Phage Therapy To Reduce Preprocessing Salmonella Infections in Market-Weight Swine

Contamination of meat products with food-borne pathogens usually results from the carcass coming in contact with the feces of an infected animal during processing. In the case of Salmonella, pigs can become colonized with the organism during transport and lairage from contaminated trailers and holding pens, resulting in increased pathogen shedding just prior to processing. Increased shedding, in turn, amplifies the likelihood of carcass contamination by magnifying the amount of bacteria that enters the processing facility. We conducted a series of experiments to test whether phage therapy could limit Salmonella infections at this crucial period. In a preliminary experiment done with small pigs (3 to 4 weeks old; 30 to 40 lb), administration of an anti-Salmonella phage cocktail at the time of inoculation with Salmonella enterica serovar Typhimurium reduced Salmonella colonization by 99.0 to 99.9% (2- to 3-log reduction) in the tonsils, ileum, and cecum. To test the efficacy of phage therapy in a production-like setting, we inoculated four market-weight pigs (in three replicates) with Salmonella enterica serovar Typhimurium and allowed the challenged pigs to contaminate a holding pen for 48 h. Sixteen naïve pigs were randomly split into two groups which received either the anti-Salmonella phage cocktail or a mock treatment. Both groups of pigs were comingled with the challenged pigs in the contaminated pen. Treatment with the anti-Salmonella phage cocktail significantly reduced cecal Salmonella concentrations (95%; P < 0.05) while also reducing (numerically) ileal Salmonella concentrations (90%; P = 0.06). Additional in vitro studies showed that the phage cocktail was also lytic against several non-Typhimurium serovars.The U.S. Centers for Disease Control and Prevention report approximately 40,000 culture-confirmed cases of salmonellosis each year in the United States, which result in approximately 400 deaths (5). Many Salmonella outbreaks are associated with meat and poultry (20), with contamination usually resulting from the carcass coming into contact with the feces of a Salmonella-infected animal during processing (22).There is an association between pork products and Salmonella, as swine are generally considered to be the second largest reservoir of the organism among food animals after poultry. Although infections in adult swine are normally asymptomatic, once colonized, pigs can shed the organism in the feces for weeks and sometimes months (7).While a great deal of research has been done on developing on-farm anti-Salmonella intervention strategies, these methods are confounded by the fact that Salmonella prevalence in pigs often increases once the animals leave the farm as a result of (i) stress-induced reactivation of preexisting infections (14), (ii) new infections from contaminated transport trailers and processing facility holding pens (12, 15, 24, 31), or (iii) both. Consequently, animals with no history of previous Salmonella infection can begin shedding the organism just prior to processing, which is highly problematic in terms of food safety.We hypothesized that phage therapy could be developed as an effective means to counteract transport- and lairage-associated increases in Salmonella colonization in swine. Phage therapy has the advantage of being natural, nontoxic, and relatively inexpensive and could be used just prior to slaughter, unlike many antibiotics (18, 28). Here we describe a series of experiments demonstrating that treating market-weight pigs with an anti-Salmonella phage cocktail prior to their comingling with Salmonella-infected pigs in a highly contaminated environment resulted in reductions in Salmonella colonization. We further show that the phage cocktail could be effectively microencapsulated, making feed or water delivery possible.     

Clustered O-Glycans of IgA1: DEFINING MACRO- AND MICROHETEROGENEITY BY USE OF ELECTRON CAPTURE/TRANSFER DISSOCIATION*

IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region (HR) O-glycans, plays a pivotal role in the pathogenesis of the disease. It is not known whether the glycosylation defect occurs randomly or preferentially at specific sites. We have described the utility of activated ion-electron capture dissociation (AI-ECD) mass spectrometric analysis of IgA1 O-glycosylation. However, locating and characterizing the entire range of O-glycan attachment sites are analytically challenging due to the clustered serine and threonine residues in the HR of IgA1 heavy chain. To address this problem, we analyzed all glycoforms of the HR glycopeptides of a Gal-deficient IgA1 myeloma protein, mimicking the aberrant IgA1 in patients with IgAN, by use of a combination of IgA-specific proteases + trypsin and AI-ECD Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS). The IgA-specific proteases provided a variety of IgA1 HR fragments that allowed unambiguous localization of all O-glycosylation sites in the six most abundant glycoforms, including the sites deficient in Gal. Additionally, this protocol was adapted for on-line liquid chromatography (LC)-AI-ECD MS/MS and LC-electron transfer dissociation MS/MS analysis. Our results thus represent a new clinically relevant approach that requires ECD/electron transfer dissociation-type fragmentation to define the molecular events leading to pathogenesis of a chronic kidney disease. Furthermore, this work offers generally applicable principles for the analysis of clustered sites of O-glycosylation.Glycosylation is one of the most common post-translational modifications of proteins. It is estimated that over half of mammalian proteins are glycosylated. Patients with several autoimmune disorders, chronic inflammatory diseases, and some infectious diseases exhibit abnormal glycosylation of serum immunoglobulins and other glycoproteins (1–5). The biological functions of these modifications in health and disease have become a significant area of interest in biomedical research (6). A subset of these glycoproteins has clustered sites of O-glycosylation with serine- and threonine-rich stretches within the amino acid sequence. Mucins, such as membrane-associated MUC1, are perhaps the best known family of proteins that are heavily O-glycosylated. Their altered expression and aberrant glycosylation have made them potential targets as biomarkers for early detection of cancer (7). Immunoglobulin A1 (IgA1)1 contains both O- and N-glycans (Fig. 1). Aberrant O-glycosylation of IgA1 is involved in the pathogenesis of IgA nephropathy (IgAN) and the closely related Henoch-Schönlein purpura nephritis (1, 8). Interestingly, the aberrantly glycosylated molecules, IgA1 in IgAN and MUC1 in cancer, are recognized by the immune system as neoepitopes as evidenced by formation of specific antibodies (9–11). Mucin-like bacterial surface proteins exhibit similar properties: the molecules have clustered bacterial O-glycans that mediate cellular adhesion, and blocking antibodies target these glycan-containing epitopes (12).Open in a separate windowFig. 1.IgA1 structural elements. IgA1 has N-linked glycans (filled circles) and O-linked glycans (open circles). The O-glycosylated sites are in the HR between the first and second constant region domains of the heavy chains. The HR is a Pro-rich segment with nine possible sites of O-glycan attachment. Underlined serine and threonine residues are usually glycosylated (31). Arrows show cleavage sites of trypsin and IgA-specific proteases.An O-glycosylated protein from a single source contains a population of variably O-glycosylated isoforms that show a distinct distribution of microheterogeneity of the O-glycan chains in terms of number, sites of attachment, and composition. Characterizing these clustered sites and understanding how the distributions change under different biological conditions or disease states are an analytical challenge. Enzymatic or chemical release of O-glycans is not selective. The heterogeneity, composition, and quantitative aspects of different O-glycan chains can be assessed and quantified by gas chromatographic and/or mass spectrometric techniques. However, the site-specific information and context of location and composition of adjacent chains are lost. Carbohydrate-specific lectin analysis of O-glycoproteins can provide information on glycan composition and comparative differences between samples, such as those from healthy controls and patients with various disease states. We have successfully demonstrated this in the analysis of IgA1 O-glycans from patients with IgAN versus healthy controls and disease controls (13–15). This included proximal assessment of sites with galactose (Gal)-deficient O-glycans after digests with IgA-specific proteases (8). Several studies have demonstrated the value of mass spectrometry (MS) in identifying Gal-deficient IgA1 in patients with IgAN (16–21), including our work that demonstrated the first direct localization of native sites of O-glycan chains in the hinge region (HR) of IgA1 by use of electron capture dissociation (ECD) (20, 22). ECD and the more recently developed electron transfer dissociation (ETD) have been used to identify sites of O-glycosylation on a variety of proteins (23–26). This includes the analysis of sites of O-glycosylation by on-line LC-ECD/ETD MS/MS methods (23, 26, 27).IgAN is the most common primary glomerulonephritis worldwide (28) with about 20–40% of patients developing end stage renal failure. It is characterized by mesangial deposits of IgA1-containing immune complexes (28). The distinctive O-glycan chains of IgA1 molecules play a pivotal role in the pathogenesis of IgAN (1, 10, 14–16, 29, 30). IgA1 contains an HR between the first and second heavy chain constant region domains with a high content of Ser, Thr, and Pro. This segment usually has three to five O-glycan chains per HR (31) (see Fig. 1). Aberrantly glycosylated IgA1, deficient in Gal in some of the O-glycans in the HR, in serum is rare in healthy individuals but is present at elevated levels in IgAN patients (13, 15). This distinctive IgA1 is in circulating immune complexes (8, 10, 15) and in the glomerular deposits of IgAN patients (16, 29). The absence of Gal apparently leads to the exposure of neoepitopes, including terminal and sialylated N-acetylgalactosamine (GalNAc) residues (9, 10). These epitopes are recognized by naturally occurring anti-glycan IgG or IgA1 antibodies and, consequently, circulating immune complexes are formed (9, 10, 15) that can deposit in the glomerular mesangia. To identify the pathogenic forms of IgA1, a thorough analysis of O-glycan microheterogeneity, including identification of the attachment sites, will be required.In this work, we demonstrate the complete analysis of O-glycoform microheterogeneity and site localization of the glycoforms in a naturally Gal-deficient IgA1 (Ale) myeloma protein that mimics the nephritogenic IgA1 in patients with IgAN (8, 9). Reversed phase (RP) LC FT-ICR MS successfully identified 10 distinct IgA1 HR fragments representing >99% of total IgA1. AI-ECD of the six most abundant IgA1 HR glycoforms (>95% of total IgA1) was accomplished with three distinct IgA-specific protease + trypsin digestions, identifying sites of Gal deficiency across four distinct IgA1 O-glycoforms. Based on the success of the ECD fragmentation of these IgA1 HR fragments, we adapted the analysis for on-line LC-MS/MS methods for both ECD and ETD. The variety of IgA1 HR proteolytic fragments provides a practical set of guidelines for the ECD/ETD analysis of clustered sites of O-glycosylation on this and other proteins. These results also provide insight into the order of attachment of the O-glycans in the IgA1 HR.     

Biocatalytic conversion of avermectin to 4'-oxo-avermectin: improvement of cytochrome p450 monooxygenase specificity by directed evolution

Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4'-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4'-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.     

The role of endothelial calcium and nitric oxide in the localisation of atherosclerosis

A mathematical model of endothelial cell calcium signalling and nitric oxide synthesis under flow conditions is presented. The model is coupled to two important environmental stimuli for endothelial cells: the frictional shear stress exerted on the cell membrane by the blood flow; and the binding of adenosine triphosphate in the bloodstream to cell surface receptors. These stimuli are closely linked to haemodynamic flow conditions and are, in general, spatially varying, allowing the cellular response in different regions of the endothelium to be evaluated. This is used to indicate which areas of the artery wall experience reduced bioavailability of nitric oxide, which is a major factor in the onset of atherosclerosis. The model thus directly addresses the key issue of the causative link, and its underlying biochemical mechanisms, between incidence of atherosclerosis and regions of low wall shear stress (WSS). Model results show that intracellular levels of free calcium and endothelial nitric oxide synthase are lower in endothelial cells adjacent to a region of recirculating flow than in cells adjacent to regions of fully developed arterial flow. This will lead to deficient levels of nitric oxide in the recirculation zone and hence a potentially elevated risk of developing atherosclerotic plaque. This is consistent with the observed spatial correlation between atherosclerosis and regions of disturbed blood flow and low WSS, and provides a mechanism for the localisation of the disease to sites such as arterial bifurcations and bends.     

Comparison of cellular strain with applied substrate strain in vitro

Strain magnitudes within tenocytes undergoing substrate tensile strain are not well defined. It was hypothesized that strain magnitudes at the cellular level would reflect those of the applied substrate (equibiaxial or uniaxial) strain. A vacuum-operated device was used to apply equibiaxial or uniaxial tension to a flexible substrate upon which tenocytes were cultured in monolayer. Images of tenocytes labeled with Fura-2, to detect free intracellular calcium ions, and MitoFluor Green, to detect mitochondria, were taken prior to strain and for 20 min during application of static strain. A custom-written, texture correlation program computed strain magnitudes in the cell based on the change in pixel pattern displacements between images of non-strained and strained cells. On average, cellular strain was approximately 37+/-8% and 63+/-11% of the applied equibiaxial and uniaxial substrate strain, respectively. The largest cell strains were detected in cells oriented parallel to the direction of applied uniaxial tensile strain. However, strain magnitudes within a cell were heterogeneous. The variance in strain magnitude within and among tenocytes is dependent on cell orientation, cell stiffness, cytoskeleton organization, subcellular organelles, or placement and type of cell-substrate contacts. Results of the present study indicate that cultured tenocytes experience a moderate fraction of the applied substrate strain.     

RAB23 mutations in Carpenter syndrome imply an unexpected role for hedgehog signaling in cranial-suture development and obesity

Carpenter syndrome is a pleiotropic disorder with autosomal recessive inheritance, the cardinal features of which include craniosynostosis, polysyndactyly, obesity, and cardiac defects. Using homozygosity mapping, we found linkage to chromosome 6p12.1-q12 and, in 15 independent families, identified five different mutations (four truncating and one missense) in RAB23, which encodes a member of the RAB guanosine triphosphatase (GTPase) family of vesicle transport proteins and acts as a negative regulator of hedgehog (HH) signaling. In 10 patients, the disease was caused by homozygosity for the same nonsense mutation, L145X, that resides on a common haplotype, indicative of a founder effect in patients of northern European descent. Surprisingly, nonsense mutations of Rab23 in open brain mice cause recessive embryonic lethality with neural-tube defects, suggesting a species difference in the requirement for RAB23 during early development. The discovery of RAB23 mutations in patients with Carpenter syndrome implicates HH signaling in cranial-suture biogenesis--an unexpected finding, given that craniosynostosis is not usually associated with mutations of other HH-pathway components--and provides a new molecular target for studies of obesity.     

WHAT MOTIVATES WOMEN TO TAKE PART IN CLINICAL AND BASIC SCIENCE ENDOMETRIOSIS RESEARCH?*

BACKGROUND: The objective of this study was to identify factors motivating women to take part in endometriosis research and to determine if these factors differ for women participating in clinical versus basic science studies. METHODS: A consecutive series of 24 women volunteering for participation in endometriosis-related research were asked to indicate, in their own words, why they chose to volunteer. In addition, the women were asked to rate, on a scale of 0 to 10, sixteen potentially motivating factors. The information was gathered in the form of an anonymous self-administered questionnaire. RESULTS: Strong motivating factors (mean score > 8) included potential benefit to other women's health, improvement to one's own condition, and participation in scientific advancement. Weak motivating factors (mean score < 3) included financial compensation, making one's doctor happy, and use of 'natural' products. No difference was detected between clinical and basic science study participants. CONCLUSION: This study is the first study to specifically investigate the factors that motivate women to take part in endometriosis research. Understanding why women choose to take part in such research is important to the integrity of the informed consent process. The factors most strongly motivating women to participate in endometriosis research related to improving personal or public health; the weakest, to financial compensation and pleasing the doctor.     

Faecal soiling and gastrointestinal helminth infection in lambs

The accumulation of faeces in the wool of the breech area (tail, perineum and anus) of lambs, known as faecal soiling, has been shown to be one of the major factors predisposing sheep to blowfly strike. However, the causes of faecal soiling of lambs in the UK are not clearly understood. Hence, in this investigation, the relationships between faecal soiling, gastrointestinal parasitic nematode infection and resultant diarrhoea were examined in a longitudinal study of 200 lambs at two farms in south-west England. Faecal egg counts, pasture worm burdens, faecal soiling and growth rates were recorded for individually tagged lambs over the summer of 2003. Grass growth and nutritional composition (protein and fibre) and weather data were also recorded over this period. Analysis using linear mixed models showed that faecal soiling was associated with higher strongyle-type egg counts, longer periods since worming, lower live-weights, female gender, lower faecal consistency and pasture quality. The data indicate that dag scoring, especially in mid- to late summer, could be used as a rapid, non-invasive technique for selecting animals, particularly lambs, with high faecal egg counts for selective drenching to reduce the incidence of anthelmintic resistance. Selective drenching of lambs with high dag scores would also be expected to aid in the control of blowfly strike.     

Human adipose-derived adult stem cells upregulate palladin during osteogenesis and in response to cyclic tensile strain

Cell morphology may be an important stimulus during differentiation of human adipose-derived adult stem (hADAS) cells, but there are limited studies that have investigated the role of the cytoskeleton or associated proteins in hADAS cells undergoing differentiation. Palladin is an actin-associated protein that plays an integral role in focal adhesion and cytoskeleton organization. In this study we show that palladin was expressed by hADAS cells and was modulated during osteogenic differentiation and in response to cyclic tensile strain. Human ADAS cells expressed the 90- and 140-kDa palladin isoforms and upregulated expression of both isoforms after culture in conditions that promoted osteogenesis. Palladin mRNA expression levels were also increased in hADAS cells subjected to cyclic tensile strain. Knockdown of the palladin gene during osteogenesis resulted in decreased actin stress fibers and decreased protein levels of Eps8, an epidermal growth factor receptor tyrosine kinase that colocalizes with actin. Silencing the palladin gene, however, did not affect hADAS cells' commitment down the osteogenic lineage.