A TALEN Genome-Editing System for Generating Human Stem Cell-Based Disease Models

SummaryTranscription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease—dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease

The Disequilibrium of Nucleosomes Distribution along Chromosomes Plays a Functional and Evolutionarily Role in Regulating Gene Expression

To further understand the relationship between nucleosome-space occupancy (NO) and global transcriptional activity in mammals, we acquired a set of genome-wide nucleosome distribution and transcriptome data from the mouse cerebrum and testis based on ChIP (H3)-seq and RNA-seq, respectively. We identified a nearly consistent NO patterns among three mouse tissues—cerebrum, testis, and ESCs—and found, through clustering analysis for transcriptional activation, that the NO variations among chromosomes are closely associated with distinct expression levels between house-keeping (HK) genes and tissue-specific (TS) genes. Both TS and HK genes form clusters albeit the obvious majority. This feature implies that NO patterns, i.e. nucleosome binding and clustering, are coupled with gene clustering that may be functionally and evolutionarily conserved in regulating gene expression among different cell types

Search for Neutral Higgs Bosons in Events with Multiple Bottom Quarks at the Tevatron

The combination of searches performed by the CDF and D0 collaborations at the Fermilab Tevatron Collider for neutral Higgs bosons produced in association with b quarks is reported. The data, corresponding to 2.6 fb-1 of integrated luminosity at CDF and 5.2 fb-1 at D0, have been collected in final states containing three or more b jets. Upper limits are set on the cross section multiplied by the branching ratio varying between 44 pb and 0.7 pb in the Higgs boson mass range 90 to 300 GeV, assuming production of a narrow scalar boson. Significant enhancements to the production of Higgs bosons can be found in theories beyond the standard model, for example in supersymmetry. The results are interpreted as upper limits in the parameter space of the minimal supersymmetric standard model in a benchmark scenario favoring this decay mode.Comment: 10 pages, 2 figure

Metadherin Contributes to the Pathogenesis of Diffuse Large B-cell Lymphoma

BACKGROUND: Metadherin (MTDH) has been demonstrated as a potentially crucial mediator of various types of human malignancies. However, the expression and role of MTDH in diffuse large-B-cell lymphoma (DLBCL) have not been reported yet. This study aimed to illuminate the role of MTDH in the pathogenesis of DLBCL. METHODOLOGY/PRINCIPAL FINDINGS: A remarkable elevation of MTDH on mRNA level was detected in DLBCL tissues by quantitative polymerase chain reaction (PCR). Using Western-blot analysis we found that the expression of MTDH protein was significantly upregulated in DLBCL cell lines and DLBCL tissues compared with peripheral blood mononuclear cells (PBMCs) from healthy samples and tissues from patients of reactive hyperplasia of lymph node. The results showed high expression of MTDH in 23 of 30 (76.67%) DLBCL tissues by using immunohistochemical analysis and the over expression of MTDH was strongly correlated to the clinical staging of patients with DLBCL (P<0.05). Furthermore, the finding suggested that the increase of MTDH in DLBCL cells could distinctly enhance cell proliferation and inhibit cell apoptosis; meanwhile, inhibition of MTDH expression by specific siRNA clearly enhanced LY8 cell apoptosis. Upregulation of MTDH elevated the protein level of total β-catenin and translocation of β-catenin to the nucleus directly or indirectly. Knockdown of MTDH decreased the level of total, cytoplasmic β-catenin and reduced nuclear accumulation of β-catenin protein. This indicated that the function of MTDH on the development of DLBCL was mediated through regulation of Wnt/β-catenin signaling pathway. CONCLUSIONS/SIGNIFICANCE: Our results suggest that MTDH contributes to the pathogenesis of DLBCL mediated by activation of Wnt/β-catenin pathway. This novel study may contribute to further investigation on the useful biomarkers and potential therapeutic target in the DLBCL patients

Voltammetric aptasensors for protein disease biomarkers detection: a review

"Available online 24 May 2016"An electrochemical aptasensor is a compact analytical device where the bioreceptor (aptamer) is coupled to a transducer surface to convert a biological interaction into a measurable signal (current) that can be easily processed, recorded and displayed. Since the discovery of the Systematic Evolution of Ligands by Enrichment (SELEX) methodology, the selection of aptamers and their application as bioreceptors has become a promising tool in the design of electrochemical aptasensors. Aptamers present several advantages that highlight their usefulness as bioreceptors such as chemical stability, cost effectiveness and ease of modification towards detection and immobilization at different transducer surfaces. In this review, a special emphasis is given to the potential use of electrochemical aptasensors for the detection of protein disease biomarkers using voltammetry techniques. Methods for the immobilization of aptamers onto electrode surfaces are discussed, as well as different electrochemical strategies that can be used for the design of aptasensors.The authors acknowledge the financial support from the Strategic funding of UID/BIO/04469/2013 unit, from Project POCI-01-0145- FEDER-006984 – Associate Laboratory LSRE-LCM funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) – and by national funds through FCT - Fundação para a Ciência e a Tecnologia and project ref. RECI/BBB-EBI/ 0179/2012 (project number FCOMP-01-0124-FEDER-027462) and S. Meirinhos's doctoral grant (ref SFRH/BD/65021/2009) funded by Fundação para a Ciência e a Tecnologia

Geminivirus-encoded TrAP suppressor inhibits the histone methyltransferase SUVH4/KYP to counter host defense

Transcriptional gene silencing (TGS) can serve as an innate immunity against invading DNA viruses throughout Eukaryotes. Geminivirus code for TrAP protein to suppress the TGS pathway. Here, we identified an Arabidopsis H3K9me2 histone methyltransferase, Su(var)3-9 homolog 4/Kryptonite (SUVH4/KYP), as a bona fide cellular target of TrAP. TrAP interacts with the catalytic domain of KYP and inhibits its activity in vitro. TrAP elicits developmental anomalies phenocopying several TGS mutants, reduces the repressive H3K9me2 mark and CHH DNA methylation, and reactivates numerous endogenous KYP-repressed loci in vivo. Moreover, KYP binds to the viral chromatin and controls its methylation to combat virus infection. Notably, kyp mutants support systemic infection of TrAP-deficient Geminivirus. We conclude that TrAP attenuates the TGS of the viral chromatin by inhibiting KYP activity to evade host surveillance. These findings provide new insight on the molecular arms race between host antiviral defense and virus counter defense at an epigenetic level. DOI: http://dx.doi.org/10.7554/eLife.06671.00

A TALEN Genome-Editing System for Generating Human Stem Cell-Based Disease Models

Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease—dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease.Stem Cell and Regenerative Biolog