UFGI publication round-up week 01/23/2017
Luminescent iridium(iii) complexes as COX-2-specific imaging agents in cancer cells.
Author information: Liu C1, Yang C2, Lu L3, Wang W1, Tan W4, Leung CH2, Ma DL1.
Date of e-pub: January 2017
Abstract: Two luminescent iridium(iii) complexes, 1 and 2, were synthesized and evaluated for their ability to probe COX-2 in human cancer cells. This is the first application of iridium(iii) complexes as imaging agents for COX-2. We demonstrate that complex 1 differentiates cancer cells from normal cells with high stability and low cytotoxicity.
Integrative FourD omics approach profiles the target network of the carbon storage regulatory system.
Author information: Sowa SW1, Gelderman G2, Leistra AN2, Buvanendiran A3, Lipp S2, Pitaktong A2, Vakulskas CA4, Romeo T4, Baldea M2, Contreras LM5.
Date of e-pub: January 2017
Abstract: Multi-target regulators represent a largely untapped area for metabolic engineering and anti-bacterial development. These regulators are complex to characterize because they often act at multiple levels, affecting proteins, transcripts and metabolites. Therefore, single omics experiments cannot profile their underlying targets and mechanisms. In this work, we used an Integrative FourD omics approach (INFO) that consists of collecting and analyzing systems data throughout multiple time points, using multiple genetic backgrounds, and multiple omics approaches (transcriptomics, proteomics and high throughput sequencing crosslinking immunoprecipitation) to evaluate simultaneous changes in gene expression after imposing an environmental stress that accentuates the regulatory features of a network. Using this approach, we profiled the targets and potential regulatory mechanisms of a global regulatory system, the well-studied carbon storage regulatory (Csr) system of Escherichia coli, which is widespread among bacteria. Using 126 sets of proteomics and transcriptomics data, we identified 136 potential direct CsrA targets, including 50 novel ones, categorized their behaviors into distinct regulatory patterns, and performed in vivo fluorescence-based follow up experiments. The results of this work validate 17 novel mRNAs as authentic direct CsrA targets and demonstrate a generalizable strategy to integrate multiple lines of omics data to identify a core pool of regulator targets.
A chemical genetic roadmap to improved tomato flavor.
Author information: Tieman D1,2, Zhu G1,3, Resende MF Jr4, Lin T1,3, Nguyen C2, Bies D2, Rambla JL5, Beltran KS5, Taylor M2, Zhang B2, Ikeda H2, Liu Z2, Fisher J6, Zemach I6, Monforte A5, Zamir D6, Granell A5, Kirst M7, Huang S8,3, Klee H8,2.
Date of e-pub: January 2017
Abstract: Modern commercial tomato varieties are substantially less flavorful than heirloom varieties. To understand and ultimately correct this deficiency, we quantified flavor-associated chemicals in 398 modern, heirloom, and wild accessions. A subset of these accessions was evaluated in consumer panels, identifying the chemicals that made the most important contributions to flavor and consumer liking. We found that modern commercial varieties contain significantly lower amounts of many of these important flavor chemicals than older varieties. Whole-genome sequencing and a genome-wide association study permitted identification of genetic loci that affect most of the target flavor chemicals, including sugars, acids, and volatiles. Together, these results provide an understanding of the flavor deficiencies in modern commercial varieties and the information necessary for the recovery of good flavor through molecular breeding.
Efficient Gene Delivery and Expression in Pancreas and Pancreatic Tumors by Capsid-optimized AAV8 Vectors.
Author information: Chen M1,2, Maeng K3, Nawab A4, Francois RA5, Bray JK6, Reinhard MK7, Boye SL8, Hauswirth WW9, Kaye FJ10, Aslanidi GV11, Srivastava A12, Zajac-Kaye M13.
Date of e-pub: January 2017
Abstract: Despite efforts to use adeno-associated viral (AAV) vector-mediated gene therapy for treatment of pancreatic ductal adenocarcinoma (PDAC), transduction efficiency remains a limiting factor and thus improvement of AAV delivery would significantly facilitate the treatment of this malignancy. Site-directed mutagenesis of specific tyrosine (Y) residues to phenylalanine (F) on the surface of various AAV serotype capsids has been reported as a method for enhancing gene transfer efficiencies. In the present studies, we determine whether Y-to-F mutations could also enhance AAV8 gene transfer in the pancreas to facilitate gene therapy for PDAC. Three different Y-to-F mutant vectors (a single-mutant, Y733F; a double-mutant, Y447F+Y733F, and a triple-mutant, Y275F+Y447F+Y733F) and wild-type AAV8 (WT-AAV8) were administered by intraperitoneal or tail-vein routes to Kras<sup>G12D+/-</sup>, Kras<sup>G12D+/-</sup>/Pten and wild-type mice. The transduction efficiency of these vectors expressing the mCherry reporter gene was evaluated two weeks post administration in pancreas or PDAC, and correlated with viral genome copy numbers. Our comparative and quantitative analyses of the transduction profiles demonstrated that the Y to F double-mutant exhibited the highest mCherry expression in pancreatic tissues (range 45-70%) compared with WT-AAV8 (7%; p<0.01). We also detected a 7-fold higher level of vector genome copy numbers in normal pancreas following transduction with the double-mutant AAV8 compared to WT-AAV8 (10,285 vs. 1,500 vector copies/μg DNA respectively, p<0.05). In addition, we observed that intraperitoneal injection of the double-mutant AAV8 led to a 15-fold enhanced transduction efficiency as compared to WT-AAV8 in mouse PDAC, with a corresponding ~14-fold increase in vector genome copy numbers (26,575 vs. 2,165 copies/μg DNA respectively, p<0.05). These findings indicate that the Y447+Y733F-AAV8 leads to a significant enhancement of transduction efficiency in both normal and malignant pancreatic tissues, suggesting the potential use of this vector in targeting pancreatic diseases in general, and PDAC in particular.
OneBac 2.0: Sf9 Cell Lines for Production of AAV1, AAV2 and AAV8 Vectors with Minimal Encapsidation of Foreign DNA.
Author information: Mietzsch M1, Hering H2, Hammer EM3, Agbandje-McKenna M4, Zolotukhin S5, Heilbronn R6.
Date of e-pub: January 2017
Abstract: Recombinant adeno-associated virus vectors (rAAV) for human gene therapy require efficient and economical production methods to keep pace with the rapidly increasing clinical demand. In addition, the manufacturing process must ensure high vector quality and biological safety. The OneBac system offers easily scalable rAAV vector production in insect Sf9-derived AAV rep/cap expressing producer cell lines infected with a single baculovirus that carries the rAAV backbone. For most AAV serotypes high burst sizes per cell were achieved, combined with high infectivity rates. OneBac2.0 represents a two-fold advancement: First, enhanced VP1 proportions in AAV5 capsids lead to vastly increased per particle infectivity rates. Secondly, collateral packaging of foreign DNA is suppressed by removal of the Rep-binding element (RBE). In this study we show that this advancement of AAV5 packaging can be translated to OneBac2.0-derived packaging systems for alternative AAV serotypes. By removal of the RBE collateral packaging of non-vector DNA was drastically reduced in all newly tested serotypes, AAV1, AAV2 and AAV8. However the splicing-based strategy to enhance VP1 expression in order to increase AAV5 infectivity hardly improved infectivity rates of AAV-1, -2, or -8 compared to the original OneBac cell lines. Our results underline that OneBac2.0 represents an advancement for scalable, high-titer production of various AAV serotypes, leading to AAV particles with minimal packaging of foreign DNA.
Phylogenomics from Whole Genome Sequences Using aTRAM.
Author information: Allen JM1, Boyd B2,3, Nguyen NP4, Vachaspati P5, Warnow T4,5,2, Huang DI6, Grady PG2, Bell KC7, Cronk QC6, Mugisha L8,9, Pittendrigh BR10, Soledad Leonardi M2, Reed DL3, Johnson KP2.
Date of e-pub: January 2017
Abstract: Novel sequencing technologies are rapidly expanding the size of datasets that can be applied to phylogenetic studies. Currently the most commonly used phylogenomic approaches involve some form of genome reduction. While these approaches make assembling phylogenomic datasets more economical for organisms with large genomes, they reduce the genomic coverage and thereby the long-term utility of the data. Currently, for organisms with moderate to small genomes (<1000 Mbp) it is feasible to sequence the entire genome at modest coverage (10-30X). Computational challenges for handling these large datasets can be alleviated by assembling targeted reads, rather than assembling the entire genome, to produce a phylogenomic data matrix.Here we demonstrate the use of automated Target Restricted Assembly Method (aTRAM) to assemble 1,107 single copy ortholog genes from whole genome sequencing of sucking lice (Anoplura) and outgroups. We developed a pipeline to extract exon sequences from the aTRAM assemblies by annotating them with respect to the original target protein. We aligned these protein sequences with the inferred amino acids and then performed phylogenetic analyses on both the concatenated matrix of genes and on each gene separately in a coalescent analysis. Finally, we tested the limits of successful assembly in aTRAM by assembling 100 genes from close to distantly related taxa at high to low levels of coverage.Both the concatenated analysis and the coalescent-based analysis produced the same tree topology, which was consistent with previously published results and resolved weakly supported nodes. These results demonstrate that this approach is successful at developing phylogenomic datasets from raw genome sequencing reads. Further, we found that with coverages above 5 – 10X, aTRAM was successful at assembling 80 – 90% of the contigs for both close and distantly related taxa. As sequencing costs continue to decline, we expect full genome sequencing will become more feasible for a wider array of organisms, and aTRAM will enable mining of these genomic datasets for an extensive variety of applications, including phylogenomics.
Membrane Ion Channels and Receptors in Animal Lifespan Modulation.
Author information: Sheng Y1, Tang L1, Kang L2, Xiao R1,3,4,5.
Date of e-pub: January 2017
Abstract: Acting in the interfaces between environment and membrane compartments, membrane ion channels and receptors transduce various physical and chemical cues into downstream signaling events. Not surprisingly, these membrane proteins play essential roles in a wide range of cellular processes such as sensory perception, synaptic transmission, cellular growth and development, fate determination, and apoptosis. However, except insulin and insulin-like growth factor receptors, the functions of membrane receptors in animal lifespan modulation have not been well appreciated. On the other hand, although ion channels are popular therapeutic targets for many age-related diseases, their potential roles in aging itself are largely neglected. In this review, we will discuss our current understanding of the conserved functions and mechanisms of membrane ion channels and receptors in the modulation of lifespan across multiple species including C. elegans, Drosophila, mouse, and human. This article is protected by copyright. All rights reserved.
Sodium chloride enhances rAAV production in a serum-free suspension manufacturing platform using the Herpes Simplex Virus System.
Author information: Adamson-Small L1, Potter M2, Byrne BJ3,4, Clement N5.
Date of e-pub: January 2017
Abstract: The increase in effective treatments utilizing recombinant adeno-associated viral (rAAV) vectors has underscored the importance of scalable, high yield manufacturing methods. Previous work from this group reported the use of recombinant herpes simplex virus type 1 (rHSV) vectors to produce rAAV in adherent HEK293 cells, demonstrating the capacity of this system and quality of the product generated. Here we report production and optimization of rAAV utilizing the rHSV system in suspension HEK293 cells (EXPI293F™) grown in serum and animal component-free media. Through adjustment of salt concentration in the media and optimization of infection conditions, titers greater than 1 x 1014 vector genomes per liter (vg/L) were observed in purified rAAV stocks produced in EXPI293F™ cells. Furthermore, this system allowed for high titer production of multiple rAAV serotypes (2, 5, and 9) as well as multiple transgenes (GFP and acid alpha-glucosidase (GAA)). A proportional increase in vector production was observed as this method was scaled, with a final 3L shaker flask production yielding an excess of 1 x 1015 vg in crude cell harvests and an average of 3.5 x 1014 total vg of purified rAAV9 material, resulting in greater than 1 x 105 vg/cell. These results support the use of this rHSV-based rAAV production method for large scale pre-clinical and clinical vector production.
ThiN as a versatile domain of transcriptional repressors and catalytic enzymes of thiamine biosynthesis.
Author information: Hwang S1, Cordova B1, Abdo M1, Pfeiffer F2, Maupin-Furlow JA3,4.
Date of e-pub: January 2017
Abstract: Thiamine biosynthesis is commonly regulated by a riboswitch mechanism; however, the enzymatic steps and regulation of this pathway in archaea are poorly understood. Haloferax volcanii, one of the representative archaea, uses a yeast-like Thi4 (thiamine thiazole synthase) for production of the thiazole ring and condenses this ring with a pyrimidine moiety synthesized by an apparent bacterial-like ThiC (HMP-phosphate synthase) branch. Here we found the archaeal Thi4 and ThiC were encoded by leaderless transcripts, ruling out a riboswitch mechanism. Instead, a novel ThiR transcription factor was identified that harbored an N-terminal HTH-DNA binding domain and C-terminal ThiN (TMP synthase) domain. In the presence of thiamine, ThiR was found to repress the expression of thi4 and thiC by a DNA operator sequence that was conserved across archaeal phyla. Despite having a ThiN domain, ThiR was found catalytically inactive in compensating for loss of ThiE (TMP synthase) function. By contrast, a bifunctional ThiDN, in which the ThiN domain is fused to an N-terminal ThiD (HMP/HMP∼P kinase) domain, was found interchangeable for ThiE function and, thus, active in thiamine biosynthesis. A conserved Met residue of an extended α-helix near the active site His of the ThiN domain was found important for ThiDN catalytic activity; whereas, the corresponding Met residue was absent and the α-helix was shorter in ThiR homologs. Thus, we provide a new insight into residues that distinguish catalytic from non-catalytic ThiN domains and reveal that thiamine biosynthesis in archaea is regulated by a transcriptional repressor ThiR, not by a riboswitch.
Thiamine pyrophosphate (TPP) is a cofactor needed for the enzymatic activity of many cellular processes including central metabolism. In archaea, thiamine biosynthesis is an apparent chimera of eukaryotic- and bacterial-type pathways that is not well defined at the level of enzymatic steps or regulatory mechanisms. Here we find ThiN to be a versatile domain of transcriptional repressors and catalytic enzymes of thiamine biosynthesis in archaea. Our study provides a new insight into residues that distinguish catalytic from non-catalytic ThiN domains and reveals archaeal thiamine biosynthesis to be regulated by a ThiN domain transcriptional repressor ThiR, not by a riboswitch.
Serum Trypsinogen Levels in Type 1 Diabetes.
Author information: Li X1,2, Campbell-Thompson M2, Wasserfall CH2, McGrail K2, Posgai A2, Schultz AR2, Brusko TM2, Shuster J3, Liang F4, Muir A5, Schatz D6, Haller MJ6, Atkinson MA7,6.
Date of e-pub: January 2017
Abstract: The pancreas in type 1 diabetes exhibits decreased size (weight/volume) and abnormal exocrine morphology. Serum trypsinogen levels are an established marker of pancreatic exocrine function. As such, we hypothesized that trypsinogen levels may be reduced in patients with pre-type 1 diabetes and type 1 diabetes compared with healthy control subjects.
Serum trypsinogen levels were determined in 100 persons with type 1 diabetes (72 new-onset, 28 established), 99 autoantibody-positive (AAb+) subjects at varying levels of risk for developing this disease, 87 AAb-negative (AAb-) control subjects, 91 AAb- relatives with type 1 diabetes, and 18 patients with type 2 diabetes.
Trypsinogen levels increased significantly with age in control subjects (r = 0.71; P < 0.0001) and were significantly lower in patients with new-onset (mean ± SD 14.5 ± 6.1 ng/mL; P < 0.0001) and established type 1 diabetes (16.7 ± 6.9 ng/mL; P < 0.05) versus AAb- control subjects (25.3 ± 11.2 ng/mL), AAb- relatives (29.3 ± 15.0 ng/mL), AAb+ subjects (26.5 ± 12.1 ng/mL), and patients with type 2 diabetes (31.5 ± 17.3 ng/mL). Multivariate analysis revealed reduced trypsinogen in multiple-AAb+ subjects (P < 0.05) and patients with type 1 diabetes (P < 0.0001) compared with AAb- subjects (control subjects and relatives combined) and single-AAb+ (P < 0.01) subjects when considering age and BMI.
These findings further support the interplay between pancreatic endocrine and exocrine dysfunction. Longitudinal studies are warranted to validate trypsinogen as a predictive biomarker of type 1 diabetes progression.
Metnase Mediates Loading of Exonuclease 1 onto Single Strand Overhang DNA for End Resection at Stalled Replication Forks.
Author information: Kim HS1, Williamson EA2, Nickoloff JA3, Hromas RA2, Lee SH4.
Date of e-pub: January 2017
Abstract: Stalling at DNA replication forks generates stretches of single-stranded (ss) DNA on both strands that are exposed to nucleolytic degradation, potentially compromising genome stability. One enzyme crucial for DNA replication fork repair and restart of stalled forks in human is Metnase (also known as SETMAR), a chimeric fusion protein consisting of a su(var)3-9, enhancer-of-zeste and trithorax (SET) histone methylase and transposase nuclease domain. We previously showed that Metnase possesses a unique fork cleavage activity necessary for its function in replication restart and that its SET domain is essential for recovery from hydroxyurea-induced DNA damage. However, its exact role in replication restart is unclear. In this study, we show that Metnase associates with exonuclease 1 (Exo1), a 5′-exonuclease crucial for 5′-end resection to mediate DNA processing at stalled forks. Metnase DNA cleavage activity was not required for Exo1 5′-exonuclease activity on the lagging strand daughter DNA, but its DNA binding activity mediated loading of Exo1 onto ssDNA overhangs. Metnase-induced enhancement of Exo1-mediated DNA strand resection required the presence of these overhangs but did not require Metnase’s DNA cleavage activity. These results suggest that Metnase enhances Exo1-mediated exonuclease activity on the lagging strand DNA by facilitating Exo1 loading onto a single strand gap at the stalled replication fork.
NOTE: These abstracts were retrieved from the U.S. National Library of Medicine website managed in collaboration with the U.S. National Library of Medicine