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The present study aimed firstly to use a set of crude oil samples and a dataset to provide new evidence for source input contribution in selected aromatic isomers for discrimination of oils from three oilfields from Tarim Basin and identify the key factor (s) controlling the isotope composition. Thus, the present research showed that the δ13C values of alkylnaphthalenes and alkylphenanthrenes plotted against P/DBT and Ga/C30H ratios is a reliable and convenient tool for discrimination of organic matter variations in different oilfields. More importantly, molecular ratios and different diagram plots revealed that the selected oil samples would be derived from a mixing of indigenous organic matter from the terrestrial (in Kuqa area) and marine (in the cratonic area) depositional environments prior the apparition of the Yakela Faulted Uplift. Thus, Daolaoba, Yakela, and Tahe oils were made up of organic materials from both marine and terrestrial sources. Furthermore, marine organic matter input dominates oils from the Tahe and Yakela, with a minor input from terrestrial sources. The oils from Daolaoba were assigned to be from a mixing of marine and terrestrial material inputs. The controlling factors assessment revealed that biodegradation has an insignificant effect on the set of oils; however, the source input and the thermal maturity together control the isotopic compositions of individual aromatic isomers from these three oilfields.

This DPR Debate is based on the contribution by Justin Lin, Chief Economist at the World Bank, and his colleague Célestin Monga, on 'Growth Identification and Facilitation: The Role of the State in the Dynamics of Structural Change'. The article under consideration is important and timely as it articulates a number of new policy implications from Justin Lin's earlier work on New Structural Economics, which was discussed in a previous DPR debate (Lin and Chang, 2009). This symposium contains the article and comments on it from five distinguished specialists, and closes with a rejoinder by Lin and Monga. This introduction discusses the article, the comments and the rejoinder. The historical record indicates that, in all successful economies, the state has always played an important role in facilitating structural change and helping the private sector sustain it across time. This article puts forward a new approach to help policy-makers in developing countries identify those industries that may hold latent comparative advantage, and recommends ways of removing binding constraints to facilitate private firms' entry into those industries. Two types of government interventions are distinguished: first, policies that facilitate structural change by overcoming information, co-ordination and externality issues, which are intrinsic to industrial upgrading and diversification; and second, policies aimed at protecting certain selected firms and industries that defy the comparative advantage determined by the existing endowment structure. [ABSTRACT FROM AUTHOR]

The authors present asymmetric turbo hybrid automatic-repeat-request (ATH-ARQ) schemes that employ component code selection and asymmetric rate compatible punctured turbo codes (ARCPT) for enhanced performance in fading channels. The paper also presents a novel low-rate ARCPT encoder structure. An analysis is presented for efficient ARCPT puncture schemes for improved throughput performance at low and high SNR values. It is shown that in certain instances, the use of conventional turbo code structures results in the best performance. [ABSTRACT FROM AUTHOR]

The C57B1/6 susceptible (Begs) and its resistant (Begr) congenic mouse, previously developed by retrogressive backcrossing, were infected with 1 × 106 colony-forming units (CFU) of Mycobacterium avium and bacterial growth and their immune responses during the early and prolonged periods of infection were examined. There was a high proliferation in the liver and spleen of Begr mice, whereas no proliferation was observed in the Begr mice. Similarly, the sizes and weights of these organs were much higher than those of their Begr counterparts. The size and number of granulomas in Begs were also found to be higher than those of Begr. The CD3+ and CD4+ subsets increased dramatically in both mice during the early stage of infection. However, in the later phase of the infection, these populations decreased dramatically in Beg' mice, but not in Begr mice, resulting in a depression in cell-mediated immune responses. No significant decrease in cell mediated immune responses was observed in Begr mice even after prolonged infection. ELISA was performed to determine the antibody levels in both mice, and it was found that serum IgG and IgM levels in Begs were comparatively higher than those in Begr mice throughout the period of infection. The Beg gene therefore may have an important role in the maintenance of resistance not only in the early phase but also in the later phase of Myco.avium infection. [ABSTRACT FROM AUTHOR]

Blood-bearing medical devices are essential for the delivery of critical care medicine and are often required to function for weeks to months. However, thrombus formation on their surfaces can lead to reduced device function and failure and expose patients to systemic thrombosis risks. While clinical anticoagulants reduce device related thrombosis, they also increase patient bleeding risk. The root cause of device thrombosis and inflammation is protein adsorption on the biomaterial surfaces of these devices. Protein adsorption activates the coagulation cascade and complement, and this, in turn, activates platelets and white blood cells. Surface modifications with zwitterionic polymers are particularly effective at reducing protein adsorption as well as conformational changes in proteins due to their hydrophilicity. Multiple coating strategies have been developed using carboxybetaine (CB), sulfobetaine (SB), and 2-methacryloyloxyethyl phosphorylcholine (MPC) zwitterionic polymers applied to the metals and hydrophobic polymers that make up the bulk of blood-bearing medical devices. These coatings have been highly successful at creating large reductions in protein adsorption and platelet adhesion during studies on the order of hours on flat surfaces and at reducing thrombus formation for up to a few days in full medical devices. Future work needs to focus on their ability to limit inflammation, particularly during hemodialysis, and in providing anticoagulation on the order of weeks, particularly in artificial lungs.

The artificial lung has provided life-saving support for pulmonary disease patients and recently afforded patients with severe cases of COVID-19 better prognostic outcomes. While it addresses a critical medical need, reducing the risk of clotting inside the device remains challenging. Herein, a two-step surface coating process of the lung circuit using Zwitterionic polysulfobetaine methacrylate is evaluated for its nonspecific protein antifouling activity. It is hypothesized that similarly applied coatings on materials integrated (IT) or nonintegrated (NIT) into the circuit will yield similar antifouling activity. The effects of human plasma preconditioned with nitric oxide-loaded liposome on platelet (plt) fouling are also evaluated. Fibrinogen antifouling activities in coated fibers are similar in the IT and NIT groups. It however decreases in coated polycarbonate (PC) in the IT group. Also, plt antifouling activity in coated fibers is similar in the IT and NIT groups and is lower in coated PC and Tygon in the IT group compared to the NIT group. Coating process optimization in the IT lung circuit may help address difference in the coating appearance of outer and inner fiber bundle fibers, and the NO-liposome significantly reduces (86%) plt fouling on fibers indicating its potential use for blood anticoagulation., (© 2023 Wiley-VCH GmbH.)

In IgE-mediated food allergies, exposure to the allergen activates systemic allergic responses. Oral immunotherapy (OIT) treats food allergies through incremental increases in oral allergen exposure. However, OIT only induces sustained clinical tolerance and decreased basophil sensitivity in a subset of individuals despite increases in circulating allergen-specific IgG in all treated individuals. Therefore, we examined the allergen-specific antibodies from 2 OIT cohorts of patients with sustained and transient responses. Here, we compared antibodies from individuals with sustained or transient responses and discovered specific tolerance-associated conformational epitopes of the immunodominant allergen Ara h 2 recognized by neutralizing antibodies. First, we identified what we believe to be previously unknown conformational, intrahelical epitopes using x-ray crystallography with recombinant antibodies. We then identified epitopes only recognized in sustained tolerance. Finally, antibodies recognizing tolerance-associated epitopes effectively neutralized allergen to suppress IgE-mediated effector cell activation. Our results demonstrate the molecular basis of antibody-mediated protection in IgE-mediated food allergy, by defining how these antibodies disrupt IgE-allergen interactions to prevent allergic reactions. Our approach to studying the structural and functional basis for neutralizing antibodies demonstrates the clinical relevance of specific antibody clones in antibody-mediated tolerance. We anticipate that our findings will form the foundation for treatments of peanut allergy using neutralizing antibodies and hypoallergens.

Mucus layers (McLs) are on the front line of the human defense system that protect us from foreign abiotic/biotic particles (e.g., airborne virus SARS-CoV-2) and lubricates our organs. Recently, the impact of McLs on human health (e.g., nutrient absorption and drug delivery) and diseases (e.g., infections and cancers) has been studied extensively, yet their mechanisms are still not fully understood due to their high variety among organs and individuals. We characterize these variances as the heterogeneity of McLs, which lies in the thickness, composition, and physiology, making the systematic research on the roles of McLs in human health and diseases very challenging. To advance mucosal organoids and develop effective drug delivery systems, a comprehensive understanding of McLs' heterogeneity and how it impacts mucus physiology is urgently needed. When the role of airway mucus in the penetration and transmission of coronavirus (CoV) is considered, this understanding may also enable a better explanation and prediction of the CoV's behavior. Hence, in this Review, we summarize the variances of McLs among organs, health conditions, and experimental settings as well as recent advances in experimental measurements, data analysis, and model development for simulations.

Biopolymers are widely accepted natural materials in regenerative medicine, and further development of their bioactivities and discoveries on their composition/function relationships could greatly advance the field. However, a concise insight on commonly investigated biopolymers, their current applications and outlook of their modifications for multibioactivity are scarce. This review bridges this gap for professionals and especially freshmen in the field who are also interested in modification methods not yet in commercial use. A series of polymeric materials in research and development uses are presented as well as challenges that limit their efficacy in tissue regeneration are discussed. Finally, their roles in the regeneration of select tissues including the skin, bone, cartilage, and tendon are highlighted along with modifiable biopolymer moieties for different bioactivities., Competing Interests: The authors declare no conflict of interest.

Campylobacter fetus is currently classified into three main subspecies, but only two of these, C. fetus subspecies fetus and C. fetus subsp. v enerealis originate principally from ruminants where they inhabit different niches and cause distinct pathogenicity. Their importance as pathogens in international trade and reporting is also different yet the criteria defining these properties have never been fully substantiated nor understood. The situation is further compromised because the ability to differentiate between these two closely related C. fetus subspecies has traditionally been performed by phenotypic characterisation of isolates, methods which are limited in scope, time-consuming, tedious, and often yield inconsistent results, thereby leading to isolate misidentification. The development of robust genetic markers that could enable rapid discrimination between C. fetus subsp. fetus and subsp. venerealis has also been challenging due to limited differences in the gene complement of their genomes, high levels of sequence repetition, the small number of closed genome sequences available and the lack of standardisation of the discriminatory biochemical tests employed for comparative purposes. To yield a better understanding of the genomic differences that define these C. fetus strains, seven isolates were exhaustively characterised phenotypically and genetically and compared with seven previously well characterised isolates. Analysis of these 14 C. fetus samples clearly illustrated that adaption by C. fetus subsp. venerealis to the bovine reproductive tract correlated with increasing genome length and plasticity due to the acquisition and propagation of several mobile elements including prophages, transposons and plasmids harbouring virulence factors. Significant differences in the repertoire of the CRISPR (clustered regularly interspersed short palindromic repeats)- cas system of all C. fetus strains was also found. We therefore suggest that a deficiency in this adaptive immune system may have permitted the emergence of extensive genome plasticity and led to changes in host tropism through gene disruption and/or changes in gene expression. Notable differences in the sub-species complement of DNA adenine methylase genes may also have an impact. These data will facilitate future studies to better understand the precise genetic differences that underlie the phenotypic and virulence differences between these animal pathogens and may identify additional markers useful for diagnosis and sub-typing., Competing Interests: The authors declare that they have no competing interests.

Most amyotrophic lateral sclerosis (ALS) cases are considered sporadic, without a known genetic basis, and environmental exposures are thought to play a causal role. To learn more about sporadic ALS etiology, we recruited n = 188 ALS patients from northern New England and Ohio and matched controls 2:1 from the general population of the same regions. Questionnaires evaluated the association between a variety of lifestyle, behavioral (ie, hobbies and activities), and occupational factors and the risk of ALS, including the duration of time between exposure and ALS onset, and exposure frequency. Head trauma was associated with increased ALS risk (adjusted odds ratio [OR] 1.60 95% confidence interval [CI] 1.04-2.45), with significantly greater effects for injuries occurring 10 or more years prior to symptom onset (P = .037). ALS risk was increased for those reporting severe electrical burns (adjusted OR 2.86, 95% CI 1.37-6.03), with odds ratios highest for burns after age 30 (OR 3.14), and for burns 10 or more years prior to symptom onset (OR 3.09). Hobbies involving lead were the most strongly associated with ALS risk (adjusted OR 2.92, 95% CI 1.45-5.91). Exposures to lead 20 or more years prior to diagnosis had larger effect sizes compared to those occurring more recently. Holding a job in mechanics, painting, or construction was associated with ALS. The identification of these specific environmental factors associated with ALS highlight the need for future prospective and laboratory studies to assess causality, biological mechanisms, and find prevention or treatment opportunities., (© 2020 The Authors. Muscle & Nerve published by Wiley Periodicals LLC.)

Cyclotrons used in nuclear medicine imaging accelerate protons, deuterons, and helium ions to bombard a target, which produces nuclear reactions that generate positron-emitting radionuclides. Secondary neutrons are nonuniformly emitted in these reactions and induce heterogeneous activation of the cyclotron components and concrete vault enclosure. This poses radioactive waste management complications when decommissioning a cyclotron facility, since the objective is to ensure that exposures are within regulatory limits and as low as reasonably achievable (ALARA). The McGovern Medical School in The University of Texas Health Science Center in Houston housed a Scanditronix MC40 cyclotron that produced short-lived radioisotopes for Positron Emission Tomography (PET) imaging from 1984 to 2001 until Tropical Storm Allison rendered it inoperable. The purpose of this study was to provide underrepresented Science, Technology, Engineering and Mathematics (STEM) students an ALARA experience with a practical problem encountered in the radiation safety profession. Gamma dose rate measurements were performed with both a Mirion InSpector 1000 spectrometer and Fluke 451P survey meter in the vault at locations identified as hotspots based on preliminary scoping surveys with the Ludlum model 44-9 detector. However, gamma spectra were measured with the spectrometer exclusively at hotspots along the west wall. Results indicated the maximum gamma dose rate of 129 ± 31 nSv h was about 2 times background near the central beam transport line of the now inoperable cyclotron. Furthermore, gamma emission peaks were identified in the spectra from trace amounts of Co and Eu in the vault's concrete walls.

The aim of the current study was to investigate the effects of mineral trioxide aggregate (MTA) on the proliferation and differentiation of human adipose-derived mesenchymal stem cells (Ad-MSCs) as a surrogate cell source in futuristic stem-cell-based endodontic therapies. Human Ad-MSCs and mesenchymal stem cells derived from bone marrow (BM-MSCs) were isolated from liposuction waste adipose tissue and femur, respectively, and the effects of MTA-conditioned media on their viability, mineralization potential, and osteo/odontogenic differentiation capacity were subsequently evaluated. Alkaline phosphatase (ALP) activity, quantitative alizarin red S staining, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses were performed to investigate and compare the osteo/odontogenic induction potential of MTA on the Ad/BM-MSCs. The results of cytotoxicity assay revealed that at different concentrations, MTA-conditioned medium was not only biocompatible toward both cell types, but also capable of promoting cell proliferation. ALP activity assay showed that 0.2 mg/mL was the optimal concentration of MTA-conditioned medium for osteo/odontogenic induction in Ad/BM-MSCs. The expression of osteo/odontogenic gene markers was increased in Ad/BM-MSCs treated with 0.2 mg/mL MTA-conditioned media. Our results indicated that MTA can efficiently enhance the osteo/odontogenic potential of Ad-MSCs, and thus they can be considered as a better cell source for dentin-pulp complex regeneration. However, further investigations are required to test these potentials in animal models.

Antimicrobial resistance (AMR) has important implications for the continued use of antibiotics to control infectious diseases in both beef cattle and humans. AMR along the One Health continuum of the beef production system is largely unknown. Here, whole genomes of presumptive extended-spectrum β-lactamase E. coli (ESBL-EC) from cattle feces ( n = 40), feedlot catch basins ( n = 42), surrounding streams ( n = 21), a beef processing plant ( n = 4), municipal sewage ( n = 30), and clinical patients ( n = 25) are described. ESBL-EC were isolated from ceftriaxone selective plates and subcultured on ampicillin selective plates. Agreement of genotype-phenotype prediction of AMR ranged from 93.2% for ampicillin to 100% for neomycin, trimethoprim/sulfamethoxazole, and enrofloxacin resistance. Overall, β-lactam (100%; bla EC , bla TEM-1 , bla SHV , bla OXA , bla CTX-M- ), tetracycline (90.1%; tet(A), tet(B) ) and folate synthesis ( sul2 ) antimicrobial resistance genes (ARGs) were most prevalent. The ARGs tet(C), tet(M), tet(32), bla CTX-M-1 , bla CTX-M-14 , bla OXA-1 , dfrA18, dfrA19, catB3 , and catB4 were exclusive to human sources, while bla TEM-150 , bla SHV-11-12 , dfrA12, cmlA1 , and cmlA5 were exclusive to beef cattle sources. Frequently encountered virulence factors across all sources included adhesion and type II and III secretion systems, while IncFIB(AP001918) and IncFII plasmids were also common. Specificity and prevalence of ARGs between cattle-sourced and human-sourced presumptive ESBL-EC likely reflect differences in antimicrobial use in cattle and humans. Comparative genomics revealed phylogenetically distinct clusters for isolates from human vs. cattle sources, implying that human infections caused by ESBL-EC in this region might not originate from beef production sources.

For a One-Health investigation of antimicrobial resistance (AMR) in Enterococcus spp., isolates from humans and beef cattle along with abattoirs, manured fields, natural streams, and wastewater from both urban and cattle feedlot sources were collected over two years. Species identification of Enterococcus revealed distinct associations across the continuum. Of the 8430 isolates collected, Enterococcus faecium and Enterococcus faecalis were the main species in urban wastewater (90%) and clinical human isolates (99%); Enterococcus hirae predominated in cattle (92%) and feedlot catch-basins (60%), whereas natural streams harbored environmental Enterococcus spp. Whole-genome sequencing of E. faecalis (n = 366 isolates) and E. faecium (n = 342 isolates), revealed source clustering of isolates, indicative of distinct adaptation to their respective environments. Phenotypic resistance to tetracyclines and macrolides encoded by tet(M) and erm(B) respectively, was prevalent among Enterococcus spp. regardless of source. For E. faecium from cattle, resistance to β-lactams and quinolones was observed among 3% and 8% of isolates respectively, compared to 76% and 70% of human clinical isolates. Clinical vancomycin-resistant E. faecium exhibited high rates of multi-drug resistance, with resistance to all β-lactam, macrolides, and quinolones tested. Differences in the AMR profiles among isolates reflected antimicrobial use practices in each sector of the One-Health continuum.

The purpose of this study was to develop a novel and robust molecular assay for the detection of human pathogenic yersiniae (i.e. Yersinia enterocolitica, Y. pseudotuberculosis and Y. pestis) in complex food samples. The assay combines multiplexed real-time PCR (qPCR) and Pyrosequencing for detecting and differentiating human pathogenic yersiniae with high confidence through sequence based confirmation. The assay demonstrated 100% specificity and inclusivity when tested against a panel of 14 Y. enterocolitica, 22 Y. pestis, 24 Y. pseudotuberculosis and a diverse selection of 17 other non-Yersinia bacteria. Pyrosequencing reads ranged from 28 to 40bp in length and had 94-100% sequence identity to the correct species in the GenBank nr database. Microbial enrichments of 48 ready-to-eat foods collected in the Greater Toronto Area from March 2014 to May 2014, including 46 fresh sprout and 2 salad products, were then tested using the assay. All samples were negative for Y. pestis and Y. pseudotuberculosis. Both salads (n=2) and 35% of sprout products (n=46) including 7.1% of alfalfa sprouts (n=14), 81% of bean sprouts (n=16), 12% of mixed sprouts (n=8) tested positive for Y. enterocolitica which was not detected in broccoli sprouts (n=5), onion sprouts (n=1), and pea sprouts (n=2). Cycle thresholds (Ct) of positive samples for Y. enterocolitica were between 23.0 and 37.9 suggesting post enrichment concentrations of approximately 1×10 2 to 1×10 6 Y. enterocolitica per 1mL of enriched broth. An internal amplification control which was coamplified with targets revealed PCR inhibition in five samples which was resolved following a one in ten dilution. Pyrosequencing of qPCR amplicons suggests monoclonality and revealed a single nucleotide polymorphism that is present in Y. enterocolitica biotype 1A suggesting low pathogenicity of the detected strains. This study is the first to combine Pyrosequencing and qPCR for the detection of human pathogenic yersiniae and is applicable to a broad range of complex samples including ready-to-eat food samples., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Salmonella enterica serovar Typhimurium strains 22495 and 22792, obtained from wild birds, were found to display different virulence attributes in an experimental chicken model. Closed genome sequences were assembled after sequencing with the Roche 454 and Illumina MiSeq platforms. An additional plasmid was present in the more virulent strain 22495., (© Crown copyright 2017.)

Clinical outcomes of Shiga toxin (stx)-producing Escherichia coli infection are largely determined by virulence gene subtypes. This study used a polymerase chain reaction (PCR)-pyrosequencing assay to analyze single-nucleotide polymorphisms for subtyping three major virulence genes (stx 1 , stx 2 , eae) of pathogenic E. coli (O157, O26, O111, and O103) isolated from cattle over a 2-year interval (n = 465) and human clinical cases (n = 42) in western Canada. Most bovine isolates were PCR positive for at least one target virulence gene (367/465), whereas 100% of human isolates harbored eae in combination with at least one stx gene. Four Shiga toxin (1a, 2a, 2c, and 2e) and four eae (λ/γ1-eae, ɛ-eae, θ/γ2-eae, and β-eae) subtypes were identified in over 25 distinct virulence genotypes. Among cattle isolates, every serogroup, but O103, presented a dominant genotype (O157: stx 1a +stx 2a +λ/γ1-eae, O26: β-eae alone, and O111: stx 1a +θ/γ2-eae). Similar patterns were found in human isolates, although it was not possible to establish a clear genotypic association between the two sources. Many O157 and non-O157 cattle isolates lacked stx genes; the absence was greater in non-O157 (75/258) and O157:non-H7 (19/40) than in O157:H7 strains (1/164). In addition, there was a greater diversity of virulence genotypes of E. coli isolated from cattle than those of human diseases, which could be due to sample characteristics (e.g., source and clinical condition). However, the majority of cattle strains had virulence profiles identical to those of clinical cases. Consequently, determining the presence of certain stx (stx 1a and stx 2a ) and eae (λ/γ1-eae) subtypes known to cause human disease would be a valuable tool for risk assessment and prediction of disease outcome along the farm-to-fork continuum.

The determination of Shiga toxin (ST) subtypes can be an important element in the risk characterization of foodborne ST-producing Escherichia coli (STEC) isolates for making risk management decisions. ST subtyping methods include PCR techniques based on electrophoretic or pyrosequencing analysis of amplicons and in silico techniques based on whole genome sequence analysis using algorithms that can be readily incorporated into bioinformatics analysis pipelines for characterization of isolates by their genetic composition. The choice of technique will depend on the performance characteristics of the method and an individual laboratory's access to specialized equipment or personnel. We developed two whole genome sequence-based ST subtyping tools: (i) an in silico PCR algorithm requiring genome assembly to replicate a reference PCR-based method developed by the Statens Serum Institut (SSI) and (ii) an assembly-independent routine in which raw sequencing results are mapped to a database of known ST subtype sequence variants (V-Typer). These tools were evaluated alongside the SSI reference PCR method and a recently described PCR-based pyrosequencing technique. The V-Typer method results corresponded closely with the reference method in the analysis of 67 STEC cultures obtained from a World Health Organization National Reference Laboratory. In contrast, the in silico PCR method failed to detect ST subtypes in several cases, a result which we attribute to assembly-induced errors typically encountered with repetitive gene sequences. The V-Typer can be readily integrated into bioinformatics protocols used in the identification and characterization of foodborne STEC isolates.

Background: There is a need to characterize genomes of the foodborne pathogen, Salmonella enterica serovar Enteritidis (SE) and identify genetic information that could be ultimately deployed for differentiating strains of the organism, a need that is yet to be addressed mainly because of the high degree of clonality of the organism. In an effort to achieve the first characterization of the genomes of SE of Canadian origin, we carried out massively parallel sequencing of the nucleotide sequence of 11 SE isolates obtained from poultry production environments (n = 9), a clam and a chicken, assembled finished genomes and investigated diversity of the SE genome., Results: The median genome size was 4,678,683 bp. A total of 4,833 chromosomal genes defined the pan genome of our field SE isolates consisting of 4,600 genes present in all the genomes, i.e., core genome, and 233 genes absent in at least one genome (accessory genome). Genome diversity was demonstrable by the presence of 1,360 loci showing single nucleotide polymorphism (SNP) in the core genome which was used to portray the genetic distances by means of a phylogenetic tree for the SE isolates. The accessory genome consisted mostly of previously identified SE prophage sequences as well as two, apparently full-sized, novel prophages namely a 28 kb sequence provisionally designated as SE-OLF-10058 (3) prophage and a 43 kb sequence provisionally designated as SE-OLF-10012 prophage., Conclusions: The number of SNPs identified in the relatively large core genome of SE is a reflection of substantial diversity that could be exploited for strain differentiation as shown by the development of an informative phylogenetic tree. Prophage sequences can also be exploited for SE strain differentiation and lineage tracking. This work has laid the ground work for further studies to develop a readily adoptable laboratory test for the subtyping of SE.

The roles of yadA, invA, and psaA genes introduced into the genetic background of the Y. pseudotuberculosis strain possessing the large p VM82 plasmid in virulence and invasion capacity were studied. Isogenic single mutants as well as double and multiple mutants of these genes were constructed and used. LD50 was used as a measure of virulence and the estimation of the ability to invade mammalian cells and the effect of infection on the weight changes of infected mice were used as additional indicators of pathogenicity. It was shown that the YadA had a major effect on the bacterial virulence when compared with the effects of PsaA and InvA. InvA appears to mediate the main pathway of the cellular invasion. YadA is responsible for the weight loss after infection of mice with sublethal doses of Y. pseudotuberculosis. The effects of YadA on virulence and of InvA on bacterial invasion were independent of the expression of the other genes studied. To our knowledge, this study showed for the first time the direct involvement of YadA in the virulence of Y. pseudotuberculosis in mice. Further pathomorphological studies are required to reveal the differences in the pathogenesis of pseudotuberculosis caused by yadA mutants or yadA+ bacteria of Y. pseudotuberculosis.

A one-step multiplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using SmartCycler technology and TaqMan probes was developed for detection and typing of bovine viral diarrhea viruses (BVDV). Common primers and type-specific (BVDV1 and BVDV2) TaqMan probes were designed in the 5'-untranslated region of the viral genome. The real-time assay was able to detect 10-100 TCID50 of virus, with correlation coefficient (r2) values of 0.998 and 0.999 for BVDV1 and BVDV2, respectively. The assay accurately typed 54 BVDV strains and field isolates and specificity of the TaqMan probes was further demonstrated by the lack of reactivity with the closely related Pestiviruses, classical swine fever virus and border disease virus. The assay was also shown to have high reproducibility. When the assay was compared with virus isolation for bovine serum samples, there was full agreement between the tests. Thus, the one-step real-time RT-PCR assay appears to be a rapid, sensitive, and specific test for detection and typing of BVDV.

We examined two variants of the genome-sequenced strain, Campylobacter jejuni NCTC11168, which show marked differences in their virulence properties including colonization of poultry, invasion of Caco-2 cells, and motility. Transcript profiles obtained from whole genome DNA microarrays and proteome analyses demonstrated that these differences are reflected in late flagellar structural components and in virulence factors including those involved in flagellar glycosylation and cytolethal distending toxin production. We identified putative sigma(28) and sigma(54) promoters for many of the affected genes and found that greater differences in expression were observed for sigma(28)-controlled genes. Inactivation of the gene encoding sigma(28), fliA, resulted in an unexpected increase in transcripts with sigma(54) promoters, as well as decreased transcription of sigma(28)-regulated genes. This was unlike the transcription profile observed for the attenuated C. jejuni variant, suggesting that the reduced virulence of this organism was not entirely due to impaired function of sigma(28). However, inactivation of flhA, an important component of the flagellar export apparatus, resulted in expression patterns similar to that of the attenuated variant. These findings indicate that the flagellar regulatory system plays an important role in campylobacter pathogenesis and that flhA is a key element involved in the coordinate regulation of late flagellar genes and of virulence factors in C. jejuni.

The endotoxins from two recently-classified subspecies of Fusobacterium, namely F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, were compared. Chemical analysis of the isolated endotoxins revealed that they were clearly different. Distinct levels of polysaccharides were demonstrated. The endotoxins isolated were devoid of heptose and 3-deoxy-D-manno-octulosonate (KDO). The endotoxins of F. n. necrophorum and F. n. funduliforme contained lipid A in a ratio of 4:1 which may account for the variations in their virulence.

An attempt was made to determine the receptor for the hemolysin of Fusobacterium necrophorum using horse erythrocyte or its membranes as target. The spectrum of erythrocyte sensitivity has indicated that horse, dog and mouse erythrocytes are highly sensitive whereas cattle, sheep, goat and chicken red blood cells are insensitive to this hemolysin. A high correlation between sensitivity and phosphatidylcholine content of the erythrocyte membranes was noted. Binding of hemolysin to horse erythrocyte membranes was reduced significantly by prior treatment of membranes with phospholipase A2 but not with phospholipase C. Pretreatment of erythrocyte membranes with pronase, proteinase K, trypsin or neuraminidase did not alter binding of hemolysin to the membranes, suggesting that protein or sialyl residues are not involved as receptors. Gas liquid chromatography analysis showed that the fatty acid profile from hydrolysis of bovine liver phosphatidylcholine by hemolysin and phospholipase A2 were similar. In conclusion, this report presents evidence that phosphatidylcholine may be acting as a possible receptor for the hemolysin of F. necrophorum.

To determine the role of the Bcg gene in the resistance and susceptibility of BCG-infected C57BL/6 (Bcg(s)) and its Bcg(r) congenic mice, the antigen presenting ability of spleen adherent cells and peritoneal macrophages, delayed type hypersensitivity (DTH) and lymphocyte blastogenic responses were investigated. The results obtained indicate that the DTH and lymphocyte blastogenic responses in Bcg(r) congenic mice were higher than in the Bcg(s) mice. Stimulation of spleen adherent cells with live Mycobacterium bovis BCG or PPD-BCG resulted in a higher antigen presenting ability in Bcg(r) than in Bcg(s) mice. However, comparatively low responses were associated with M. avium stimulation, with those in Bcg(r) being higher than in Bcg(s). I-A expression was also comparatively higher in Bcg(r) than in Bcg(s) mice. This study demonstrates that the Bcg gene seems to exhibit some effect on the antigen presenting ability of macrophages in immune responses of the congenic mice.

The interactions between the hemolysin of Fusobacterium necrophorum subsp. necrophorum, erythrocytes and erythrocyte membranes were studied as an attempt to determine the initial characteristics leading to hemolysis. The spectrum of erythrocyte sensitivity indicated that horse, dog and mouse erythrocytes were highly sensitive whereas those of cattle, sheep, goat and chicken were insensitive to the hemolysin. Binding of hemolysin to horse and dog erythrocytes or their ghosts was more pronounced than to those of cattle and sheep as detected by a decrease of hemolytic activity from hemolysin preparations. The kinetics of hemolysis revealed that lysis is preceded by a prelytic phase characterized by binding of hemolysin to erythrocytes. Treatment of horse erythrocytes with hemolysin at various temperatures prior to incubation at 37 degrees C also revealed that this binding prelytic phase is temperature independent. This was followed by a temperature dependent lytic stage since erythrocytes pretreated with hemolysin and incubated at 4 degrees C showed no hemolysis. An inverse relation was found between erythrocyte concentration and hemolytic activity suggesting a multiple-hit mechanism of hemolysis.

The effect of various agents as enhancers or inhibitors of hemolysin secretion by Fusobacterium necrophorum was investigated. The hemolysin secreted in phosphate buffered saline (PBS) alone was inactivated shortly after secretion. Tween-80 or albumin preserved the hemolytic activity in PBS in which cultures of F. necrophorum had been suspended. Hemoglobin was found to enhance hemolysin secretion. However, higher concentrations diminished secretion. Chloramphenicol, an inhibitor of protein synthesis, exhibited no effect on the hemolytic activity. However, the addition of sodium azide, an energy metabolic inhibitor, significantly reduced the hemolytic activity. Lower temperatures and pH above 9 and below 6 all yielded a low hemolytic activity. Cells suspended in Tween-80 prior to sonication yielded a substantial amount of extracellular hemolytic activity with low intracellular activity detected. However, cells suspended in PBS alone yielded a low extracellular activity but rather a high intracellular activity. The same spectrum of red blood cells of different species were found to be sensitive to both the extracellular and intracellular hemolysins.

The stability and stabilization of the hemolytic activity of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme were monitored over a period of four weeks using culture supernatants. The hemolytic activity was completely lost after one week at room temperature and 37 degrees C. After a two-week storage at 4 degrees C and -80 degrees C only trace activity was detected with -80 degrees C being the better of the two conditions. The addition of cysteine monohydrochloride, bovine serum albumin or Tween 80 as stabilizers, however, led to the detection of a considerable amount of the hemolytic activity in the sample stored at 4 degrees C and - 80 degrees C throughout the period investigated. The hemolytic activity appeared to be more stable in the presence of Tween 80 at -80 degrees C. Cysteine monohydrochloride was found to crystallize at - 80 degrees C and was therefore ineffective as a stabilizer at this temperature. Hemoglobin was also ineffective as a stabilizer.

Bcg congenic mice were developed by using C57BL/6 and DBA/2 strains of mice as progenitors. They were obtained by introgressively backcrossing the Bcgr marker of DBA/2 onto C57BL/6. After twenty successive backcrossings, the heterozygous resistant mice were mated with each other to obtain homozygous mice as the Bcgr congenic mice. The results of immunogenic and genetic markers coupled with those of an mixed lymphocyte reaction, all confirmed that the newly developed mice were highly congenic. These congenic mice were found to be resistant to in vivo infections by Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium bovis BCG.

The in vitro activity of the hemolysin of Fusobacterium necrophorum was determined using the hemolysis of horse erythrocytes as an assay. The effects of medium composition and pH on hemolysin production were investigated. Calf serum and casitone stimulated a comparatively higher hemolytic activity in F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, respectively. However, sugars, such as glucose, galactose and fructose were inhibitors of hemolytic activity. The spectrum of erythrocyte sensitivity to the hemolysin indicated that horse and quail erythrocytes were more sensitive to the hemolysin of both F. necrophorum subsp. necrophorum and subsp. funduliforme, than were cat, dog, rabbit, pigeon and human erythrocytes. Cat erythrocytes were however insensitive to the hemolysin of subsp. funduliforme. Cattle, sheep and chicken erythrocytes were insensitive to the hemolysin of the two subspecies. Medium pH near neutral were more effective in enhancing hemolytic activity, and hemolytic activity was positively correlated with growth. In general, F. necrophorum subsp. necrophorum was more hemolytic than subsp. funduliforme.

A total of 10 strains each of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme were tested for the production of 13 extracellular enzymes. DNase, alkaline phosphatase, and lipase were predominantly associated with all the strains of F. necrophorum subsp. necrophorum, with DNase not detected in any of the strains of F. necrophorum subsp. funduliforme. In addition, the strains of F. necrophorum subsp. necrophorum were generally more hemolytic than those of F. necrophorum subsp. funduliforme. Lecithinase, beta-lactamase, elastase, hyaluronidase, chondroitin sulfatase, and coagulase were not detected in any of the strains. DNase may be used to differentiate between the two subspecies.

We used isolated rat hearts subjected to coronary artery ligation and reperfusion to study the antiarrhythmic activity of 5-hydroxypropafenone (5OHP) and N-depropylpropafenone (NDPP), major metabolites of propafenone (P) in humans, and of the two enantiomers (R)- and (S)-propafenone. 5OHP suppressed reperfusion arrhythmias similar to the parent drug in a concentration-dependent manner. The concentration of 5OHP needed to prevent ventricular fibrillation in 50% of experiments (EC50) was significantly higher than that of P (0.186 +/- 0.05 vs. 0.153 +/- 0.005 mg/L, mean +/- SEM, p less than 0.05). 5OHP had a relative potency of 80% compared to P. When 5OHP and P were administered together, their antiarrhythmic effect appeared to be supra-additive. The NDPP metabolite showed very little antiarrhythmic potency and was about four times less active than P. The two enantiomers (R) and (S) were equipotent and showed antiarrhythmic activities similar to racemic P.

We investigated the antiarrhythmic activity of two major metabolites of quinidine in human, 3-hydroxyquinidine and quinidine-N-oxide, alone and in combination with the parent drug in an experimental model using reperfusion arrhythmias in an isolated rat heart preparation. No definite pharmacological activity could be shown for quinidine-N-oxide up to concentrations of 16 mg/l. Quinidine and 3-hydroxyquinidine prevented ventricular fibrillation and ventricular tachycardia after coronary reperfusion in a concentration-dependent manner. The relationship between the drug concentration in the perfusate and the fractional suppression of arrhythmia could be described adequately for both compounds by the Hill equation. Whereas no difference was found for the Hill coefficient, the estimates of the concentration associated with 50% arrhythmia suppression was significantly higher for 3-hydroxyquinidine (10.7 +/- 0.3 mg/l vs. 2.2 +/- 0.25 mg/l), indicating that the relative potency of the metabolite was only about 20% compared to the parent compound. To investigate the pharmacodynamic interaction of the two compounds the concentration-response curve was determined for quinidine also in the presence of 3-hydroxyquinidine at a constant concentration of 4 mg/l. A method has been derived that allows quantitative assessment of the pharmacodynamic interaction of two compounds for which the concentration-effect relationship can be described by the Hill equation. The results indicate that the antiarrhythmic effects of 3-hydroxyquinidine and quinidine are additive.