Short-Chain Inulin Modulates the Cecal Microbiota Structure of Leptin Knockout Mice in High-Fat Diet.

The aim of this study was to explore the effect of short-chain inulin on cecal microbiota of high-fat diet-fed leptin knockout mice and the different influences of cecal microbiota on wild-type and leptin knockout mice. A total of 18 specific pathogen-free male C57BL/6J wild-type mice and 18 C57BL/6J leptin knockout mice (OB/OB mice) were selected. Mice were divided into six groups according to their genotype: wild-type mice have three groups, including the normal diet group (CT), 60% high-fat diet group (CH), and 60% high fat with 10% short-chain inulin group (CHI); OB/OB mice were also divided into three groups, including the normal diet group (OT), 60% high-fat diet group (OH), and 60% high fat with 10% short-inulin group (OHI). The mice were fed for 8 weeks to analyze the diversity of cecal microbiota. The results show that compared with CH and OH, the variety of cecal microbiota was significantly reduced in CH and OH and further reduced in CHI and OHI. Bifidobacterium and Lactobacillus are the biomarkers in genus level. Dietary short-chain inulin significantly enhanced Bifidobacterium in OHI compared with OH (p < 0.01) and significantly reduced in CHI and compared with CH (p < 0.01). Lactobacillus was significantly enhanced in CHI and OHI compared with CH and OH, respectively (p < 0.01). Blautia was significantly enhanced in CH and OH compared with other groups (p < 0.01). Both Escherichia-Shigella and Enterococcus were significantly reduced in CHI and OHI, compared with CH and OH, respectively (p < 0.05). Escherichia-Shigella was even lower than CT and OT in CHI and OHI. Functional prediction of microbial communities showed that the abundance of amino acid sugar and nucleotide sugar metabolism pathways were significantly enhanced (p < 0.05) in CH and OH, and OH was significantly higher than CH (p < 0.05). Among the leptin knockout groups, PICRUSt2 function prediction showed that the fatty acid metabolism pathway significantly reduced (p < 0.05) in OHI and OT compared with OH. In conclusion, short-chain inulin modulated the dysbiosis induced by high-fat diet, improved probiotics growth and inhibited conditioned pathogenic bacteria, and the influences were significantly different in wild-type and leptin knockout mice.

TALEN-Mediated Knockout of CCR5 Confers Protection Against Infection of Human Immunodeficiency Virus.

Transcription activator-like effector nuclease (TALEN) represents a valuable tool for genomic engineering due to its single-nucleotide precision, high nuclease activity, and low cytotoxicity. We report here systematic design and characterization of 28 novel TALENs targeting multiple regions of CCR5 gene (CCR5-TALEN) which encodes the co-receptor critical for entry of human immunodeficiency virus type I (HIV-1). By systemic characterization of these CCR5-TALENs, we have identified one (CCR5-TALEN-515) with higher nuclease activity, specificity, and lower cytotoxicity compared with zinc-finger nuclease (CCR5-ZFN) currently undergoing clinical trials. Sequence analysis of target cell line GHOST-CCR5-CXCR4 and human primary CD4 T cells showed that the double-strand breaks at the TALEN targeted sites resulted in truncated or nonfunctional CCR5 proteins thereby conferring protection against HIV-1 infection in vitro. None of the CCR5-TALENs had detectable levels of off-target nuclease activity against the homologous region in CCR2 although substantial level was identified for CCR5-ZFN in the primary CD4 T cells. Our results suggest that the CCR5-TALENs identified here are highly functional nucleases that produce protective genetic alterations to human CCR5. Application of these TALENs directly to the primary CD4 T cells and CD34 hematopoietic stem cells (HSCs) of infected individuals could help to create an immune system resistant to HIV-1 infection, recapitulating the success of "Berlin patient" and serving as an essential first step towards a "functional" cure of AIDS.

RNAe in a transgenic growth hormone mouse model shows potential for use in gene therapy.

OBJECTIVE: RNAe is a new method that enhances protein expression at the post-transcriptional level. RNAe utility was further explored to improve endogenous protein expression. RESULTS: Transgenic mice were created by targeting RNAe to growth hormone gene into the C57/BL mouse genome by transposon mediated integration; the mice showed a heavier body weight and longer body length compared with normal mice. RNAe can also be used for gene therapy through the delivery of in vitro transcribed RNA. CONCLUSION: This study takes a further step towards applying RNAe in pharmaceutical approaches by transposon-based transgenic mice model construction and the use of in vitro transcribed RNA transfection assay.

Whole-exome sequencing implicates UBE3D in age-related macular degeneration in East Asian populations.

Age-related macular degeneration (AMD) is a leading cause of irreversible central blindness among the elderly worldwide. We use exome sequencing to analyse nonsynonymous single-nucleotide variants (SNVs) across the whole genome of 216 neovascular AMD cases and 1,553 controls. As a follow-up validation, we evaluate 3,772 neovascular AMD cases and 6,942 controls from five independent cohorts in the East Asian population. Here we show strong evidence of an association at a novel, missense SNV, rs7739323, which is located in the ubiquitin protein ligase E3D (UBE3D) gene (Pmeta=1.46 × 10(-9), odds ratio (OR)=0.74, 95% confidence interval (CI): 0.63-0.88). Furthermore, ablation of the UBE3D protein lead to an abnormal amount of pigment granules deposited in retinal pigment epithelium microvilli area and an abnormal response on electroretinography (ERG) in UBE3D(+/-) heterozygous mice. Our findings indicate that the ubiquitin-proteasome system may play a role in the pathogenesis of neovascular AMD.

Engineered TAL Effector modulators for the large-scale gain-of-function screening.

Recent effective use of TAL Effectors (TALEs) has provided an important approach to the design and synthesis of sequence-specific DNA-binding proteins. However, it is still a challenging task to design and manufacture effective TALE modulators because of the limited knowledge of TALE-DNA interactions. Here we synthesized more than 200 TALE modulators and identified two determining factors of transcription activity in vivo: chromatin accessibility and the distance from the transcription start site. The implementation of these modulators in a gain-of-function screen was successfully demonstrated for four cell lines in migration/invasion assays and thus has broad relevance in this field. Furthermore, a novel TALE-TALE modulator was developed to transcriptionally inhibit target genes. Together, these findings underscore the huge potential of these TALE modulators in the study of gene function, reprogramming of cellular behaviors, and even clinical investigation.

AEG-1 Promotes Metastasis Through Downstream AKR1C2 and NF1 in Liver Cancer.

Liver cancer is one of the most lethal cancers, but our knowledge of the molecular mechanism underlying this process remains insufficient. Through deep sequencing and expression regulation analysis in liver cancer cells, we identified two novel factors, AKR1C2 (positive factor) and NF1 (negative factor), as the AEG-1 downstream players in the process of metastasis in liver cancer. They were experimentally validated to have the capacities of regulating cell migration, cell invasion, cell proliferation, and EMT. Further clinic expression and animal model evidence confirmed their functions. Together, our findings provide a new insight into the pharmaceutical and therapeutic use of AEG-1 and downstream AKR1C2 and NF1.