Enhanced Actin Pedestal Formation by Enterohemorrhagic Escherichia coli O157:H7 Adapted to the Mammalian Host

Upon intestinal colonization, enterohemorrhagic Escherichia coli (EHEC) induces epithelial cells to generate actin “pedestals” beneath bound bacteria, lesions that promote colonization. To induce pedestals, EHEC utilizes a type III secretion system to translocate into the mammalian cell bacterial effectors such as translocated intimin receptor (Tir), which localizes in the mammalian cell membrane and functions as a receptor for the bacterial outer membrane protein intimin. Whereas EHEC triggers efficient pedestal formation during mammalian infection, EHEC cultured in vitro induces pedestals on cell monolayers with relatively low efficiency. To determine whether growth within the mammalian host enhances EHEC pedestal formation, we compared in vitro-cultivated bacteria with EHEC directly isolated from infected piglets. Mammalian adaptation by EHEC was associated with a dramatic increase in the efficiency of cell attachment and pedestal formation. The amounts of intimin and Tir were significantly higher in host-adapted than in in vitro-cultivated bacteria, but increasing intimin or Tir expression, or artificially increasing the level of bacterial attachment to mammalian cells, did not enhance pedestal formation by in vitro-cultivated EHEC. Instead, a functional assay suggested that host-adapted EHEC translocate Tir much more efficiently than does in vitro-cultivated bacteria. These data suggest that adaptation of EHEC to the mammalian intestine enhances bacterial cell attachment, expression of intimin and Tir, and translocation of effectors that promote actin signaling

In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism

<p>Abstract</p> <p>Background</p> <p><it>Shigella dysenteriae </it>serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of <it>Shigella dysenteriae </it>serotype 1 (SD1) <it>in vitro </it>(derived from LB cell cultures) and <it>in vivo </it>(derived from gnotobiotic piglets) was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology.</p> <p>Results</p> <p>Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate), including 1480 and 1505 from <it>in vitro </it>and <it>in vivo </it>samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM) proteins (38% of <it>in silico </it>predicted SD1 membrane proteome) contributed to the most extensive survey of the <it>Shigella </it>membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under <it>in vivo </it>conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High <it>in vivo </it>abundances of proteins involved in acid resistance (GadB, AdiA) and mixed acid fermentation (PflA/PflB) indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB) implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB) were increased, while β-barrel OM proteins (OmpA), OM lipoproteins (NlpD), chaperones involved in OM protein folding pathways (YraP, NlpB) and lipopolysaccharide biosynthesis (Imp) were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers <it>in vivo</it>. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins) required for invasion of colonic epithelial cells, and release of bacteria into the host cell cytosol were increased <it>in vivo</it>.</p> <p>Conclusions</p> <p>Global proteomic profiling of SD1 comparing <it>in vivo vs. in vitro </it>proteomes revealed differential expression of proteins geared towards survival of the pathogen in the host gut environment, including increased abundance of proteins involved in anaerobic energy respiration, acid resistance and virulence. The immunogenic OspC2, OspC3 and IpgA virulence proteins were detected solely under <it>in vivo </it>conditions, lending credence to their candidacy as potential vaccine targets.</p

Comparison of two label-free global quantitation methods, APEX and 2D gel electrophoresis, applied to the Shigella dysenteriae proteome

The in vitro stationary phase proteome of the human pathogen Shigella dysenteriae serotype 1 (SD1) was quantitatively analyzed in Coomassie Blue G250 (CBB)-stained 2D gels. More than four hundred and fifty proteins, of which 271 were associated with distinct gel spots, were identified. In parallel, we employed 2D-LC-MS/MS followed by the label-free computationally modified spectral counting method APEX for absolute protein expression measurements. Of the 4502 genome-predicted SD1 proteins, 1148 proteins were identified with a false positive discovery rate of 5% and quantitated using 2D-LC-MS/MS and APEX. The dynamic range of the APEX method was approximately one order of magnitude higher than that of CBB-stained spot intensity quantitation. A squared Pearson correlation analysis revealed a reasonably good correlation (R2 = 0.67) for protein quantities surveyed by both methods. The correlation was decreased for protein subsets with specific physicochemical properties, such as low Mr values and high hydropathy scores. Stoichiometric ratios of subunits of protein complexes characterized in E. coli were compared with APEX quantitative ratios of orthologous SD1 protein complexes. A high correlation was observed for subunits of soluble cellular protein complexes in several cases, demonstrating versatile applications of the APEX method in quantitative proteomics

Footprints- In the Footprints of Squier and Davis: Archeological Fieldwork in Ross County, Ohio

TABLE OF CONTENTS Acknowledgements....................................................................iList of Tables...................................................................vii List of Figures...................................................................ix Contributors .........................................................................xiii Chapter 1 In the Footprints of Squier and Davis : Archeological Fieldwork in Ross County, Ohio Mark J. Lynott................................................................................1 Previous Studies in Ohio ...................................................1 Hopewell Studies Today ................................................................4 Recent Field Research ..........................................................................6 Goal of this Volume ................................................................................12 Chapter 2 In Non-mound Space at the Hopewell Mound Group Jennifer Pederson Weinberger .........................................13 Geophysical Survey .......................................................15Western Village Area.....................................................................16 Near the East Village .....................................................................18 Central Area ............................................................................19 Discussion .......................................................................................19 Chapter 3 Field Studies of the Octagon and Great Circle, High Bank Earthworks Ross County, Ohio N’omi B. Greber and Orrin C. Shane III ..............................................23 Excavations at the Octagon ............................................................25 Geophysics at the Great Circle ............................................................30 Excavations at the Great Circle ..........................................................33 Radiocarbon Assays ...........................................................................41 Comments ....................................................................................44 Addendum ..........................................................................................46 Chapter 4 Spruce Hill Earthworks: The 1995-1996 National Park Service Investigations Bret J. Ruby .............................................................................................49 Background .......................................................................................49 The Spruce Hill Earthworks .......................................................................49 Spruce Hill Revisited ......................................................................53 The 1995-1996 National Park Service Investigations ............................53 Discussion and Conclusions ..............................................................61 Chapter 5 Falling Through a Crack in the Core: The Surprise and Demise of Anderson Earthwork William H. Pickard and Jeffrey W. Weinberger ........................................ 67 History and Setting ..................................................................................68 1993 Excavations ..................................................................................70 Discussion ..............................................................................................72 Conclusion ............................................................................................74 Chapter 6 Middle Woodland and Other Settlement Remains in the Overly Tract Near The Hopeton Earthwork, Ross County, Ohio William S. Dancey .....................................................................................................77 Research Design.............................................................................77 Artifact Categories and their Distributions..............................................80 Discussion............................................................................................92 Chapter 7 Hopewell Occupation at the Hopeton Earthworks: Large Scale Surface Survey Using GPS Technology Jarrod Burks and Dawn Walter Gagliano................................................97 The Survey Area ................................................................................98 Survey Methodology .............................................................................99 Survey Results ...................................................................................99 The Surface Data: A Siteless Approach .................................................103 Discussion and Conclusion .........................................................................106 Notes ...................................................................................................107 Chapter 8 Hopewellian Centers in Context: Investigations In and Around the Hopeton Earthworks Bret J. Ruby And Mark J. Lynott .................................................................109 Surface Survey ......................................................................................110 Redwing Site ...........................................................................................111 Comparisons .........................................................................................118 Conclusions ......................................................................................122 Chapter 9 Searching for Hopewell Settlements: The Triangle Site at the Hopeton Earthworks Mark Lynott ...................................................................................... 125 Field Investigations ............................................................................127 Geophysical Survey ............................................................................128 1998 Season ....................................................................................128 Features, Artifacts and Radiocarbon Dating ........................................130 Animal Remains ...............................................................................137 Plant Remains ................................................................................138 Interpretations ..............................................................................139 Chapter 10 Geophysical Investigations at the Hopeton Earthworks John Weymouth, Bruce Bevan, and Rinita Dalan ...............................145 The Cesium Gradiometer Survey ...........................................................146 Cesium Gradiometer Results ...................................................................146 Geoscan Instrument Surveys ..............................................................148 Comparison of Cesium Magnetic and Resistance Data ........................148 Small Circles...........................................................................................149 Trench Excavations ...................................................................................149 Discussion ................................................................................................152 Conclusions ......................................................................................157 Chapter 11 Archeological and Geoarcheological Study of the Rectangular Enclosure at the Hopeton Works Mark J. Lynott and Rolfe D. Mandel ......................................................159 The Study of the Rectangular Earthwork ................................................161 Geophysical Survey ..............................................................................163 Trench Excavations .............................................................................164 Chronology ........................................................................................170 Geoarcheological Analysis of Trench 1 .................................................172 Interpretations ................................................................................174 Chapter 12 Ohio Hopewell Ritual Craft Production Katherine A. Spielmann ............................................................ 179Raw Material Procurement ....................................................................180 Hopewell Crafting ................................................................................181 Deposition ...........................................................................................186 Conclusions ....................................................................................188 References Cited .....................................................................................................18

A 1.5-million-year record of orbital and millennial climate variability in the North Atlantic

Climate during the last glacial period was marked by abrupt instability on millennial timescales that included large swings of temperature in and around Greenland (Daansgard-Oeschger events) and smaller, more gradual changes in Antarctica (AIM events). Less is known about the existence and nature of similar variability during older glacial periods, especially during the early Pleistocene when glacial cycles were dominantly occurring at 41 kyr intervals compared to the much longer and deeper glaciations of the more recent period. Here, we report a continuous millennially resolved record of stable isotopes of planktic and benthic foraminifera at IODP Site U1385 (the "Shackleton Site") from the southwestern Iberian margin for the last 1.5 million years, which includes the Middle Pleistocene Transition (MPT). Our results demonstrate that millennial climate variability (MCV) was a persistent feature of glacial climate, both before and after the MPT. Prior to 1.2 Ma in the early Pleistocene, the amplitude of MCV was modulated by the 41 kyr obliquity cycle and increased when axial tilt dropped below 23.5° and benthic δ18O exceeded ∼3.8 ‰ (corrected to Uvigerina), indicating a threshold response to orbital forcing. Afterwards, MCV became focused mainly on the transitions into and out of glacial states (i.e. inceptions and terminations) and during times of intermediate ice volume. After 1.2 Ma, obliquity continued to play a role in modulating the amplitude of MCV, especially during times of glacial inceptions, which are always associated with declining obliquity. A non-linear role for obliquity is also indicated by the appearance of multiples (82, 123 kyr) and combination tones (28 kyr) of the 41 kyr cycle. Near the end of the MPT (∼0.65 Ma), obliquity modulation of MCV amplitude wanes as quasi-periodic 100 kyr and precession power increase, coinciding with the growth of oversized ice sheets on North America and the appearance of Heinrich layers in North Atlantic sediments. Whereas the planktic δ18O of Site U1385 shows a strong resemblance to Greenland temperature and atmospheric methane (i.e. Northern Hemisphere climate), millennial changes in benthic δ18O closely follow the temperature history of Antarctica for the past 800 kyr. The phasing of millennial planktic and benthic δ18O variation is similar to that observed for MIS 3 throughout much of the record, which has been suggested to mimic the signature of the bipolar seesaw - i.e. an interhemispheric asymmetry between the timing of cooling in Antarctica and warming in Greenland. The Iberian margin isotopic record suggests that bipolar asymmetry was a robust feature of interhemispheric glacial climate variations for at least the past 1.5 Ma despite changing glacial boundary conditions. A strong correlation exists between millennial increases in planktic δ18O (cooling) and decreases in benthic δ13C, indicating that millennial variations in North Atlantic surface temperature are mirrored by changes in deep-water circulation and remineralization of carbon in the abyssal ocean. We find strong evidence that climate variability on millennial and orbital scales is coupled across different timescales and interacts in both directions, which may be important for linking internal climate dynamics and external astronomical forcing

Using Whole Genome Sequences to Investigate Adenovirus Outbreaks in a Hematopoietic Stem Cell Transplant Unit

A recent surge in human mastadenovirus (HAdV) cases, including five deaths, amongst a haematopoietic stem cell transplant population led us to use whole genome sequencing (WGS) to investigate. We compared sequences from 37 patients collected over a 20-month period with sequences from GenBank and our own database of HAdVs. Maximum likelihood trees and pairwise differences were used to evaluate genotypic relationships, paired with the epidemiological data from routine infection prevention and control (IPC) records and hospital activity data. During this time period, two formal outbreaks had been declared by IPC, while WGS detected nine monophyletic clusters, seven were corroborated by epidemiological evidence and by comparison of single-nucleotide polymorphisms. One of the formal outbreaks was confirmed, and the other was not. Of the five HAdV-associated deaths, three were unlinked and the remaining two considered the source of transmission. Mixed infection was frequent (10%), providing a sentinel source of recombination and superinfection. Immunosuppressed patients harboring a high rate of HAdV positivity require comprehensive surveillance. As a consequence of these findings, HAdV WGS is being incorporated routinely into clinical practice to influence IPC policy contemporaneously

AVURT: aspirin versus placebo for the treatment of venous leg ulcers a Phase II pilot randomised controlled trial

Background Venous leg ulcers (VLUs) are the most common cause of leg ulceration, affecting 1 in 100 adults. VLUs may take many months to heal (25% fail to heal). Estimated prevalence is between 1% and 3% of the elderly population. Compression is the mainstay of treatment and few additional therapies exist to improve healing. Two previous trials have indicated that low-dose aspirin, as an adjunct to standard care, may improve healing time, but these trials were insufficiently robust. Aspirin is an inexpensive, widely used medication but its safety and efficacy in the treatment of VLUs remains to be established. Objectives Primary objective – to assess the effects of 300 mg of aspirin (daily) versus placebo on the time to healing of the reference VLU. Secondary objectives – to assess the feasibility of leading into a larger pragmatic Phase III trial and the safety of aspirin in this population. Design A multicentred, pilot, Phase II randomised double-blind, parallel-group, placebo-controlled efficacy trial. Setting Community leg ulcer clinics or services, hospital outpatient clinics, leg ulcer clinics, tissue viability clinics and wound clinics in England, Wales and Scotland. Participants Patients aged ≥ 18 years with a chronic VLU (i.e. the VLU is > 6 weeks in duration or the patient has a history of VLU) and who are not regularly taking aspirin. Interventions 300 mg of daily oral aspirin versus placebo. All patients were offered care in accordance with Scottish Intercollegiate Guidelines Network (SIGN) guidance with multicomponent compression therapy aiming to deliver 40 mmHg at the ankle when possible. Randomisation Participants were allocated in a 1 : 1 (aspirin : placebo) ratio by the Research Pharmacy, St George’s University Hospitals NHS Foundation Trust, using a randomisation schedule generated in advance by the investigational medicinal product manufacturer. Randomisation was stratified according to ulcer size (≤ 5cm2 or > 5cm2). Main outcome measure The primary outcome was time to healing of the largest eligible ulcer (reference ulcer). Feasibility results – recruitment 27 patients were recruited from eight sites over a period of 8 months. The target of 100 patients was not achieved and two sites did not recruit. Barriers to recruitment included a short recruitment window and a large proportion of participants failing to meet the eligibility criteria. Results The average age of the 27 randomised participants (placebo, n = 13; aspirin, n = 14) was 62 years (standard deviation 13 years), and two-thirds were male (n = 18). Participants had their reference ulcer for a median of 15 months, and the median size of ulcer was 17.1 cm2. There was no evidence of a difference in time to healing of the reference ulcer between groups in an adjusted analysis for log-ulcer area and duration (hazard ratio 0.58, 95% confidence interval 0.18 to 1.85; p = 0.357). One expected, related serious adverse event was recorded for a participant in the aspirin group. Limitations The trial under-recruited because many patients did not meet the eligibility criteria. Conclusions There was no evidence that aspirin was efficacious in hastening the healing of chronic VLUs. It can be concluded that a larger Phase III (effectiveness) trial would not be feasible. Trial registration Clinical Trials.gov NCT02333123; European Clinical Trials Database (EudraCT) 2014-003979-39. Funding This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 22, No. 55. See the NIHR Journals Library website for further project information

BOB CAT: a Large-Scale Review and Delphi Consensus for Management of Barrett’s Esophagus With No Dysplasia, Indefinite for, or Low-Grade Dysplasia

OBJECTIVES: Barrett’s esophagus (BE) is a common premalignant lesion for which surveillance is recommended. This strategy is limited by considerable variations in clinical practice. We conducted an international, multidisciplinary, systematic search and evidence-based review of BE and provided consensus recommendations for clinical use in patients with nondysplastic, indefinite, and low-grade dysplasia (LGD). METHODS: We defined the scope, proposed statements, and searched electronic databases, yielding 20,558 publications that were screened, selected online, and formed the evidence base. We used a Delphi consensus process, with an 80% agreement threshold, using GRADE (Grading of Recommendations Assessment, Development and Evaluation) to categorize the quality of evidence and strength of recommendations. RESULTS: In total, 80% of respondents agreed with 55 of 127 statements in the final voting rounds. Population endoscopic screening is not recommended and screening should target only very high-risk cases of males aged over 60 years with chronic uncontrolled reflux. A new international definition of BE was agreed upon. For any degree of dysplasia, at least two specialist gastrointestinal (GI) pathologists are required. Risk factors for cancer include male gender, length of BE, and central obesity. Endoscopic resection should be used for visible, nodular areas. Surveillance is not recommended for <5 years of life expectancy. Management strategies for indefinite dysplasia (IND) and LGD were identified, including a de-escalation strategy for lower-risk patients and escalation to intervention with follow-up for higher-risk patients. CONCLUSIONS: In this uniquely large consensus process in gastroenterology, we made key clinical recommendations for the escalation/de-escalation of BE in clinical practice. We made strong recommendations for the prioritization of future research

Metabolic Deficiences Revealed in the Biotechnologically Important Model Bacterium Escherichia coli BL21(DE3)

The Escherichia coli B strain BL21(DE3) has had a profound impact on biotechnology through its use in the production of recombinant proteins. Little is understood, however, regarding the physiology of this important E. coli strain. We show here that BL21(DE3) totally lacks activity of the four [NiFe]-hydrogenases, the three molybdenum- and selenium-containing formate dehydrogenases and molybdenum-dependent nitrate reductase. Nevertheless, all of the structural genes necessary for the synthesis of the respective anaerobic metalloenzymes are present in the genome. However, the genes encoding the high-affinity molybdate transport system and the molybdenum-responsive transcriptional regulator ModE are absent from the genome. Moreover, BL21(DE3) has a nonsense mutation in the gene encoding the global oxygen-responsive transcriptional regulator FNR. The activities of the two hydrogen-oxidizing hydrogenases, therefore, could be restored to BL21(DE3) by supplementing the growth medium with high concentrations of Ni2+ (Ni2+-transport is FNR-dependent) or by introducing a wild-type copy of the fnr gene. Only combined addition of plasmid-encoded fnr and high concentrations of MoO42− ions could restore hydrogen production to BL21(DE3); however, to only 25–30% of a K-12 wildtype. We could show that limited hydrogen production from the enzyme complex responsible for formate-dependent hydrogen evolution was due solely to reduced activity of the formate dehydrogenase (FDH-H), not the hydrogenase component. The activity of the FNR-dependent formate dehydrogenase, FDH-N, could not be restored, even when the fnr gene and MoO42− were supplied; however, nitrate reductase activity could be recovered by combined addition of MoO42− and the fnr gene. This suggested that a further component specific for biosynthesis or activity of formate dehydrogenases H and N was missing. Re-introduction of the gene encoding ModE could only partially restore the activities of both enzymes. Taken together these results demonstrate that BL21(DE3) has major defects in anaerobic metabolism, metal ion transport and metalloprotein biosynthesis

Dystropathology increases energy expenditure and protein turnover in the mdx mouse model of Duchenne muscular dystrophy

The skeletal muscles in Duchenne muscular dystrophy and the mdx mouse model lack functional dystrophin and undergo repeated bouts of necrosis, regeneration, and growth. These processes have a high metabolic cost. However, the consequences for whole body energy and protein metabolism, and on the dietary requirements for these macronutrients at different stages of the disease, are not well-understood. This study used juvenile (4- to 5- wk-old) and adult (12- to 14-wk-old) male dystrophic C57BL/10ScSn-mdx/J and age-matched C57BL/10ScSn/J control male mice to measure total and resting energy expenditure, food intake, spontaneous activity, body composition, whole body protein turnover, and muscle protein synthesis rates. In juvenile mdx mice that have extensive muscle damage, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were higher than in age-matched controls. Adaptations in food intake and decreased activity were insufficient to meet the increased energy and protein needs of juvenile mdx mice and resulted in stunted growth. In (non-growing) adult mdx mice with less severe dystropathology, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were also higher than in age-matched controls. Food intake was sufficient to meet their protein and energy needs, but insufficient to result in fat deposition. These data show that dystropathology impacts the protein and energy needs of mdx mice and that tailored dietary interventions are necessary to redress this imbalance. If not met, the resultant imbalance blunts growth, and may limit the benefits of therapies designed to protect and repair dystrophic muscles