾病和湖北省肿瘤医院2006年1 月至2013年7 月甲状腺癌的临床病理资料。结果:湖北省人民医院自2008年以来,甲状腺癌发病明显升高,由14.94% 上升至18.10% ,其中甲状腺乳头状癌所占比例明显上升,由2008年的15.23%（46/302）上升至2012年的19.32%（166/859）。 两家医院中,甲状腺乳头状癌所占比例明显上升,由85.33%（378/443）上升至90.89%（1 038/1 142）;微小乳头状癌比例由9.26%（35/378）上升至22.83%（237/1 038）;确诊甲状腺乳头状癌患者1 416 例,男女比例为1:3.75;颈部淋巴结阳性检出率在性别、年龄方面相比均有显著性差异（P<0.05）,多病灶甲状腺乳头状癌患者颈部淋巴结阳性检出率为77.94% ,与单病灶甲状腺乳头状癌患者相比有显著性差异（P<0.05）。 单纯甲状腺乳头状癌患者颈部淋巴结阳性检出率为72.29% ,与合并结节性甲状腺肿者及合并桥本氏甲状腺炎者相比有显著性差异（P<0.05）。 结论:甲状腺乳头状癌的发病呈增长趋势,男性、年龄<45岁肿瘤直径>1 cm、多病灶肿瘤、单纯PTC 更易并发颈部淋巴结转移。      

Typhoid fever complicated by multiple organ involvement: report of two cases

Typhoid fever complicated by multiple organ involvement has been rarely mentioned in the literature. We reported two cases of typhoid fever with several unusual manifestations, including acute renal failure, acute hepatitis, acute pancreatitis, disseminated intravascular coagulation, and lower gastrointestinal bleeding. A renal biopsy in the first case showed no pathological change. Bone marrow biopsy showed focal necrosis of matrix, which might have been due to severe illness. A liver biopsy in the second case showed a predominantly histiocytic proliferation with occasional neutrophilic infiltration in the portal areas and hepatic sinusoids. Focal necrosis, bile duct injury, and multiple eosinophilic bodies were also noted. After appropriate antimicrobial therapy, both patients recovered without any sequelae. The potential of multiple organ involvement is highlighted in typhoid fever, which, on rare occasions, may occur simultaneously in the same patient.     

The Mood Stabilizer Lithium Potentiates the Antidepressant-Like Effects and Ameliorates Oxidative Stress Induced by Acute Ketamine in a Mouse Model of Stress

Background:

Evidence suggests that mammalian target of rapamycin activation mediates ketamine’s rapid but transient antidepressant effects and that glycogen synthase kinase-3β inhibits this pathway. However, ketamine has associated psychotomimetic effects and a high risk of abuse. The mood stabilizer lithium is a glycogen synthase kinase-3 inhibitor with strong antisuicidal properties. Here, we used a mouse stress model to investigate whether adjunct lithium treatment would potentiate ketamine’s antidepressant-like effects.

Methods:

Mice received chronic restraint stress and long-term pre- or postketamine lithium treatment in drinking water. The effects of lithium on ketamine-induced antidepressant-like effects, activation of the mammalian target of rapamycin/brain-derived neurotrophic factor signaling pathways, oxidative stress, and dendritic spine density in the brain of mice were investigated.

Results:

Subtherapeutic (600mg/L) lithium-pretreated mice exhibited an antidepressant-like response to an ineffective ketamine (2.5mg/kg, intraperitoneally) challenge in the forced swim test. Both the antidepressant-like effects and restoration of dendritic spine density in the medial prefrontal cortex of stressed mice induced by a single ketamine (50mg/kg) injection were sustained by postketamine treatment with 1200mg/L of lithium for at least 2 weeks. These benefits of lithium treatments were associated with activation of the mammalian target of rapamycin/brain-derived neurotrophic factor signaling pathways in the prefrontal cortex. Acute ketamine (50mg/kg) injection also significantly increased lipid peroxidation, catalase activity, and oxidized glutathione levels in stressed mice. Notably, these oxidative stress markers were completely abolished by pretreatment with 1200mg/L of lithium.

Conclusions:

Our results suggest a novel therapeutic strategy and justify the use of lithium in patients who benefit from ketamine.