Shovel Test Pit Paperwork of Transect 8 from Quarterman (8BR223)

This document contains the field notes taken during phase 1 survey for transect 8

Dietary Isoflavones Alter Gut Microbiota and Lipopolysaccharide Biosynthesis to Reduce Inflammation

The etiopathogenesis of multiple sclerosis (MS) is strongly affected by environmental factors such as diet and the gut microbiota. An isoflavone-rich (ISO) diet was previously shown to reduce the severity of MS in the animal model experimental autoimmune encephalomyelitis (EAE). Translation of this concept to clinical trial where dietary isoflavones may be recommended for MS patients will require preliminary evidence that providing the isoflavone-rich diet to people with MS (PwMS) who lack phytoestrogen-metabolizing bacteria has beneficial effects. We have previously shown that the gut microbiota of PwMS resembles the gut microbiota of mice raised under a phytoestrogen-free (phyto-free) diet in that it lacks phytoestrogen-metabolizing bacteria. To investigate the effects of phytoestrogens on the microbiota inflammatory response and EAE disease severity we switched the diet of mice raised under a phyto-free (PF) diet to an isoflavone-rich diet. Microbiota analysis showed that the change in diet from one that is ISO to one that is PF reduces beneficial bacteria such as Bifidobacterium species. In addition we observed functional differences in lipopolysaccharide (LPS) biosynthesis pathways. Moreover LPS extracted from feces of mice fed an ISO diet induced increased production of anti-inflammatory cytokines from bone marrow-derived macrophages relative to fecal-LPS isolated from mice fed a PF diet. Eventually mice whose diet was switched from a PF diet to an ISO diet trended toward reduced EAE severity and mortality. Overall we show that an isoflavone-rich diet specifically modulates LPS biosynthesis of the gut microbiota imparts an anti-inflammatory response and decreases disease severity

A simple method for construction of pir+ Enterobacterial hosts for maintenance of R6K replicon plasmids

<p>Abstract</p> <p>Background</p> <p>The R6K replicon is one of the best studied bacterial plasmid replicons. Replication of the R6K plasmid and derivatives harboring its γ origin of replication (<it>ori</it>_R6Kγ) is dependent on the <it>pir </it>gene-encoded π protein. Originally encoded by R6K, this protein is usually provided <it>in trans </it>in hosts engineered to support replication of plasmids harboring <it>ori</it>_R6Kγ. In <it>Escherichia coli </it>this is commonly achieved by chromosomal integration of <it>pir </it>either via lysogenization with a λ<it>pir </it>phage or homologous recombination at a pre-determined locus.</p> <p>Findings</p> <p>Current methods for construction of host strains for <it>ori</it>_R6Kγ-containing plasmids involve procedures that do not allow selection for presence of the <it>pir </it>gene and require cumbersome and time-consuming screening steps. In this study, we established a mini-Tn<it>7</it>-based method for rapid and reliable construction of <it>pir</it>⁺host strains. Using a curable mini-Tn<it>7 </it>delivery plasmid, <it>pir </it>expressing derivatives of several commonly used <it>E. coli </it>cloning and mobilizer strains were isolated using both the wild-type <it>pir⁺</it>gene as well as the copy-up <it>pir-116 </it>allele. In addition, we isolated <it>pir</it>⁺and <it>pir-116 </it>expressing derivatives of a clinical isolate of <it>Salmonella enterica </it>serovar Typhimurium. In both <it>E. coli </it>and <it>S. enterica </it>serovar Typhimurium, the presence of the <it>pir⁺</it>wild-type or <it>pir-116 </it>alleles allowed the replication of <it>ori</it>_R6Kγ-containing plasmids.</p> <p>Conclusions</p> <p>A mini-Tn<it>7 </it>system was employed for rapid and reliable engineering of <it>E. coli </it>and <it>S. enterica </it>serovar Typhimurium host strains for plasmids containing <it>ori</it>_R6Kγ. Since mini-Tn7 elements transpose in most, if not all, Gram negative bacteria, we anticipate that with relatively minor modifications this newly established method will for the first time allow engineering of other bacterial species to enable replication of plasmids with <it>ori</it>_R6Kγ.</p

Wholly Rickettsia! Reconstructed Metabolic Profile of the Quintessential Bacterial Parasite of Eukaryotic Cells

Reductive genome evolution has purged many metabolic pathways from obligate intracellular Rickettsia (Alphaproteobacteria; Rickettsiaceae). While some aspects of host-dependent rickettsial metabolism have been characterized, the array of host-acquired metabolites and their cognate transporters remains unknown. This dearth of information has thwarted efforts to obtain an axenic Rickettsia culture, a major impediment to conventional genetic approaches. Using phylogenomics and computational pathway analysis, we reconstructed the Rickettsia metabolic and transport network, identifying 51 host-acquired metabolites (only 21 previously characterized) needed to compensate for degraded biosynthesis pathways. In the absence of glycolysis and the pentose phosphate pathway, cell envelope glycocon- jugates are synthesized from three imported host sugars, with a range of additional host-acquired metabolites fueling the tricarboxylic acid cycle. Fatty acid and glycero- phospholipid pathways also initiate from host precursors, and import of both iso- prenes and terpenoids is required for the synthesis of ubiquinone and the lipid car- rier of lipid I and O-antigen. Unlike metabolite-provisioning bacterial symbionts of arthropods, rickettsiae cannot synthesize B vitamins or most other cofactors, accen- tuating their parasitic nature. Six biosynthesis pathways contain holes (missing en- zymes); similar patterns in taxonomically diverse bacteria suggest alternative en- zymes that await discovery. A paucity of characterized and predicted transporters emphasizes the knowledge gap concerning how rickettsiae import host metabolites, some of which are large and not known to be transported by bacteria. Collectively, our reconstructed metabolic network offers clues to how rickettsiae hijack host met- abolic pathways. This blueprint for growth determinants is an important step toward the design of axenic media to rescue rickettsiae from the eukaryotic cell

The Grizzly, September 13, 2001

Eating in Wismer: The Crunch at Lunch • America. No Longer the Beautiful • Collegeville Police Crack Down on Ursinus Students • A New Look and New Menu at Wismer • Service Woes for Select Residents • Opinions: Wismer Bussing: A Major Problem; New Social Host Policy a Wet Blanket • International Film Festival Brings Foreign Flair to Ursinus College Campus • Review of the Restaurant La Fontana • Unconventional Fringe Fest Takes Over Philly • Like Old Movies? Then the Colonial Theater is the Place to Be • Pizza: Where\u27s the Best Buy for Your Money? • Ursinus Webpage is Getting a Makeover • Colonization of Sigma Sigma Sigma • Duncan Breaks Record as Ursinus Downs Waynesburg • Cross Country Breezes Through LBV Invitational • Two Tough Teams Equal First Two Losses for Men\u27s Soccer • UC Women\u27s Volleyball Defeats Wilkes for Third Win • Bears Fall to Montclair; Tie with Widener • UC Field Hockey Takes Slap Shot • Soccer Teams Without a Home Fieldhttps://digitalcommons.ursinus.edu/grizzlynews/1493/thumbnail.jp

Volume 01

Introduction from Dean Dr. Charles Ross Three Decades of Digging: Undergraduate Archeology at Longwood by Jessica Fields and Stephanie Neeley Interactions of Allelopathy and Heat Stress in Plants by Derek W. Hambright and Mary E. Lehman Inertial Electrostatic Confinement D-D Fusion Device: Construction and Simulation by Andrew R. Grzankowski Shackled Nim by Zachary Johnson Development of GC-MS and Chemometric Methods for the Analysis of Accelerants in Arson Cases by Boone M. Prentice A Comparison of Image Analysis Methods in cDNA Microarrays by Ashley M. Swandby Perceived Sexual Activity of Short and Long-Term Relationships by Victoria Morgan and Katie Williamson Elderly Male Communication by Kristine G. Bender Three Poems: “Adam and Eve and an Orange Tree”, “The Name of Everything Before Dying”, and “The ‘Poet Voice’” by Katelyn N. Romaine There\u27s Nothing Like Dancing, After All : Marriage and Gender in the Dance Scenes of Jane Austen\u27s Novels by D. Nicole Swann Two Poems: “Age Nine with Mother” and “The Apple That Crawls Away From the Tree” by Jessica Fox Untitled by Mike McAteer Room 9 by Alex Grabiec Two Photographs: “Gracie” and “Emily” by Laura Nodtvedt Bowling Lanes Night by Nick Costa Two Paintings: “Can and Kettle” and “Scarecrow” by Rachel Wolfe Exploring Henrik Ibsen\u27s “Perr Gynt” by Zack Dalton Creative Writing Scholarship at Longwood University Music Scholarship at Longwood – Senior Recital Arianne K. Burrus Longwood University Theater – Peer Gyn

Structural Insight into How Bacteria Prevent Interference between Multiple Divergent Type IV Secretion Systems

Prokaryotes use type IV secretion systems (T4SSs) to translocate substrates (e.g., nucleoprotein, DNA, and protein) and/or elaborate surface structures (i.e., pili or adhesins). Bacterial genomes may encode multiple T4SSs, e.g., there are three functionally divergent T4SSs in some Bartonella species (vir, vbh, and trw). In a unique case, most rickettsial species encode a T4SS (rvh) enriched with gene duplication. Within single genomes, the evolutionary and functional implications of cross-system interchangeability of analogous T4SS protein components remains poorly understood. To lend insight into cross-system interchangeability, we analyzed the VirB8 family of T4SS channel proteins. Crystal structures of three VirB8 and two TrwG Bartonella proteins revealed highly conserved C-terminal periplasmic domain folds and dimerization interfaces, despite tremendous sequence divergence. This implies remarkable structural constraints for VirB8 components in the assembly of a functional T4SS. VirB8/TrwG heterodimers, determined via bacterial two-hybrid assays and molecular modeling, indicate that differential expression of trw and vir systems is the likely barrier to VirB8-TrwG interchangeability. We also determined the crystal structure of Rickettsia typhi RvhB8-II and modeled its coexpressed divergent paralog RvhB8-I. Remarkably, while RvhB8-I dimerizes and is structurally similar to other VirB8 proteins, the RvhB8-II dimer interface deviates substantially from other VirB8 structures, potentially preventing RvhB8-I/RvhB8-II heterodimerization. For the rvh T4SS, the evolution of divergent VirB8 paralogs implies a functional diversification that is unknown in other T4SSs. Collectively, our data identify two different constraints (spatio-temporal for Bartonella trw and vir T4SSs and structural for rvh T4SSs) that mediate the functionality of multiple divergent T4SSs within a single bacterium. IMPORTANCE Assembly of multiprotein complexes at the right time and at the right cellular location is a fundamentally important task for any organism. In this respect, bacteria that express multiple analogous type IV secretion systems (T4SSs), each composed of around 12 different components, face an overwhelming complexity. Our work here presents the first structural investigation on factors regulating the maintenance of multiple T4SSs within a single bacterium. The structural data imply that the T4SS-expressing bacteria rely on two strategies to prevent cross-system interchangeability: (i) tight temporal regulation of expression or (ii) rapid diversification of the T4SS components. T4SSs are ideal drug targets provided that no analogous counterparts are known from eukaryotes. Drugs targeting the barriers to cross-system interchangeability (i.e., regulators) could dysregulate the structural and functional independence of discrete systems, potentially creating interference that prevents their efficient coordination throughout bacterial infection.Peer reviewe

eXtraembryonic ENdoderm (XEN) Stem Cells Produce Factors that Activate Heart Formation

Initial specification of cardiomyocytes in the mouse results from interactions between the extraembryonic anterior visceral endoderm (AVE) and the nascent mesoderm. However the mechanism by which AVE activates cardiogenesis is not well understood, and the identity of specific cardiogenic factors in the endoderm remains elusive. Most mammalian studies of the cardiogenic potential of the endoderm have relied on the use of cell lines that are similar to the heart-inducing AVE. These include the embryonal-carcinoma-derived cell lines, END2 and PYS2. The recent development of protocols to isolate eXtraembryonic ENdoderm (XEN) stem cells, representing the extraembryonic endoderm lineage, from blastocyst stage mouse embryos offers new tools for the genetic dissection of cardiogenesis.Here, we demonstrate that XEN cell-conditioned media (CM) enhances cardiogenesis during Embryoid Body (EB) differentiation of mouse embryonic stem (ES) cells in a manner comparable to PYS2-CM and END2-CM. Addition of CM from each of these three cell lines enhanced the percentage of EBs that formed beating areas, but ultimately, only XEN-CM and PYS2-CM increased the total number of cardiomyocytes that formed. Furthermore, our observations revealed that both contact-independent and contact-dependent factors are required to mediate the full cardiogenic potential of the endoderm. Finally, we used gene array comparison to identify factors in these cell lines that could mediate their cardiogenic potential.These studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation. Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts. These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm

A Comparative Analysis of Extra-Embryonic Endoderm Cell Lines

Prior to gastrulation in the mouse, all endodermal cells arise from the primitive endoderm of the blastocyst stage embryo. Primitive endoderm and its derivatives are generally referred to as extra-embryonic endoderm (ExEn) because the majority of these cells contribute to extra-embryonic lineages encompassing the visceral endoderm (VE) and the parietal endoderm (PE). During gastrulation, the definitive endoderm (DE) forms by ingression of cells from the epiblast. The DE comprises most of the cells of the gut and its accessory organs. Despite their different origins and fates, there is a surprising amount of overlap in marker expression between the ExEn and DE, making it difficult to distinguish between these cell types by marker analysis. This is significant for two main reasons. First, because endodermal organs, such as the liver and pancreas, play important physiological roles in adult animals, much experimental effort has been directed in recent years toward the establishment of protocols for the efficient derivation of endodermal cell types in vitro. Conversely, factors secreted by the VE play pivotal roles that cannot be attributed to the DE in early axis formation, heart formation and the patterning of the anterior nervous system. Thus, efforts in both of these areas have been hampered by a lack of markers that clearly distinguish between ExEn and DE. To further understand the ExEn we have undertaken a comparative analysis of three ExEn-like cell lines (END2, PYS2 and XEN). PYS2 cells are derived from embryonal carcinomas (EC) of 129 strain mice and have been characterized as parietal endoderm-like [1], END2 cells are derived from P19 ECs and described as visceral endoderm-like, while XEN cells are derived from blastocyst stage embryos and are described as primitive endoderm-like. Our analysis suggests that none of these cell lines represent a bona fide single in vivo lineage. Both PYS2 and XEN cells represent mixed populations expressing markers for several ExEn lineages. Conversely END2 cells, which were previously characterized as VE-like, fail to express many markers that are widely expressed in the VE, but instead express markers for only a subset of the VE, the anterior visceral endoderm. In addition END2 cells also express markers for the PE. We extended these observations with microarray analysis which was used to probe and refine previously published data sets of genes proposed to distinguish between DE and VE. Finally, genome-wide pathway analysis revealed that SMAD-independent TGFbeta signaling through a TAK1/p38/JNK or TAK1/NLK pathway may represent one mode of intracellular signaling shared by all three of these lines, and suggests that factors downstream of these pathways may mediate some functions of the ExEn. These studies represent the first step in the development of XEN cells as a powerful molecular genetic tool to study the endodermal signals that mediate the important developmental functions of the extra-embryonic endoderm. Our data refine our current knowledge of markers that distinguish various subtypes of endoderm. In addition, pathway analysis suggests that the ExEn may mediate some of its functions through a non-classical MAP Kinase signaling pathway downstream of TAK1