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Articles 1 - 30 of 455

Full-Text Articles in Molecular Biology

Promoter Library Designed For Fine-Tuned Gene Expression In Pichia Pastoris, Franz S. Hartner, Claudia Ruth, David Langenegger, Sabrina N. Johnson, Petr Hyka, Geoff P. Lin-Cereghino, Joan Lin-Cereghino, Karin Kovar, James Cregg, Anton Glieder Mar 2019

Promoter Library Designed For Fine-Tuned Gene Expression In Pichia Pastoris, Franz S. Hartner, Claudia Ruth, David Langenegger, Sabrina N. Johnson, Petr Hyka, Geoff P. Lin-Cereghino, Joan Lin-Cereghino, Karin Kovar, James Cregg, Anton Glieder

Joan Lin-Cereghino

Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (PAOX1) sequence. This first library initially spanned an activity range between ∼6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing a green ...


Condensed Protocol For Competent Cell Preparation And Transformation Of The Methylotrophic Yeast Pichia Pastoris, Joan Lin-Cereghino, William Wong, See Xiong, William Giang, Linda Luong, Jane Vu, Sabrina Johnson, Geoff P. Lin-Cereghino Mar 2019

Condensed Protocol For Competent Cell Preparation And Transformation Of The Methylotrophic Yeast Pichia Pastoris, Joan Lin-Cereghino, William Wong, See Xiong, William Giang, Linda Luong, Jane Vu, Sabrina Johnson, Geoff P. Lin-Cereghino

Joan Lin-Cereghino

No abstract provided.


Endosome To Golgi Retrieval Of The Vacuolar Protein Sorting Receptor, Vps10p, Requires The Function Of The Vps29, Vps30, And Vps35 Gene Products, Matthew N. J. Seaman, Eric G. Marcusson, Joan Lin-Cereghino, Scott D. Emr Mar 2019

Endosome To Golgi Retrieval Of The Vacuolar Protein Sorting Receptor, Vps10p, Requires The Function Of The Vps29, Vps30, And Vps35 Gene Products, Matthew N. J. Seaman, Eric G. Marcusson, Joan Lin-Cereghino, Scott D. Emr

Joan Lin-Cereghino

Mutations in the S. cerevisiae VPS29 and VPS30 genes lead to a selective protein sorting defect in which the vacuolar protein carboxypeptidase Y (CPY) is missorted and secreted from the cell, while other soluble vacuolar hydrolases like proteinase A (PrA) are delivered to the vacuole. This phenotype is similar to that seen in cells with mutations in the previously characterized VPS10 and VPS35 genes. Vps10p is a late Golgi transmembrane protein that acts as the sorting receptor for soluble vacuolar hydrolases like CPY and PrA, while Vps35p is a peripheral membrane protein which cofractionates with membranes enriched in Vps10p. The ...


An Automated Bayesian Pipeline For Rapid Analysis Of Single-Molecule Binding Data, Carlas Smith, Karina Jouravleva, Maximiliaan Huisman, Samson M. Jolly, Phillip D. Zamore, David Grünwald Mar 2019

An Automated Bayesian Pipeline For Rapid Analysis Of Single-Molecule Binding Data, Carlas Smith, Karina Jouravleva, Maximiliaan Huisman, Samson M. Jolly, Phillip D. Zamore, David Grünwald

David Grünwald

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. In addition, our method enables state estimation on multivariate Gaussian signals. We validate our approach using simulated data, and benchmark the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We ...


The Cytoplasmic Tail Domain Of The Vacuolar Protein Sorting Receptor Vps10p And A Subset Of Vps Gene Products Regulate Receptor Stability, Function, And Localization, Joan Lin-Cereghino, Eric G. Marcusson, Scott D. Emr Mar 2019

The Cytoplasmic Tail Domain Of The Vacuolar Protein Sorting Receptor Vps10p And A Subset Of Vps Gene Products Regulate Receptor Stability, Function, And Localization, Joan Lin-Cereghino, Eric G. Marcusson, Scott D. Emr

Joan Lin-Cereghino

VPS10 of Saccharomyces cerevisiae encodes a type I transmembrane receptor protein required for the sorting of the soluble vacuolar hydrolase carboxypeptidase Y (CPY). To characterize the essential structural features and intercompartmental transport itinerary of the CPY receptor, we have constructed mutant forms of Vps10p that alter the carboxyterminal cytoplasmic tail of the protein. In addition, we have analyzed the effect these mutations as well as mutations in several VPS genes have on the function, stability, and localization of Vps10p. Although wild-type Vps10p is very stable over a 3-h chase period, overproduction of Vps10p results in PEP4-dependent degradation of the receptor ...


Identification Of Novel Δnp63Α-Regulated Mirnas Using An Optimized Small Rna-Seq Analysis Pipeline, Suraj Sakaram, Michael P. Craig, Natasha T. Hill, Amjad Aljiagthmi, Christian Garrido, Oleg Paliy, Michael Bottomley, Michael L. Raymer, Madhavi Kadakia Feb 2019

Identification Of Novel Δnp63Α-Regulated Mirnas Using An Optimized Small Rna-Seq Analysis Pipeline, Suraj Sakaram, Michael P. Craig, Natasha T. Hill, Amjad Aljiagthmi, Christian Garrido, Oleg Paliy, Michael Bottomley, Michael L. Raymer, Madhavi Kadakia

Michael L. Raymer

Advances in high-throughput sequencing have enabled profiling of microRNAs (miRNAs), however, a consensus pipeline for sequencing of small RNAs has not been established. We built and optimized an analysis pipeline using Partek Flow, circumventing the need for analyzing data via scripting languages. Our analysis assessed the effect of alignment reference, normalization method, and statistical model choice on biological data. The pipeline was evaluated using sequencing data from HaCaT cells transfected with either a non-silencing control or siRNA against ΔNp63α, a p53 family member protein which is highly expressed in non-melanoma skin cancer and shown to regulate a number of miRNAs ...


Generating High-Order Optical And Spin Harmonics From Ferromagnetic Monolayers, G.P. Zhang, M.S. Si, M. Murakami, Y.H. Bai, Thomas F. George Nov 2018

Generating High-Order Optical And Spin Harmonics From Ferromagnetic Monolayers, G.P. Zhang, M.S. Si, M. Murakami, Y.H. Bai, Thomas F. George

Thomas George

High-order harmonic generation (HHG) in solids has entered a new phase of intensive research, with envisioned band-structure mapping on an ultrashort time scale. This partly benefits from a flurry of new HHG materials discovered, but so far has missed an important group. HHG in magnetic materials should have profound impact on future magnetic storage technology advances. Here we introduce and demonstrate HHG in ferromagnetic monolayers. We find that HHG carries spin information and sensitively depends on the relativistic spin–orbit coupling; and if they are dispersed into the crystal momentum k space, harmonics originating from real transitions can be k-resolved ...


Spatial Mapping And Profiling Of Metabolite Distributions During Germination, Adam D. Feenstra, Liza E. Alexander, Zhihong Song, Andrew R. Korte, Marna D. Yandeau-Nelson, Basil J. Nikolau, Young-Jin Lee Nov 2018

Spatial Mapping And Profiling Of Metabolite Distributions During Germination, Adam D. Feenstra, Liza E. Alexander, Zhihong Song, Andrew R. Korte, Marna D. Yandeau-Nelson, Basil J. Nikolau, Young-Jin Lee

Robbyn Anand

Germination is a highly complex process by which seeds begin to develop and establish themselves as viable organisms. In this paper, we utilize a combination of GC-MS, LC-fluorescence, and mass spectrometry imaging (MSI) approaches to profile and visualize the metabolic distributions of germinating seeds from two different inbreds of maize seeds, B73 and Mo17. GC and LC analyses demonstrate that the two inbreds are highly differentiated in their metabolite profiles throughout the course of germination, especially with regard to amino acids, sugar alcohols, and small organic acids. Crude dissection of the seed followed by GC-MS analysis of polar metabolites also ...


Alignment Of Mitotic Chromosomes In Human Cells Involves Sr-Like Splicing Factors Btf And Trap150, Sapna Varia, Divya Cheedu, Michael P. Markey, Keshia Torres-Shafer, Vishnu P. Battini, Athanasios Bubulya, Paula A. Bubulya Aug 2018

Alignment Of Mitotic Chromosomes In Human Cells Involves Sr-Like Splicing Factors Btf And Trap150, Sapna Varia, Divya Cheedu, Michael P. Markey, Keshia Torres-Shafer, Vishnu P. Battini, Athanasios Bubulya, Paula A. Bubulya

Michael P. Markey

Serine-arginine-rich (SR) or SR-like splicing factors interact with exon junction complex proteins during pre-mRNA processing to promote mRNA packaging into mature messenger ribonucleoproteins (mRNPs) and to dictate mRNA stability, nuclear export, and translation. The SR protein family is complex, and while many classical SR proteins have well-defined mRNA processing functions, those of other SR-like proteins is unclear. Here, we show that depletion of the homologous non-classical serine-arginine-rich (SR) splicing factors Bcl2-associated transcription factor (Btf or BCLAF) and thyroid hormone receptor-associated protein of 150 kDa (TRAP150) causes mitotic defects. We hypothesized that the depletion of these SR-like factors affects mitosis indirectly ...


Copy Number Variation In Archival Melanoma Biopsies Versus Benign Melanocytic Lesions, Ahmed Mahas, Keerti Potluri, Michael N. Kent, Sameep Naik, Michael P. Markey Aug 2018

Copy Number Variation In Archival Melanoma Biopsies Versus Benign Melanocytic Lesions, Ahmed Mahas, Keerti Potluri, Michael N. Kent, Sameep Naik, Michael P. Markey

Michael P. Markey

BACKGROUND: Skin melanocytes can give rise to benign and malignant neoplasms. Discrimination of an early melanoma from an unusual/atypical benign nevus can represent a significant challenge. However, previous studies have shown that in contrast to benign nevi, melanoma demonstrates pervasive chromosomal aberrations. OBJECTIVE: This substantial difference between melanoma and benign nevi can be exploited to discriminate between melanoma and benign nevi. METHODS: Array-comparative genomic hybridization (aCGH) is an approach that can be used on DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues to assess the entire genome for the presence of changes in DNA copy number. In this study, high ...


Characterization Of Receptor Expression And Aberrant Gli2 Signaling In Osteosarcoma Cells, Chanelle Hunter, Erik Beadle M.S., Julie Sterling Aug 2018

Characterization Of Receptor Expression And Aberrant Gli2 Signaling In Osteosarcoma Cells, Chanelle Hunter, Erik Beadle M.S., Julie Sterling

Chanelle Hunter

Osteosarcoma is the most prevalent primary bone tumor diagnosed in adolescents and young adults and is known for poor prognosis in late stages of detection. Although, there have been many advances in the development of chemotherapy, inhibitors, and combination therapy, there are few existing targeted therapies against the onset of osteosarcoma. Given the complex nature of the bone microenvironment in relation to tumor formation, it is necessary to gain insight into the molecular variations in activation, function, and regulation in order to identify and develop new methods of targeting and drug design. Our objective is to characterize the oncogenic pathway ...


Mapping Molecular Datasets Back To The Brain Regions They Are Extracted From: Remembering The Native Countries Of Hypothalamic Expatriates And Refugees, Arshad M. Khan, Alice H. Grant, Anais Martinez, Gully Apc Burns, Brendan S. Thatcher, Vishwanath T. Anekonda, Benjamin W. Thompson, Zachary S. Roberts, Daniel H. Moralejo, James E. Blevins Jun 2018

Mapping Molecular Datasets Back To The Brain Regions They Are Extracted From: Remembering The Native Countries Of Hypothalamic Expatriates And Refugees, Arshad M. Khan, Alice H. Grant, Anais Martinez, Gully Apc Burns, Brendan S. Thatcher, Vishwanath T. Anekonda, Benjamin W. Thompson, Zachary S. Roberts, Daniel H. Moralejo, James E. Blevins

Arshad M. Khan, Ph.D.

This article, which includes novel unpublished data along with commentary and analysis,
focuses on approaches to link transcriptomic, proteomic, and peptidomic datasets mined from
brain tissue to the original locations within the brain that they are derived from using digital atlas
mapping techniques. We use, as an example, the transcriptomic, proteomic and peptidomic
analyses conducted in the mammalian hypothalamus. Following a brief historical overview, we
highlight studies that have mined biochemical and molecular information from the hypothalamus
and then lay out a strategy for how these data can be linked spatially to the mapped locations in a
canonical brain atlas ...


Mutagenic Potential Of Phthalates In Mammalian Cells, Chanelle Hunter, Virginia Lane, Adrian Torres, Shahd Mohamed, Alicia Hawthorne, Emily Bradshaw Apr 2018

Mutagenic Potential Of Phthalates In Mammalian Cells, Chanelle Hunter, Virginia Lane, Adrian Torres, Shahd Mohamed, Alicia Hawthorne, Emily Bradshaw

Chanelle Hunter

Several popular industrial chemicals, such as Phthalates and Bisphenol A (BPA), are commonly found in many of the personal products used in daily life. This includes plastics present in water bottles, medical equipment such as IV fluid transporters, storage containers of food, PVC piping, children’s toys, cooking utensils, printing inks, and even baby pacifiers. Although plastic enhances the quality of life, phthalates and BPA have both been linked to genetic mutations. BPA can cause increased chromosomal mutations, and phthalates can cause germline mutations in sperm. These genetic changes can result in adverse effects on human health in areas including ...


A Genome Scan To Detect Qtl Affecting Growth, Composition, And Meat Quality Traits In A Berkshire X Yorkshire Cross, Jack Dekkers, Jong-Joo Kim, Massoud Malek, Hauke Thomsen, Hakkyo Lee, Hong-Hua Zhao, Max Rothschild Mar 2018

A Genome Scan To Detect Qtl Affecting Growth, Composition, And Meat Quality Traits In A Berkshire X Yorkshire Cross, Jack Dekkers, Jong-Joo Kim, Massoud Malek, Hauke Thomsen, Hakkyo Lee, Hong-Hua Zhao, Max Rothschild

Max Rothschild

The techniques of molecular biology and molecular genetics have rapidly progressed. These methods, coupled with advances in human genetics, have opened new vistas for investigators wishing to identify genes that control quantitative traits (quantitative trait loci or QTL). Also, over the past years, a great deal of progress has been made in development of genetic maps in the pig. For example, a large international mapping effort (Archibald et al., 1994), a USDA/ARS effort (Rohrer et al., 1996) and a U.S. coordinated effort (Rothschild, 1994) have produced several genetic linkage maps for the pig. Based on these linkage maps ...


Identification Of Genes For Carcass Merit And Meat Quality In The Pig, Max Rothschild, Daniel Ciobanu, Steven Lonergan, Jack Dekkers, Ken Stalder Mar 2018

Identification Of Genes For Carcass Merit And Meat Quality In The Pig, Max Rothschild, Daniel Ciobanu, Steven Lonergan, Jack Dekkers, Ken Stalder

Max Rothschild

Advances in the fields of molecular genetics and genomics have been considerable over the past several years. Recent discoveries in human genetics and in molecular biology have led to the development of a very useful genetic map of the pig. Several recent quantitative trait loci (QTL) scans and candidate gene analyses have identified important chromosomal regions and individual genes associated with carcass and meat quality traits. The causative mutations for porcine stress syndrome (HAL or RYR1) and the acid meat (RN) disorder, both with effects on meat quality are now known. Candidate genes for carcass merit (MC4R) and meat quality ...


In Silico Modeling Of Epigenetic-Induced Changes In Photoreceptor Cis-Regulatory Elements, Reafa A. Hossain, Nicholas R. Dunham, Raymond A. Enke, Christopher E. Berndsen Dec 2017

In Silico Modeling Of Epigenetic-Induced Changes In Photoreceptor Cis-Regulatory Elements, Reafa A. Hossain, Nicholas R. Dunham, Raymond A. Enke, Christopher E. Berndsen

Ray Enke Ph.D.

No abstract provided.


Jennifer Maurer Phd Thesis.Pdf, Jennifer Maurer Nov 2017

Jennifer Maurer Phd Thesis.Pdf, Jennifer Maurer

Jennifer Maurer


Signaling cascades, such as the extracellular signal-regulated kinase (ERK) pathway, play vital roles in early vertebrate development. Signals through these pathways are initiated by a growth factor or hormone, are transduced through a kinase cascade, and result in the expression of specific downstream genes that promote cellular proliferation, growth, or differentiation.Tight regulation of these signals is provided by positive or negative modulators at varying levels in the pathway, and is required for proper development and function. Two members of the dual-specificity phosphatase (Dusp) family, dusp6 and dusp2, are believed to be negative regulators of the ERK pathway and are ...


C. Elegans Avoids Toxin-Producing Streptomyces Using A Seven Transmembrane Domain Chemosensory Receptor, Alan Tran, Angelina Tang, Colleen T. O’Loughlin, Anthony Balistreri, Eric Chang, Doris Coto Villa, Joy Li, Aruna Varshney, Vanessa Jimenez, Jacqueline Pyle, Bryan Tsujimoto, Christopher Wellbrook, Christopher Vargas, Alex Duong, Nebat Ali, Sarah Y. Matthews, Samantha Levinson, Sarah Woldemariam, Sami Khuri, Martina Bremer, Daryl K. Eggers, Noelle L’Etoile, Laura C. Miller Conrad, Miri Vanhoven Sep 2017

C. Elegans Avoids Toxin-Producing Streptomyces Using A Seven Transmembrane Domain Chemosensory Receptor, Alan Tran, Angelina Tang, Colleen T. O’Loughlin, Anthony Balistreri, Eric Chang, Doris Coto Villa, Joy Li, Aruna Varshney, Vanessa Jimenez, Jacqueline Pyle, Bryan Tsujimoto, Christopher Wellbrook, Christopher Vargas, Alex Duong, Nebat Ali, Sarah Y. Matthews, Samantha Levinson, Sarah Woldemariam, Sami Khuri, Martina Bremer, Daryl K. Eggers, Noelle L’Etoile, Laura C. Miller Conrad, Miri Vanhoven

Daryl K. Eggers

Predators and prey co-evolve, each maximizing their own fitness, but the effects of predator–prey interactions on cellular and molecular machinery are poorly understood. Here, we study this process using the predator Caenorhabditis elegans and the bacterial prey Streptomyces, which have evolved a powerful defense: the production of nematicides. We demonstrate that upon exposure to Streptomyces at their head or tail, nematodes display an escape response that is mediated by bacterially produced cues. Avoidance requires a predicted G-protein-coupled receptor, SRB-6, which is expressed in five types of amphid and phasmid chemosensory neurons. We establish that species of Streptomyces secrete dodecanoic ...


C. Elegans Avoids Toxin-Producing Streptomyces Using A Seven Transmembrane Domain Chemosensory Receptor, Alan Tran, Angelina Tang, Colleen T. O’Loughlin, Anthony Balistreri, Eric Chang, Doris Coto Villa, Joy Li, Aruna Varshney, Vanessa Jimenez, Jacqueline Pyle, Bryan Tsujimoto, Christopher Wellbrook, Christopher Vargas, Alex Duong, Nebat Ali, Sarah Y. Matthews, Samantha Levinson, Sarah Woldemariam, Sami Khuri, Martina Bremer, Daryl K. Eggers, Noelle L’Etoile, Laura C. Miller Conrad, Miri Vanhoven Sep 2017

C. Elegans Avoids Toxin-Producing Streptomyces Using A Seven Transmembrane Domain Chemosensory Receptor, Alan Tran, Angelina Tang, Colleen T. O’Loughlin, Anthony Balistreri, Eric Chang, Doris Coto Villa, Joy Li, Aruna Varshney, Vanessa Jimenez, Jacqueline Pyle, Bryan Tsujimoto, Christopher Wellbrook, Christopher Vargas, Alex Duong, Nebat Ali, Sarah Y. Matthews, Samantha Levinson, Sarah Woldemariam, Sami Khuri, Martina Bremer, Daryl K. Eggers, Noelle L’Etoile, Laura C. Miller Conrad, Miri Vanhoven

Miri VanHoven

Predators and prey co-evolve, each maximizing their own fitness, but the effects of predator–prey interactions on cellular and molecular machinery are poorly understood. Here, we study this process using the predator Caenorhabditis elegans and the bacterial prey Streptomyces, which have evolved a powerful defense: the production of nematicides. We demonstrate that upon exposure to Streptomyces at their head or tail, nematodes display an escape response that is mediated by bacterially produced cues. Avoidance requires a predicted G-protein-coupled receptor, SRB-6, which is expressed in five types of amphid and phasmid chemosensory neurons. We establish that species of Streptomyces secrete dodecanoic ...


Expression And Purification Of Dna Polymerase 1 (Klenow Fragment) For Single-Molecule Nanotube Dna Sequencing, Chanelle Hunter, Kristin Gabriel, Gregory Weiss Aug 2017

Expression And Purification Of Dna Polymerase 1 (Klenow Fragment) For Single-Molecule Nanotube Dna Sequencing, Chanelle Hunter, Kristin Gabriel, Gregory Weiss

Chanelle Hunter

Errors during DNA synthesis can introduce genomic mutations, and can lead to cancer and other genetic diseases. DNA sequencing can identify these errors at the molecular level, but wide spread application of this technique requires breakthroughs in DNA sequencing. This research examines DNA synthesis using a single molecule of the Klenow Fragment (KF) of DNA Polymerase 1 wired to a single-walled carbon nanotube field effect transistor (SWCNT-FET). If successful, this approach could revolutionize DNA sequencing. KF catalyzes incorporation of deoxynucleotidetriphosphates (dNTPs) into new strands of DNA. During this catalysis, conformational changes in the protein could generate signals specific to each ...


Biophysical And Computational Studies Of The Vcci:Vmip-Ii Complex, Anna Nguyen, Nai-Wei Kuo, Laura Showalter, Ricardo Ramos, Cynthia Dupureur, Michael Colvin, Patricia Liwang Aug 2017

Biophysical And Computational Studies Of The Vcci:Vmip-Ii Complex, Anna Nguyen, Nai-Wei Kuo, Laura Showalter, Ricardo Ramos, Cynthia Dupureur, Michael Colvin, Patricia Liwang

Cynthia Dupureur

Certain viruses have the ability to subvert the mammalian immune response, including interference in the chemokine system. Poxviruses produce the chemokine binding protein vCCI (viral CC chemokine inhibitor; also called 35K), which tightly binds to CC chemokines. To facilitate the study of vCCI, we first provide a protocol to produce folded vCCI from Escherichia coli (E. coli.) It is shown here that vCCI binds with unusually high affinity to viral Macrophage Inflammatory Protein-II (vMIP-II), a chemokine analog produced by the virus, human herpesvirus 8 (HHV-8). Fluorescence anisotropy was used to investigate the vCCI:vMIP-II complex and shows that vCCI binds ...


Crystal Structure Of Apobec3a Bound To Single-Stranded Dna Reveals Structural Basis For Cytidine Deamination And Specificity, Takahide Kouno, Tania V. Silvas, Brendan J. Hilbert, Shivender Shandilya, Markus-Frederik Bohn, Brian A. Kelch, William E. Royer, Mohan Somasundaran, Nese Kurt Yilmaz, Hiroshi Matsuo, Celia A. Schiffer Jul 2017

Crystal Structure Of Apobec3a Bound To Single-Stranded Dna Reveals Structural Basis For Cytidine Deamination And Specificity, Takahide Kouno, Tania V. Silvas, Brendan J. Hilbert, Shivender Shandilya, Markus-Frederik Bohn, Brian A. Kelch, William E. Royer, Mohan Somasundaran, Nese Kurt Yilmaz, Hiroshi Matsuo, Celia A. Schiffer

Celia A. Schiffer

Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 A. This structure not only visualizes the active ...


A Single Residue Switch Converts Abietadiene Synthase Into A Pimaradiene Specific Cyclase, P. Ross Wilderman, Reuben J. Peters Jun 2017

A Single Residue Switch Converts Abietadiene Synthase Into A Pimaradiene Specific Cyclase, P. Ross Wilderman, Reuben J. Peters

Reuben J. Peters

Terpene synthases often catalyze complex cyclization reactions that typically represent the committed step in particular biosynthetic pathways, leading to great interest in their enzymatic mechanisms. We have recently demonstrated that substitution of a specific Ile with Thr was sufficient to “short circuit” the complex cyclization reaction normally catalyzed by ent-kaurene synthases to instead produce ent-pimaradiene. Here we report that the complex cyclization/rearrangement reaction catalyzed by abietadiene synthase can be similarly cut short to produce pimaradienes by an analogous Ser for Ala change, albeit with a slight shift in active site location to accommodate the difference in substrate stereochemistry. This ...


Strand-Specific Libraries For High Throughput Rna Sequencing (Rna-Seq) Prepared Without Poly(A) Selection, Zhao Zhang, William E. Theurkauf, Zhiping Weng, Phillip D. Zamore Jun 2017

Strand-Specific Libraries For High Throughput Rna Sequencing (Rna-Seq) Prepared Without Poly(A) Selection, Zhao Zhang, William E. Theurkauf, Zhiping Weng, Phillip D. Zamore

Zhao Zhang

BACKGROUND: High throughput DNA sequencing technology has enabled quantification of all the RNAs in a cell or tissue, a method widely known as RNA sequencing (RNA-Seq). However, non-coding RNAs such as rRNA are highly abundant and can consume >70% of sequencing reads. A common approach is to extract only polyadenylated mRNA; however, such approaches are blind to RNAs with short or no poly(A) tails, leading to an incomplete view of the transcriptome. Another challenge of preparing RNA-Seq libraries is to preserve the strand information of the RNAs. DESIGN: Here, we describe a procedure for preparing RNA-Seq libraries from 1 ...


Terpenoid Synthase Structures: A So Far Incomplete View Of Complex Catalysis, Yang Gao, Richard B. Honzatko, Reuben J. Peters Jun 2017

Terpenoid Synthase Structures: A So Far Incomplete View Of Complex Catalysis, Yang Gao, Richard B. Honzatko, Reuben J. Peters

Reuben J. Peters

The complexity of terpenoid natural products has drawn significant interest, particularly since their common (poly)isoprenyl origins were discovered. Notably, much of this complexity is derived from the highly variable cyclized and/or rearranged nature of the observed hydrocarbon skeletal structures. Indeed, at least in some cases it is difficult to immediately recognize their derivation from poly-isoprenyl precursors. Nevertheless, these diverse structures are formed by sequential elongation to acyclic precursors, most often with subsequent cyclization and/or rearrangement. Strikingly, the reactions used to assemble and diversify terpenoid backbones share a common carbocationic driven mechanism, although the means by which the ...


Two Rings In Them All: The Labdane-Related Diterpenoids, Reuben J. Peters Jun 2017

Two Rings In Them All: The Labdane-Related Diterpenoids, Reuben J. Peters

Reuben J. Peters

In his original exposition of the biogenetic isoprenoid rule, L. Ruzicka noted the structural identity between the fused A/B rings of triterpenoids/sterols and certain multicyclic diterpenoids as part of the impetus leading to that profound insight. His prescient hypothesis that this chemical structure relationship reflects similarities in the initial cyclization of these diterpenoids with that occurring in triterpenoid biosynthesis has since been verified. However, this chemical structure relationship does not continue to hold true for the additional rings found in many of these di- and tri- terpenoid natural products. This is now appreciated to arise from differences in ...


Microarray Analysis Of Aging-Associated Immune System Alterations In The Rostral Ventrolateral Medulla Of F344 Rats, Sivasai Balivada, Chanran K. Ganta, Yongqing Zhang, Hitesh N. Pawar, Richard J. Ortiz, Kevin G. Becker, Arshad M. Khan, Michael J. Kenney Jun 2017

Microarray Analysis Of Aging-Associated Immune System Alterations In The Rostral Ventrolateral Medulla Of F344 Rats, Sivasai Balivada, Chanran K. Ganta, Yongqing Zhang, Hitesh N. Pawar, Richard J. Ortiz, Kevin G. Becker, Arshad M. Khan, Michael J. Kenney

Arshad M. Khan, Ph.D.

The rostral ventrolateral medulla (RVLM) is an area of the brain stem that contains diverse neural substrates that are involved in systems critical for physiological function. There is evidence that aging affects some neural substrates within the RVLM, although age-related changes in RVLM molecular mechanisms are not well established. The goal of the present study was to characterize the transcriptomic profile of the aging RVLM and to test the hypothesis that aging is associated with altered gene expression in the RVLM, with an emphasis on immune system associated gene transcripts. RVLM tissue punches from young, middle-aged, and aged F344 rats ...


Central Role Of Il-23 And Il-17 Producing Eosinophils As Immunomodulatory Effector Cells In Acute Pulmonary Aspergillosis And Allergic Asthma, Evelyn V. Santos Guerra, Chrono K. Lee, Charles A. Specht, Bhawna Yadav, Haibin Huang, Ali Akalin, Jun R. Huh, Christian Mueller, Stuart M. Levitz May 2017

Central Role Of Il-23 And Il-17 Producing Eosinophils As Immunomodulatory Effector Cells In Acute Pulmonary Aspergillosis And Allergic Asthma, Evelyn V. Santos Guerra, Chrono K. Lee, Charles A. Specht, Bhawna Yadav, Haibin Huang, Ali Akalin, Jun R. Huh, Christian Mueller, Stuart M. Levitz

Christian Mueller

Aspergillus fumigatus causes invasive pulmonary disease in immunocompromised hosts and allergic asthma in atopic individuals. We studied the contribution of lung eosinophils to these fungal diseases. By in vivo intracellular cytokine staining and confocal microscopy, we observed that eosinophils act as local sources of IL-23 and IL-17. Remarkably, mice lacking eosinophils had a >95% reduction in the percentage of lung IL-23p19+ cells as well as markedly reduced IL-23 heterodimer in lung lavage fluid. Eosinophils killed A. fumigatus conidia in vivo. Eosinopenic mice had higher mortality rates, decreased recruitment of inflammatory monocytes, and decreased expansion of lung macrophages after challenge with ...


Hepatitis C Virus-Induced Monocyte Differentiation Into Polarized M2 Macrophages Promotes Stellate Cell Activation Via Tgf-Beta, Banishree Saha, Karen Kodys, Gyongyi Szabo May 2017

Hepatitis C Virus-Induced Monocyte Differentiation Into Polarized M2 Macrophages Promotes Stellate Cell Activation Via Tgf-Beta, Banishree Saha, Karen Kodys, Gyongyi Szabo

Gyongyi Szabo

BACKGROUND and AIMS: Monocyte and macrophage (MPhi) activation contributes to the pathogenesis of chronic hepatitis C virus (HCV) infection. Disease pathogenesis is regulated by both liver-resident MPhis and monocytes recruited as precursors of MPhis into the damaged liver. Monocytes differentiate into M1 (classic/proinflammatory) or M2 (alternative/anti-inflammatory) polarized MPhis in response to tissue microenvironment. We hypothesized that HCV-infected hepatoma cells (infected with Japanese fulminant hepatitis-1 [Huh7.5/JFH-1]) induce monocyte differentiation into polarized MPhis. METHODS: Healthy human monocytes were co-cultured with Huh7.5/JFH-1 cells or cell-free virus for 7 days and analyzed for MPhi markers and cytokine levels ...


Genetic Variation In Concentration Of The 33-Mer Protein Subcomponent In Wheat, Robert L. Paris, Kaleb M. Pauley, Ryan K. Lokkesmoe, Sarah E. Lyon, James C. Dunlap, Julia M. Pierre, Timothy Vanwingerden, Finny J. Johns, Kyle J. Kilchrist, Tyler J. Reid, Caleb M. Winn Apr 2017

Genetic Variation In Concentration Of The 33-Mer Protein Subcomponent In Wheat, Robert L. Paris, Kaleb M. Pauley, Ryan K. Lokkesmoe, Sarah E. Lyon, James C. Dunlap, Julia M. Pierre, Timothy Vanwingerden, Finny J. Johns, Kyle J. Kilchrist, Tyler J. Reid, Caleb M. Winn

Robert L. Paris, Ph.D.

Celiac Disease is a hypersensitive response to gluten caused by HLA-DQ2 or HLA-DQ8 T-cell presentation, initiating destruction of intestinal epithelial cells. Currently, the only remedy for those suffering from celiac disease is elimination of all gluten from the diet. Studies indicate that an indigestible fragment of the gluten molecule, alpha-gliadin subcomponent 33-mer, rich in proline and glutamine, is responsible for the hypersensitivity response. Determination of 33-mer concentration in wheat lines could be beneficial to future development of wheat lines with reduced 33-mer concentration. Protein from wheat flour was extracted and subjected to ELISA techniques in order to quantify the concentration ...