PROBer Provides a General Toolkit for Analyzing Sequencing-Based Toeprinting Assays.

Cell Syst. 2017 May 09;:

Authors: Li B, Tambe A, Aviran S, Pachter L

Abstract
A number of sequencing-based transcriptase drop-off assays have recently been developed to probe post-transcriptional dynamics of RNA-protein interaction, RNA structure, and RNA modification. Although these assays survey a diverse set of epitranscriptomic marks, we use the term toeprinting assays since they share methodological similarities. Their interpretation is predicated on addressing a similar computational challenge: how to learn isoform-specific chemical modification profiles in the face of complex read multi-mapping. We introduce PROBer, a statistical model and associated software, that addresses this challenge for the analysis of toeprinting assays. PROBer takes sequencing data as input and outputs estimated transcript abundances and isoform-specific modification profiles. Results on both simulated and biological data demonstrate that PROBer significantly outperforms individual methods tailored for specific toeprinting assays. Since the space of toeprinting assays is ever expanding and these assays are likely to be performed and analyzed together, we believe PROBer's unified data analysis solution will be valuable to the RNA community.

PMID: 28501650 [PubMed - as supplied by publisher]

Higher viral load and genetic diversity of HIV-1 in the seminal compartments than in the blood of seven Chinese homosexual men with early HIV-1 infection.

Microbiol Immunol. 2017 May 13;:

Authors: Jiao YM, Chen GL, Zhu WJ, Huang HH, Fu JL, Chen WW, Shi M, Zhang T, Wu H, Wang FS

Abstract
To date, there have been no reports characterizing the Human Immunodeficiency Virus-1 (HIV-1) in the semen of Chinese men having sex with men (MSM) with early infection of HIV-1.We examined genetic diversity and viral load of HIV-1 in the seminal compartments and blood of Chinese MSM with early HIV-1 infection in this study. Viral load and genetic diversity of HIV-1 in paired samples of semen and blood were analyzed in seven homosexual patients who were with early HIV-1 infection. Levels of HIV-1 RNA and DNA were tested by real-time PCR assays. Through sequencing the C2-V5 region of the HIV-1 env gene we determined the HIV-1 genotype and genetic diversity based on V3 loop amino acid sequences by using Geno2pheno and PSSM programs theco-receptor usage was predicated. We found that in seminal plasma the HIV-1 RNA levels were higher compared with those in blood plasma and total, the levels of 2-LTR circular and integrated HIV-1 DNA were higher in seminal cells compared with those in peripheral blood mononuclear cells from all seven early HIV-infected patients. There was higher HIV-1 genetic diversity in seminal compartments as compared to blood compartments. HIV-1 in plasma displayed higher genetic diversity than in cells from the blood and semen, respectively. In addition, the V3 loop central motifs, which present some key neutralizing antibodies epitopes, varied between the blood and the semen. So virological parameters in semen may be more representative to evaluate the transmission risk in early HIV infection.

PMID: 28500746 [PubMed - as supplied by publisher]

Intranasal drug delivery of small interfering RNA targeting Beclin1 encapsulated with polyethylenimine (PEI) in mouse brain to achieve HIV attenuation.

Sci Rep. 2017 May 12;7(1):1862

Authors: Rodriguez M, Lapierre J, Ojha CR, Kaushik A, Batrakova E, Kashanchi F, Dever SM, Nair M, El-Hage N

Abstract
We previously reported that activation of the host autophagic protein, Beclin1, by HIV-1 infection represents an essential mechanism in controlling HIV replication and viral-induced inflammatory responses in microglial cells. Existing antiretroviral therapeutic approaches have been limited in their ability to cross the blood-brain barrier effectively and recognize and selectively eliminate persistent HIV-infected brain reservoirs. In the present study and for the first time, the bio-distribution and efficacy of noninvasive intranasal delivery of small interfering RNA (siRNA) against the Beclin1 gene using the cationic linear polyethylenimines (PEI) as a gene carrier was investigated in adult mouse brain. Fluorescein isothiocyanate (FITC)-labeled control siRNA delivered intranasally was found in the cytoplasm of neurons and glial cells of the prefrontal cortex at 4 and 24 hours post-delivery, with no major adverse immune reaction encountered. Intranasal delivery of the siRNA targeting Beclin1 significantly depleted the target protein expression levels in brain tissues with no evidence of toxicity. Binding of siRNA to PEI-polymer was characterized and confirmed by Raman spectroscopy. These results indicate that the intranasal drug delivery allows for the direct delivery of the PEI-siRNA nano-complex to the central nervous system, which could potentially offer an efficient means of gene silencing-mediated therapy in the HIV-infected brain.

PMID: 28500326 [PubMed - in process]

Combined HMG-COA reductase and prenylation inhibition in treatment of CCM.

Proc Natl Acad Sci U S A. 2017 May 12;:

Authors: Nishimura S, Mishra-Gorur K, Park J, Surovtseva YV, Sebti SM, Levchenko A, Louvi A, Gunel M

Abstract
Cerebral cavernous malformations (CCMs) are common vascular anomalies that develop in the central nervous system and, more rarely, the retina. The lesions can cause headache, seizures, focal neurological deficits, and hemorrhagic stroke. Symptomatic lesions are treated according to their presentation; however, targeted pharmacological therapies that improve the outcome of CCM disease are currently lacking. We performed a high-throughput screen to identify Food and Drug Administration-approved drugs or other bioactive compounds that could effectively suppress hyperproliferation of mouse brain primary astrocytes deficient for CCM3. We demonstrate that fluvastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase and the N-bisphosphonate zoledronic acid monohydrate, an inhibitor of protein prenylation, act synergistically to reverse outcomes of CCM3 loss in cultured mouse primary astrocytes and in Drosophila glial cells in vivo. Further, the two drugs effectively attenuate neural and vascular deficits in chronic and acute mouse models of CCM3 loss in vivo, significantly reducing lesion burden and extending longevity. Sustained inhibition of the mevalonate pathway represents a potential pharmacological treatment option and suggests advantages of combination therapy for CCM disease.

PMID: 28500274 [PubMed - as supplied by publisher]

ZBTB48 is both a vertebrate telomere-binding protein and a transcriptional activator.

EMBO Rep. 2017 May 12;:

Authors: Jahn A, Rane G, Paszkowski-Rogacz M, Sayols S, Bluhm A, Han CT, Draškovič I, Londoño-Vallejo JA, Kumar AP, Buchholz F, Butter F, Kappei D

Abstract
Telomeres constitute the ends of linear chromosomes and together with the shelterin complex form a structure essential for genome maintenance and stability. In addition to the constitutive binding of the shelterin complex, other direct, yet more transient interactions are mediated by the CST complex and HOT1/HMBOX1, while subtelomeric variant repeats are recognized by NR2C/F transcription factors. Recently, the Kruppel-like zinc finger protein ZBTB48/HKR3/TZAP has been described as a novel telomere-associated factor in the vertebrate lineage. Here, we show that ZBTB48 binds directly both to telomeric and to subtelomeric variant repeat sequences. ZBTB48 is found at telomeres of human cancer cells regardless of the mode of telomere maintenance and it acts as a negative regulator of telomere length. In addition to its telomeric function, we demonstrate through a combination of RNAseq, ChIPseq and expression proteomics experiments that ZBTB48 acts as a transcriptional activator on a small set of target genes, including mitochondrial fission process 1 (MTFP1). This discovery places ZBTB48 at the interface of telomere length regulation, transcriptional control and mitochondrial metabolism.

PMID: 28500257 [PubMed - as supplied by publisher]

Characterization of a splice-site mutation in the tumor suppressor gene FLCN associated with renal cancer.

BMC Med Genet. 2017 May 12;18(1):53

Authors: Bartram MP, Mishra T, Reintjes N, Fabretti F, Gharbi H, Adam AC, Göbel H, Franke M, Schermer B, Haneder S, Benzing T, Beck BB, Müller RU

Abstract
BACKGROUND: Renal cell carcinoma is among the most prevalent malignancies. It is generally sporadic. However, genetic studies of rare familial forms have led to the identification of mutations in causative genes such as VHL and FLCN. Mutations in the FLCN gene are the cause of Birt-Hogg-Dubé syndrome, a rare tumor syndrome which is characterized by the combination of renal cell carcinoma, pneumothorax and skin tumors.
METHODS: Using Sanger sequencing we identify a heterozygous splice-site mutation in FLCN in lymphocyte DNA of a patient suffering from renal cell carcinoma. Furthermore, both tumor DNA and DNA from a metastasis are analyzed regarding this mutation. The pathogenic effect of the sequence alteration is confirmed by minigene assays and the biochemical consequences on the protein are examined using TALEN-mediated transgenesis in cultured cells.
RESULTS: Here we describe an FLCN mutation in a 55-year-old patient who presented himself with progressive weight loss, bilateral kidney cysts and renal tumors. He and members of his family had a history of recurrent pneumothorax during the last few decades. Histology after tumor nephrectomy showed a mixed kidney cancer consisting of elements of a chromophobe renal cell carcinoma and dedifferentiated small cell carcinoma component. Subsequent FLCN sequencing identified an intronic c.1177-5_-3delCTC alteration that most likely affected the correct splicing of exon 11 of the FLCN gene. We demonstrate skipping of exon 11 to be the consequence of this mutation leading to a shift in the reading frame and the insertion of a premature stop codon. Interestingly, the truncated protein was still expressed both in cell culture and in tumor tissue, though it was strongly destabilized and its subcellular localization differed from wild-type FLCN. Both, altered protein stability and subcellular localization could be partly reversed by blocking proteasomal and lysosomal degradation.
CONCLUSIONS: Identification of disease-causing mutations in BHD syndrome requires the analysis of intronic sequences. However, biochemical validation of the consecutive alterations of the resulting protein is especially important in these cases. Functional characterization of the disease-causing mutations in BHD syndrome may guide further research for the development of novel diagnostic and therapeutic strategies.

PMID: 28499369 [PubMed - in process]

The dynamics of early-state transcriptional changes and aggregate formation in a Huntington's disease cell model.

BMC Genomics. 2017 May 12;18(1):373

Authors: van Hagen M, Piebes DGE, de Leeuw WC, Vuist IM, van Roon-Mom WMC, Moerland PD, Verschure PJ

Abstract
BACKGROUND: Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG expansion in the Huntingtin (HTT) gene. Proteolytic cleavage of mutant huntingtin (Htt) protein with an expanded polyglutamine (polyQ) stretch results in production of Htt fragments that aggregate and induce impaired ubiquitin proteasome, mitochondrial functioning and transcriptional dysregulation. To understand the time-resolved relationship between aggregate formation and transcriptional changes at early disease stages, we performed temporal transcriptome profiling and quantification of aggregate formation in living cells in an inducible HD cell model.
RESULTS: Rat pheochromocytoma (PC12) cells containing a stably integrated, doxycycline-inducible, eGFP-tagged N-terminal human Htt fragment with an expanded polyQ domain were used to analyse gene expression changes at different stages of mutant Htt aggregation. At earliest time points after doxycycline induction no detectable aggregates and few changes in gene expression were observed. Aggregates started to appear at intermediate time points. Aggregate formation and subsequent enlargement of aggregates coincided with a rapid increase in the number of differentially expressed (DE) genes. The increase in number of large aggregates coincided with a decrease in the number of smaller aggregates whereas the transcription profile reverted towards the profile observed before mutant Htt induction. Cluster-based analysis of the 2,176 differentially expressed genes revealed fourteen distinct clusters responding differently over time. Functional enrichment analysis of the two major gene clusters revealed that genes in the up-regulated cluster were mainly involved in metabolic (antioxidant activity and cellular ketone metabolic processes) and genes in the down-regulated cluster in developmental processes, respectively. Promoter-based analysis of the identified gene clusters resulted in identification of a transcription factor network of which several previously have been linked to HD.
CONCLUSIONS: We demonstrate a time-resolved relationship between Htt aggregation and changes in the transcriptional profile. We identified two major gene clusters showing involvement of (i) mitochondrial dysfunction and (ii) developmental processes implying cellular homeostasis defects. We identified novel and known HD-linked transcription factors and show their interaction with known and predicted regulatory proteins. Our data provide a novel resource for hypothesis building on the role of transcriptional key regulators in early stages of HD and possibly other polyQ-dependent diseases.

PMID: 28499347 [PubMed - in process]

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"systems biology"

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"systems biology"

These pubmed results were generated on 2017/05/02

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Single Cell Restriction Enzyme-Based Analysis of Methylation at Genomic Imprinted Regions in Preimplantation Mouse Embryos.

Methods Mol Biol. 2017;1605:171-189

Authors: Ling KY, Cheow LF, Quake SR, Burkholder WF, Messerschmidt DM

Abstract
The methylation of cytosines in DNA is a fundamental epigenetic regulatory mechanism. During preimplantation development, mammalian embryos undergo extensive epigenetic reprogramming, including the global erasure of germ cell-specific DNA methylation marks, to allow for the establishment of the pluripotent state of the epiblast. However, DNA methylation marks at specific regions, such as imprinted gene regions, escape this reprogramming process, as their inheritance from germline to soma is paramount for proper development. To study the dynamics of DNA methylation marks in single blastomeres of mouse preimplantation embryos, we devised a new approach-single cell restriction enzyme analysis of methylation (SCRAM). SCRAM allows for reliable, fast, and high-throughput analysis of DNA methylation states of multiple regions of interest from single cells. In the method described below, SCRAM is specifically used to address loss of DNA methylation at genomic imprints or other highly methylated regions of interest.

PMID: 28456965 [PubMed - in process]

Structural pharmacological studies on EGFR T790M/C797S.

Biochem Biophys Res Commun. 2017 Apr 26;:

Authors: Kong LL, Ma R, Yao MY, Yan XE, Zhu SJ, Zhao P, Yun CH

Abstract
Drug-resistance is a major challenge in targeted therapy of EGFR mutated non-small cell lung cancers (NSCLCs). The third-generation irreversible inhibitors such as AZD9291, CO-1686 and WZ4002 can overcome EGFR T790M drug-resistance mutant through covalent binding through Cys 797, but ultimately lose their efficacy upon emergence of the new mutation C797S. To develop new reversible inhibitors not relying on covalent binding through Cys 797 is therefore urgently demanded. Gö6976 is a staurosporine-like reversible inhibitor targeting T790M while sparing the wild-type EGFR. In the present work, we reported the complex crystal structures of EGFR T790M/C797S + Gö6976 and T790M + Gö6976, along with enzyme kinetic data of EGFR wild-type, T790M and T790M/C797S. These data showed that the C797S mutation does not significantly alter the structure and function of the EGFR kinase, but increases the local hydrophilicity around residue 797. The complex crystal structures also elucidated the detailed binding mode of Gö6976 to EGFR and explained why this compound prefers binding to T790M mutant. These structural pharmacological data would facilitate future drug development studies.

PMID: 28456628 [PubMed - as supplied by publisher]

Inference in the age of big data: Future perspectives on neuroscience.

Neuroimage. 2017 Apr 26;:

Authors: Bzdok D, Yeo BTT

Abstract
Neuroscience is undergoing faster changes than ever before. Over 100 years our field qualitatively described and invasively manipulated single or few organisms to gain anatomical, physiological, and pharmacological insights. In the last 10 years neuroscience spawned quantitative datasets of unprecedented breadth (e.g., microanatomy, synaptic connections, and optogenetic brain-behavior assays) and size (e.g., cognition, brain imaging, and genetics). While growing data availability and information granularity have been amply discussed, we direct attention to a less explored question: How will the unprecedented data richness shape data analysis practices? Statistical reasoning is becoming more important to distill neurobiological knowledge from healthy and pathological brain measurements. We argue that large-scale data analysis will use more statistical models that are non-parametric, generative, and mixing frequentist and Bayesian aspects, while supplementing classical hypothesis testing with out-of-sample predictions.

PMID: 28456584 [PubMed - as supplied by publisher]

Degradation of indoor limonene by outdoor ozone: A cascade of secondary organic aerosols.

Environ Pollut. 2017 Apr 26;:

Authors: Rösch C, Wissenbach DK, Franck U, Wendisch M, Schlink U

Abstract
In indoor air, terpene-ozone reactions can form secondary organic aerosols (SOA) in a transient process. 'Real world' measurements conducted in a furnished room without air conditioning were modelled involving the indoor background of airborne particulate matter, outdoor ozone infiltrated by natural ventilation, repeated transient limonene evaporations, and different subsequent ventilation regimes. For the given setup, we disentangled the development of nucleated, coagulated, and condensed SOA fractions in the indoor air and calculated the time dependence of the aerosol mass fraction (AMF) by means of a process model. The AMF varied significantly between 0.3 and 5.0 and was influenced by the ozone limonene ratio and the background particles which existed prior to SOA formation. Both influencing factors determine whether nucleation or adsorption processes are preferred; condensation is strongly intensified by particulate background. The results provide evidence that SOA levels in natural indoor environments can surpass those known from chamber measurements. An indicator for the SOA forming potential of limonene was found to be limona ketone. Multiplying its concentration (in μg/m(3)) by 450(±100) provides an estimate of the concentration of the reacted limonene. This can be used to detect a high particle formation potential due to limonene pollution, e.g. in epidemiological studies considering adverse health effects of indoor air pollutants.

PMID: 28456415 [PubMed - as supplied by publisher]

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