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1.
Adam?E. Tuer Margarete?K. Akens Serguei Krouglov Daaf Sandkuijl Brian?C. Wilson Cari?M. Whyne Virginijus Barzda 《Biophysical journal》2012,103(10):2093-2105
The second-order nonlinear polarization properties of fibrillar collagen in various rat tissues (vertebrae, tibia, tail tendon, dermis, and cornea) are investigated with polarization-dependent second-harmonic generation (P-SHG) microscopy. Three parameters are extracted: the second-order susceptibility ratio, R = ; a measure of the fibril distribution asymmetry, |A|; and the weighted-average fibril orientation, 〈δ〉. A hierarchical organizational model of fibrillar collagen is developed to interpret the second-harmonic generation polarization properties. Highlights of the model include: collagen type (e.g., type-I, type-II), fibril internal structure (e.g., straight, constant-tilt), and fibril architecture (e.g., parallel fibers, intertwined, lamellae). Quantifiable differences in internal structure and architecture of the fibrils are observed. Occurrence histograms of R and |A| distinguished parallel from nonparallel fibril distributions. Parallel distributions possessed low parameter values and variability, whereas nonparallel distributions displayed an increase in values and variability. From the P-SHG parameters of vertebrae tissue, a three-dimensional reconstruction of lamellae of intervertebral disk is presented. 相似文献
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《Journal of liposome research》2013,23(1):315-324
AbstractThe incubation of platelet-rich rat plasma with liposomes has changed cell functional activity. Phospholipids of liposomal membrane was modulated collagen - mediated platelet aggregation. Vesicles containing phosphatidylserine (1.6 mg/ml) have inhibited platelet aggregation induced by 1 mcg/ml collagen completely. Phoshatidylinositol liposomes didn't show considerable effect on the maximum value of aggregation but they reduced the speed and the level of disaggregation. The impairment of aggregation in platelet-rich plasma can be explained by the interactions of liposomes with plasma coagulation factors and the following formation of the inactive complexes. 相似文献
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Alan Veliz-Cuba Andrew J. Hirning Adam A. Atanas Faiza Hussain Flavia Vancia Kre?imir Josi? Matthew R. Bennett 《PLoS computational biology》2015,11(12)
Synthetic gene oscillators are small, engineered genetic circuits that produce periodic variations in target protein expression. Like other gene circuits, synthetic gene oscillators are noisy and exhibit fluctuations in amplitude and period. Understanding the origins of such variability is key to building predictive models that can guide the rational design of synthetic circuits. Here, we developed a method for determining the impact of different sources of noise in genetic oscillators by measuring the variability in oscillation amplitude and correlations between sister cells. We first used a combination of microfluidic devices and time-lapse fluorescence microscopy to track oscillations in cell lineages across many generations. We found that oscillation amplitude exhibited high cell-to-cell variability, while sister cells remained strongly correlated for many minutes after cell division. To understand how such variability arises, we constructed a computational model that identified the impact of various noise sources across the lineage of an initial cell. When each source of noise was appropriately tuned the model reproduced the experimentally observed amplitude variability and correlations, and accurately predicted outcomes under novel experimental conditions. Our combination of computational modeling and time-lapse data analysis provides a general way to examine the sources of variability in dynamic gene circuits. 相似文献
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Qian Xiao Steven C. Moore Simina M. Boca Charles E. Matthews Nathaniel Rothman Rachael Z. Stolzenberg-Solomon Rashmi Sinha Amanda J. Cross Joshua N. Sampson 《PloS one》2014,9(5)
Background
The application of metabolomics in epidemiological studies would potentially allow researchers to identify biomarkers associated with exposures and diseases. However, within-individual variability of metabolite levels caused by temporal variation of metabolites, together with technical variability introduced by laboratory procedures, may reduce the study power to detect such associations. We assessed the sources of variability of metabolites from urine samples and the implications for designing epidemiologic studies.Methods
We measured 539 metabolites in urine samples from the Navy Colon Adenoma Study using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectroscopy (GC-MS). The study collected 2–3 samples per person from 17 male subjects (age 38–70) over 2–10 days. We estimated between-individual, within-individual, and technical variability and calculated expected study power with a specific focus on large case-control and nested case-control studies.Results
Overall technical reliability was high (median intraclass correlation = 0.92), and for 72% of the metabolites, the majority of total variance can be attributed to between-individual variability. Age, gender and body mass index explained only a small proportion of the total metabolite variability. For a relative risk (comparing upper and lower quartiles of “usual” levels) of 1.5, we estimated that a study with 500, 1,000, and 5,000 individuals could detect 1.0%, 4.5% and 75% of the metabolite associations.Conclusions
The use of metabolomics in urine samples from epidemiological studies would require large sample sizes to detect associations with moderate effect sizes. 相似文献6.
Assessing Adhesion Forces of Type I and Type IV Pili of Xylella fastidiosa Bacteria by Use of a Microfluidic Flow Chamber 总被引:1,自引:0,他引:1
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Leonardo De La Fuente Emilie Montanes Yizhi Meng Yaxin Li Thomas J. Burr H. C. Hoch Mingming Wu 《Applied microbiology》2007,73(8):2690-2696
Xylella fastidiosa, a bacterium responsible for Pierce's disease in grapevines, possesses both type I and type IV pili at the same cell pole. Type IV pili facilitate twitching motility, and type I pili are involved in biofilm development. The adhesiveness of the bacteria and the roles of the two pili types in attachment to a glass substratum were evaluated using a microfluidic flow chamber in conjunction with pilus-defective mutants. The average adhesion force necessary to detach wild-type X. fastidiosa cells was 147 ± 11 pN. Mutant cells possessing only type I pili required a force of 204 ± 22 pN for removal, whereas cells possessing only type IV pili required 119 ± 8 pN to dislodge these cells. The experimental results demonstrate that microfluidic flow chambers are useful and convenient tools for assessing the drag forces necessary for detaching bacterial cells and that with specific pilus mutants, the role of the pilus type can be further assessed. 相似文献
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Ewoud R.E. Schmidt Francesca Morello R. Jeroen Pasterkamp 《Journal of visualized experiments : JoVE》2012,(61)
Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or βIII tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues. 相似文献
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Patricia Rousselle Douglas R. Keene Florence Ruggiero Marie-France Champliaud Michel van der Rest Robert E. Burgeson 《The Journal of cell biology》1997,138(3):719-728
Mutational analyses of genes that encode components of the anchoring complex underlying the basolateral surface of external epithelia indicate that this structure is the major element providing for resistance to external friction. Ultrastructurally, laminin 5 (α3β3γ2; a component of the anchoring filament) appears as a thin filament bridging the hemidesmosome with the anchoring fibrils. Laminin 5 binds the cell surface through hemidesmosomal integrin α6β4. However, the interaction of laminin 5 with the anchoring fibril (type VII collagen) has not been elucidated. In this study we demonstrate that monomeric laminin 5 binds the NH2-terminal NC-1 domain of type VII collagen. The binding is dependent upon the native conformation of both laminin 5 and type VII collagen NC-1. Laminin 6 (α3β1γ1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the β3 and/or γ2 chains of laminin 5. Approximately half of the laminin 5 solubilized from human amnion or skin is covalently complexed with laminins 6 or 7 (α3β2γ1). The adduction occurs between the NH2 terminus of laminin 5 and the branch point of the short arms of laminins 6 or 7. The results are consistent with the presumed orientation of laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH2-terminal domains within the lamina densa. The results also support a model predicting that monomeric laminin 5 constitutes the anchoring filaments and bridges integrin α6β4 with type VII collagen, and the laminin 5–6/7 complexes are present within the interhemidesmosomal spaces bound at least by integrin α3β1 where they may mediate basement membrane assembly or stability, but contribute less significantly to epithelial friction resistance. 相似文献
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Steen G. Stahlhut Sujay Chattopadhyay Carsten Struve Scott J. Weissman Pavel Aprikian Stephen J. Libby Ferric C. Fang Karen Angeliki Krogfelt Evgeni V. Sokurenko 《Journal of bacteriology》2009,191(6):1941-1950
FimH is an adhesive subunit of type 1 fimbriae expressed by different enterobacterial species. The enteric bacterium Klebsiella pneumoniae is an environmental organism that is also a frequent cause of sepsis, urinary tract infection (UTI), and liver abscess. Type 1 fimbriae have been shown to be critical for the ability of K. pneumoniae to cause UTI in a murine model. We show here that the K. pneumoniae fimH gene is found in 90% of strains from various environmental and clinical sources. The fimH alleles exhibit relatively low nucleotide and structural diversity but are prone to frequent horizontal-transfer events between different bacterial clones. Addition of the fimH locus to multiple-locus sequence typing significantly improved the resolution of the clonal structure of pathogenic strains, including the K1 encapsulated liver isolates. In addition, the K. pneumoniae FimH protein is targeted by adaptive point mutations, though not to the same extent as FimH from uropathogenic Escherichia coli or TonB from the same K. pneumoniae strains. Such adaptive mutations include a single amino acid deletion from the signal peptide that might affect the length of the fimbrial rod by affecting FimH translocation into the periplasm. Another FimH mutation (S62A) occurred in the course of endemic circulation of a nosocomial uropathogenic clone of K. pneumoniae. This mutation is identical to one found in a highly virulent uropathogenic strain of E. coli, suggesting that the FimH mutations are pathoadaptive in nature. Considering the abundance of type 1 fimbriae in Enterobacteriaceae, our present finding that fimH genes are subject to adaptive microevolution substantiates the importance of type 1 fimbria-mediated adhesion in K. pneumoniae.Klebsiella pneumoniae is recognized as an important opportunistic pathogen that frequently causes urinary tract infections (UTI), septicemia, or pneumonia, particularly in immunocompromised individuals (25). K. pneumoniae is responsible for up to 10% of all nosocomial bacterial infections (12, 35). In recent years, a high incidence of community-acquired K. pneumoniae pyogenic liver abscess with a high mortality rate has been reported, especially from Taiwan, but also from other Asian countries, Europe, and North America (6, 8, 19, 27, 44). Furthermore, 15% to 30% of K. pneumoniae isolates are resistant to broad-spectrum cephalosporins via plasmid-encoded extended-spectrum β-lactamases (5).In contrast to many other bacterial pathogens, K. pneumoniae is ubiquitous in nature. Its nonclinical habitats include environmental locations, such as vegetation, soil, and surface waters, as well as transient commensal colonization of mucosal surfaces in humans and other animals (1). Several studies have reported K. pneumoniae isolates of environmental origin to be nearly identical to clinical isolates with respect to several phenotypic properties (16, 22, 23, 25, 30). It has been suggested that environmental isolates of K. pneumoniae may be as virulent as clinical isolates (24, 39).Several virulence factors have been identified in K. pneumoniae (25, 38). The prominent polysaccharide capsule expressed by most isolates, together with the lipopolysaccharide layer, protects the bacteria against phagocytosis and the bactericidal activity of serum. Fimbrial adhesins expressed by the bacteria are protein structures able to recognize molecular receptors and to facilitate adherence to specific tissue surfaces in the host. K. pneumoniae produces two major fimbrial adhesion organelles, type 1 and type 3 fimbriae (9). Type 1 fimbriae have mannose-sensitive hemagglutinins, while type 3 fimbriae have mannose-resistant hemagglutinins (21).Type 1 fimbriae are the most common adhesive organelle in Enterobacteriaceae and have been most extensively studied in Escherichia coli. The type 1 fimbrial structures of K. pneumoniae are homologous to those of E. coli with regard to genetic composition and regulation (37). Type 1 fimbriae and the adhesive subunit FimH, in particular, play an important role in UTI caused by both K. pneumoniae and E. coli (3, 15, 17, 30, 37). Analysis of E. coli fimH variation at the population level has revealed that the FimH adhesin in urinary E. coli isolates accumulates amino acid replacements that increase its tropism toward the uroepithelium and various components of basement membranes (14, 26, 31, 33, 46). Most of the replacements increase the monomannose binding capability of FimH under low shear by altering allosteric catch bond properties of the protein (40). The natural FimH mutants were shown to provide an advantage in colonization of the urinary tract in a mouse model (32) and correlate with the overall extraintestinal virulence of E. coli (11). Thus, FimH mutations are pathoadaptive in nature. No such population-wide analysis has been performed for K. pneumoniae fimH.Population genetic analysis involves comparison of the nucleotide and structural variability of the locus of interest across multiple bacterial strains of different clonalities and geographic origins. The clonal structure of the strains can be determined by multiple-locus sequence typing (MLST), in which 400- to 500-bp sequences of multiple genetically unlinked loci are determined in order to define the phylogenetic relationship of the strains and the extent of interclonal gene recombination (horizontal gene transfer). MLST has been used to reveal the epidemiological relationship of ceftazidime- and ciprofloxacin-resistant K. pneumoniae isolates of nosocomial origin (4). In addition, the analysis of gene variability enables the determination of the type of selection processes acting on loci of interest, with possible identification of mutational changes of functional significance that could enhance the organism''s ability to cause disease, i.e., that could be of a pathoadaptive nature.In this study, the population dynamics of the K. pneumoniae FimH adhesin were determined by analysis of fimH allelic diversity in strains of environmental and various clinical origins in the context of K. pneumoniae clonal structure based on the allelic diversity of three loci—tonB, mdh and fumC—commonly used for MLST. 相似文献
11.
《Genomics》1995,29(3)
Genes that encode the vertebrate fibrillar collagen types I–III have previously been shown to share a highly conserved intron/exon organization, thought to reflect common ancestry and evolutionary pressures at the protein level. We report here the complete intron/exon organization ofCOL5A1,the human gene that encodes the α1 chain of fibrillar collagen type V. The structure ofCOL5A1is shown to be considerably diverged from the conserved structure of the genes for fibrillar collagen types I–III.COL5A1has 66 exons, which is greater than the number of exons found in the genes for collagen types I–III. The increased number of exons is partly due to the increased size of the pro-α1(V) N-propeptide, relative to the sizes of the N-propeptides of the types I–III procollagen molecules. In addition, however, the increased number of exons is due to differences in the intron/exon organization of the triple-helix coding region ofCOL5A1compared to the organization of the triple-helix coding regions of the genes for collagen types I–III. Of particular interest is the increase of 54 bp exons in this region ofCOL5A1,strongly supporting the proposal that the triple-helix coding regions of fibrillar collagen genes evolved from duplication of a 54 bp primordial genetic element. Moreover, comparison of the structure ofCOL5A1to the highly conserved structure of the genes of collagen types I–III provides insights into the probable structure of the ancestral gene that gave rise to what appears to be two classes of vertebrate fibrillar collagen genes. 相似文献
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C. Luke Williams Tyler L. Westover Rachel M. Emerson Jaya Shankar Tumuluru Chenlin Li 《Bioenergy Research》2016,9(1):1-14
Terrestrial lignocellulosic biomass has the potential to be a carbon neutral and domestic source of fuels and chemicals. However, the innate variability of biomass resources, such as herbaceous and woody materials, and the inconsistency within a single resource due to disparate growth and harvesting conditions, presents challenges for downstream processes which often require materials that are physically and chemically consistent. Intrinsic biomass characteristics, including moisture content, carbohydrate and ash compositions, bulk density, and particle size/shape distributions are highly variable and can impact the economics of transforming biomass into value-added products. For instance, ash content increases by an order of magnitude between woody and herbaceous feedstocks (from ~0.5 to 5 %, respectively) while lignin content drops by a factor of two (from ~30 to 15 %, respectively). This increase in ash and reduction in lignin leads to biofuel conversion consequences, such as reduced pyrolysis oil yields for herbaceous products as compared to woody material. In this review, the sources of variability for key biomass characteristics are presented for multiple types of biomass. Additionally, this review investigates the major impacts of the variability in biomass composition on four conversion processes: fermentation, hydrothermal liquefaction, pyrolysis, and direct combustion. Finally, future research processes aimed at reducing the detrimental impacts of biomass variability on conversion to fuels and chemicals are proposed.© 2015 Battelle Energy Alliance, LLC, contract manager for Idaho National Laboratory. 相似文献
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Karen Gilio Matthew T. Harper Judith M. E. M. Cosemans Olga Konopatskaya Imke C. A. Munnix Lenneke Prinzen Michael Leitges Qinghang Liu Jeffery D. Molkentin Johan W. M. Heemskerk Alastair W. Poole 《The Journal of biological chemistry》2010,285(30):23410-23419
Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen. 相似文献
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Y Inoue H Itoh M Aoki S Ogawa T Yamane T Baba N Tachibana M Kohno Y Oishi K Kobayashi-Hattori 《Bioscience, biotechnology, and biochemistry》2012,76(8):1549-1551
Two weeks of feeding soy peptides containing 2% collagen peptides increased the levels of type I and III tropocollagen and their mRNAs. In contrast, the diet did not increase the mRNA levels of rat hyaluronan synthases, serine palmitoyltransferase (the rate-limiting enzyme of ceramide synthesis), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (the key enzyme of cholesterol synthesis). These results suggest that feeding of soy peptides with collagen peptides specifically enhanced the tropocollagen level in the skin. 相似文献
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The Activity of Collagenase-1 Is Required for Keratinocyte Migration on a Type I Collagen Matrix 总被引:27,自引:0,他引:27
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Brian K. Pilcher Jo Ann Dumin Barry D. Sudbeck Stephen M. Krane Howard G. Welgus William C. Parks 《The Journal of cell biology》1997,137(6):1445-1457
We have shown in a variety of human wounds that collagenase-1 (MMP-1), a matrix metalloproteinase that cleaves fibrillar type I collagen, is invariably expressed by basal keratinocytes migrating across the dermal matrix. Furthermore, we have demonstrated that MMP-1 expression is induced in primary keratinocytes by contact with native type I collagen and not by basement membrane proteins or by other components of the dermal or provisional (wound) matrix. Based on these observations, we hypothesized that the catalytic activity of MMP-1 is necessary for keratinocyte migration on type I collagen. To test this idea, we assessed keratinocyte motility on type I collagen using colony dispersion and colloidal gold migration assays. In both assays, primary human keratinocytes migrated efficiently on collagen. The specificity of MMP-1 in promoting cell movement was demonstrated in four distinct experiments. One, keratinocyte migration was completely blocked by peptide hydroxymates, which are potent inhibitors of the catalytic activity of MMPs. Two, HaCaTs, a line of human keratinocytes that do not express MMP-1 in response to collagen, did not migrate on a type I collagen matrix but moved efficiently on denatured type I collagen (gelatin). EGF, which induces MMP-I production by HaCaT cells, resulted in the ability of these cells to migrate across a type I collagen matrix. Three, keratinocytes did not migrate on mutant type I collagen lacking the collagenase cleavage site, even though this substrate induced MMP-1 expression. Four, cell migration on collagen was completely blocked by recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) and by affinity-purified anti–MMP-1 antiserum. In addition, the collagen-mediated induction of collagenase-1 and migration of primary keratinocytes on collagen was blocked by antibodies against the α2 integrin subunit but not by antibodies against the α1 or α3 subunits. We propose that interaction of the α2β1 integrin with dermal collagen mediates induction of collagenase-1 in keratinocytes at the onset of healing and that the activity of collagenase-1 is needed to initiate cell movement. Furthermore, we propose that cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization. 相似文献
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A critical assessment is presented of the literature on variabilityin even-aged stands of plants. The variance of weight per plantaccumulates during two physiologically distinct periods of growth,pre- and post-seedling emergence, and can be expressed as afunction of the variances and covariances of three physiologicalvariables - the rate of growth, the duration of growth, andthe size per plant at the start of the period. This providesa quantitative framework for analyzing the physiological sourcesof variability in plant stands. Variation, competition, self-thinning, growth 相似文献
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Nagarajan Usharani Gladstone Christopher Jayakumar Swarna V. Kanth Jonnalagadda Raghava Rao Bangaru Chandrasekaran Balachandran Unni Nair 《International journal of peptide research and therapeutics》2013,19(4):357-364
The interaction of bilirubin with collagen in the significance of jaundice incidence have been previously reported and investigated. The novel peptide sequences containing bilirubin binding domain was identified and located to develop a basis for further studies investigating the interactions of collagen with bilirubin in the present study. In this study an intricate interaction between bilirubin and collagen was characterized and their binding domain has been established using in-gel digestion and LC–MS/MS analysis based on the collagen sequencing and peptide mass fingerprinting. The biotinylated bilirubin derivatives bind to α1(I) chain but not to α2(I) chains which clearly designates that bilirubin shows greater affinity to α1 chains of collagen. The intact proteins collected after analyzing the resulting complex mixture of peptides was used for peptide mapping. Using the electrospray method, among the other peptide sequence information obtained, the molecular weight of collagen alpha-2(I) chain was obtained by locating a 130 kDa weight peptide sequences with greater pi value (9.14) with 1,364 amino acid residues and collagen alpha-1(I) chain with 1,463 amino acid residues with 138.9 kDa molecular weight. This information leads to locate the exact sequence of these helices focussing on the domain identification. The total charge of the peptide domain sequences infers that the bilirubin participates in the electrostatic mode of interaction with collagen peptide. Moreover, other modes of interactions such as hydrogen bonding, covalent interactions and hydrophobic interactions are possible. 相似文献
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Canada geese (Branta canadensis) are prevalent in North America and may contribute to fecal pollution of water systems where they congregate. This work provides two novel real-time PCR assays (CGOF1-Bac and CGOF2-Bac) allowing for the specific and sensitive detection of Bacteroides 16S rRNA gene markers present within Canada goose feces.The Canada goose (Branta canadensis) is a prevalent waterfowl species in North America. The population density of Canada geese has doubled during the past 15 years, and the population was estimated to be close to 3 million in 2007 (4). Canada geese often congregate within urban settings, likely due to available water sources, predator-free grasslands, and readily available food supplied by humans (6). They are suspected to contribute to pollution of aquatic environments due to the large amounts of fecal matter that can be transported into the water. This can create a public health threat if the fecal droppings contain pathogenic microorganisms (6, 7, 9, 10, 12, 13, 19). Therefore, tracking transient fecal pollution of water due to fecal inputs from waterfowl, such as Canada geese, is of importance for protecting public health.PCR detection of host-specific 16S rRNA gene sequences from Bacteroidales of fecal origin has been described as a promising microbial source-tracking (MST) approach due to its rapidity and high specificity (2, 3). Recently, Lu et al. (15) characterized the fecal microbial community from Canada geese by constructing a 16S rRNA gene sequence database using primers designed to amplify all bacterial 16S rRNA gene sequences. The authors reported that the majority of the 16S rRNA gene sequences obtained were related to Clostridia or Bacilli and to a lesser degree Bacteroidetes, which represent possible targets for host-specific source-tracking assays.The main objective of this study was to identify novel Bacteroidales 16S rRNA gene sequences that are specific to Canada goose feces and design primers and TaqMan fluorescent probes for sensitive and specific quantification of Canada goose fecal contamination in water sources.Primers 32F and 708R from Bernhard and Field (2) were used to construct a Bacteroidales-specific 16S rRNA gene clone library from Canada goose fecal samples (n = 15) collected from grass lawns surrounding Wascana Lake (Regina, SK, Canada) in May 2009 (for a detailed protocol, see File S1 in the supplemental material). Two hundred eighty-eight clones were randomly selected and subjected to DNA sequencing (at the Plant Biotechnology Institute DNA Technologies Unit, Saskatoon, SK, Canada). Representative sequences of each operational taxonomic unit (OTU) were recovered using an approach similar to that described by Mieszkin et al. (16). Sequences that were less than 93% similar to 16S rRNA gene sequences from nontarget host species in GenBank were used in multiple alignments to identify regions of DNA sequence that were putatively goose specific. Subsequently, two TaqMan fluorescent probe sets (targeting markers designated CGOF1-Bac and CGOF2-Bac) were designed using the RealTimeDesign software provided by Biosearch Technologies (http://www.biosearchtech.com/). The newly designed primer and probe set for the CGOF1-Bac assay included CG1F (5′-GTAGGCCGTGTTTTAAGTCAGC-3′) and CG1R (5′-AGTTCCGCCTGCCTTGTCTA-3′) and a TaqMan probe (5′-6-carboxyfluorescein [FAM]-CCGTGCCGTTATACTGAGACACTTGAG-Black Hole Quencher 1 [BHQ-1]-3′), and the CGOF2-Bac assay had primers CG2F (5′-ACTCAGGGATAGCCTTTCGA-3′) and CG2R (5′-ACCGATGAATCTTTCTTTGTCTCC-3′) and a TaqMan probe (5′-FAM-AATACCTGATGCCTTTGTTTCCCTGCA-BHQ-1-3′). Oligonucleotide specificities for the Canada goose-associated Bacteroides 16S rRNA primers were verified through in silico analysis using BLASTN (1) and the probe match program of the Ribosomal Database Project (release 10) (5). Host specificity was further confirmed using DNA extracts from 6 raw human sewage samples from various geographical locations in Saskatchewan and 386 fecal samples originating from 17 different animal species in Saskatchewan, including samples from Canada geese (n = 101) (Table (Table1).1). An existing nested PCR assay for detecting Canada goose feces (15) (targeting genetic marker CG-Prev f5) (see Table S1 in the supplemental material) was also tested for specificity using the individual fecal and raw sewage samples (Table (Table1).1). All fecal DNA extracts were obtained from 0.25 g of fecal material by using the PowerSoil DNA extraction kit (Mo Bio Inc., Carlsbad, CA) (File S1 in the supplemental material provides details on the sample collection).
Open in a separate windowaThe 6 goose samples that tested negative for the All-Bac marker also tested negative for the three goose markers.The majority of the Canada goose feces analyzed in this study (94%; 95 of 101) carried the Bacteroidales order-specific genetic marker designated All-Bac, with a relatively high median concentration of 8.2 log10 copies g−1 wet feces (Table (Table11 and Fig. Fig.1).1). The high prevalence and abundance of Bacteroidales in Canada goose feces suggested that detecting members of this order could be useful in identifying fecal contamination associated with Canada goose populations.Open in a separate windowFIG. 1.Concentrations of the Bacteroidales (All-Bac, CGOF1-Bac, and CGOF2-Bac) genetic markers in feces from various individual Canada geese.The composition of the Bacteroidales community in Canada goose feces (n = 15) was found to be relatively diverse since 52 OTUs (with a cutoff of 98% similarity) were identified among 211 nonchimeric 16S rRNA gene sequences. Phylogenetic analysis of the 52 OTUs (labeled CGOF1 to CGOF52) revealed that 43 (representing 84% of the 16S rRNA gene sequences) were Bacteroides like and that 9 (representing 16% of the 16S rRNA gene sequences) were likely to be members of the Prevotella-specific cluster (see Fig. S2 in the supplemental material). Similarly, Jeter et al. (11) reported that 75.7% of the Bacteroidales 16S rRNA clone library sequences generated from goose fecal samples were Bacteroides like. The majority of the Bacteroides- and Prevotella-like OTUs were dispersed among a wide range of previously characterized sequences from various hosts and did not occur in distinct clusters suitable for the design of Canada goose-associated real-time quantitative PCR (qPCR) assays (see Fig. S2 in the supplemental material). However, two single Bacteroides-like OTU sequences (CGOF1 and CGOF2) contained putative goose-specific DNA regions that were identified by in silico analysis (using BLASTN, the probe match program of the Ribosomal Database Project, and multiple alignment). The primers and probe for the CGOF1-Bac and CGOF2-Bac assays were designed with no mismatches to the clones CGOF1 and CGOF2, respectively.The CGOF2-Bac assay demonstrated no cross-amplification with fecal DNA from other host groups, while cross-amplification for the CGOF1-Bac assay was limited to one pigeon fecal sample (1 of 25, i.e., 4% of the samples) (Table (Table1).1). Since the abundance in the pigeon sample was low (3.3 log10 marker copies g−1 feces) and detection occurred late in the qPCR (with a threshold cycle [CT] value of 37.1), it is unlikely that this false amplification would negatively impact the use of the assay as a tool for detection of Canada goose-specific fecal pollution in environmental samples. In comparison, the nested PCR CG-Prev f5 assay described by Lu and colleagues (15) demonstrated non-host-specific DNA amplification with fecal DNA samples from several animals, including samples from humans, pigeons, gulls, and agriculturally relevant pigs and chickens (Table (Table11).Both CGOF1-Bac and CGOF2-Bac assays showed limits of quantification (less than 10 copies of target DNA per reaction) similar to those of other host-specific Bacteroidales real-time qPCR assays (14, 16, 18). The sensitivities of the CGOF1-Bac and CGOF2-Bac assays were 57% (with 58 of 101 samples testing positive) and 50% (with 51 of 101 samples testing positive) for Canada goose feces, respectively (Table (Table1).1). A similar sensitivity of 58% (with 59 of 101 samples testing positive) was obtained using the CG-Prev f5 PCR assay. The combined use of the three assays increased the detection level to 72% (73 of 101) (Fig. (Fig.2).2). Importantly, all markers were detected within groups of Canada goose feces collected each month from May to September, indicating relative temporal stability of the markers. The CG-Prev f5 PCR assay is an end point assay, and therefore the abundance of the gene marker in Canada goose fecal samples could not be determined. However, development of the CGOF1-Bac and CGOF2-Bac qPCR approach allowed for the quantification of the host-specific CGOF1-Bac and CGOF2-Bac markers. In the feces of some individual Canada geese, the concentrations of CGOF1-Bac and CGOF2-Bac were high, reaching levels up to 8.8 and 7.9 log10 copies g−1, respectively (Fig. (Fig.11).Open in a separate windowFIG. 2.Venn diagram for Canada goose fecal samples testing positive with the CGOF1-Bac, CGOF2-Bac, and/or CG-Prev f5 PCR assay. The number outside the circles indicates the number of Canada goose fecal samples for which none of the markers were detected.The potential of the Canada goose-specific Bacteroides qPCR assays to detect Canada goose fecal pollution in an environmental context was tested using water samples collected weekly during September to November 2009 from 8 shoreline sampling sites at Wascana Lake (see File S1 and Fig. S1 in the supplemental material). Wascana Lake is an urban lake, located in the center of Regina, that is routinely frequented by Canada geese. In brief, a single water sample of approximately 1 liter was taken from the surface water at each sampling site. Each water sample was analyzed for Escherichia coli enumeration using the Colilert-18/Quanti-Tray detection system (IDEXX Laboratories, Westbrook, ME) (8) and subjected to DNA extraction (with a PowerSoil DNA extraction kit [Mo Bio Inc., Carlsbad, CA]) for the detection of Bacteroidales 16S rRNA genetic markers using the Bacteroidales order-specific (All-Bac) qPCR assay (14), the two Canada goose-specific (CGOF1-Bac and CGOF2-Bac) qPCR assays developed in this study, and the human-specific (BacH) qPCR assay (17). All real-time and conventional PCR procedures as well as subsequent data analysis are described in the supplemental material and methods. The E. coli and All-Bac quantification data demonstrated that Wascana Lake was regularly subjected to some form of fecal pollution (Table (Table2).2). The All-Bac genetic marker was consistently detected in high concentrations (6 to 7 log10 copies 100 ml−1) in all the water samples, while E. coli concentrations fluctuated according to the sampling dates and sites, ranging from 0 to a most probable number (MPN) of more than 2,000 100 ml−1. High concentrations of E. coli were consistently observed when near-shore water experienced strong wave action under windy conditions or when dense communities of birds were present at a given site and time point.
Open in a separate windowaMin, minimum; max, maximum.The frequent detection of the genetic markers CGOF1-Bac (in 65 of 75 water samples [87%]), CGOF2-Bac (in 55 of 75 samples [73%]), and CG-Prev f5 (in 60 of 75 samples [79%]) and the infrequent detection of the human-specific Bacteroidales 16S rRNA gene marker BacH (17) (in 5 of 75 water samples [7%[) confirmed that Canada geese significantly contributed to the fecal pollution in Wascana Lake during the sampling period. Highest mean concentrations of both CGOF1-Bac and CGOF2-Bac markers were obtained at the sampling sites W3 (3.8 and 3.9 log10 copies 100 ml−1) and W4 (3.4 log10 copies 100 ml−1 for both), which are heavily frequented by Canada geese (Table (Table2),2), further confirming their significant contribution to fecal pollution at these particular sites. It is worth noting that concentrations of the CGOF1-Bac and CGOF2-Bac markers in water samples displayed a significant positive relationship with each other (correlation coefficient = 0.87; P < 0.0001), supporting the accuracy of both assays for identifying Canada goose-associated fecal pollution in freshwater.In conclusion, the CGOF1-Bac and CGOF2-Bac qPCR assays developed in this study are efficient tools for estimating freshwater fecal inputs from Canada goose populations. Preliminary results obtained during the course of the present study also confirmed that Canada geese can serve as reservoirs of Salmonella and Campylobacter species (see Fig. S3 in the supplemental material). Therefore, future work will investigate the cooccurence of these enteric pathogens with the Canada goose fecal markers in the environment. 相似文献
TABLE 1.
Specificities of the CGOF1-Bac, CGOF2-Bac, and CG-Prev f5 PCR assays for different species present in Saskatchewan, CanadaHost group or sample type | No. of samples | No. positive for Bacteroidales marker: | |||
---|---|---|---|---|---|
CGOF1-Bac | CGOF2-Bac | CG-Prev f5 | All-Bac | ||
Individual human feces | 25 | 0 | 0 | 1 | 25 |
Raw human sewage | 6 | 0 | 0 | 0 | 6 |
Cows | 41 | 0 | 0 | 0 | 41 |
Pigs | 48 | 0 | 0 | 1 | 48 |
Chickens | 34 | 0 | 0 | 8 | 34 |
Geese | 101 | 58 | 51 | 59 | 95a |
Gulls | 16 | 0 | 0 | 6 | 14 |
Pigeons | 25 | 1 | 0 | 2 | 22 |
Ducks | 10 | 0 | 0 | 0 | 10 |
Swans | 1 | 0 | 0 | 0 | 1 |
Moose | 10 | 0 | 0 | 0 | 10 |
Deer | |||||
White tailed | 10 | 0 | 0 | 0 | 10 |
Mule | 10 | 0 | 0 | 0 | 10 |
Fallow | 10 | 0 | 0 | 0 | 10 |
Caribou | 10 | 0 | 0 | 0 | 10 |
Bison | 10 | 0 | 0 | 0 | 10 |
Goats | 10 | 0 | 0 | 0 | 10 |
Horses | 15 | 0 | 0 | 0 | 15 |
Total | 392 | 59 | 51 | 77 | 381 |
TABLE 2.
Levels of E. coli and incidences of the Canada goose-specific (CGOF1-Bac and CGOF2-Bac), human-specific (BacH), and generic (All-Bac) Bacteroidales 16S rRNA markers at the different Wascana Lake sites sampled weeklyaSite | E. coli | All-Bac | CGOF1-Bac | CGOF2-Bac | BacH | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
No. of positive water samples/total no. of samples analyzed (%) | Min level-max level (MPN 100 ml−1) | Mean level (MPN 100 ml−1) | No. of positive water samples/total no. of samples analyzed (%) | Min level-max level (log copies 100 ml−1) | Mean level (log copies 100 ml−1) | No. of positive water samples/total no. of samples analyzed (%) | Min level-max level (log copies 100 ml−1) | Mean level (log copies 100 ml−1) | No. of positive water samples/total no. of samples analyzed (%) | Min level-max level (log copies 100 ml−1) | Mean level (log copies 100 ml−1) | No. of positive water samples/total no. of samples analyzed | Min level-max level (log copies 100 ml−1) | Mean level (log copies 100 ml−1) | |
W1 | 8/8 (100) | 6-196 | 71.1 | 8/8 (100) | 6.2-8.1 | 6.9 | 6/8 (75) | 0-4.7 | 2.4 | 4/8 (50) | 0-4 | 1.7 | 2/8 | 0-3.7 | 1.7 |
W2 | 9/10 (90) | 0-1,120 | 194 | 10/10 (100) | 5.8-6.8 | 6.4 | 9/10 (90) | 0-3.7 | 2.6 | 8/10 (80) | 0-3.3 | 2.2 | 0/10 | 0 | 0 |
W3 | 10/10 (100) | 6-1,550 | 534 | 10/10 (100) | 6-7.8 | 7 | 10/10 (100) | 2.9-4.8 | 3.8 | 10/10 (100) | 2-4.5 | 3.4 | 0/10 | 0 | 0 |
W4 | 10/10 (100) | 16-1,732 | 529 | 10/10 (100) | 6.4-7.6 | 7 | 10/10 (100) | 3.2-4.6 | 3.9 | 10/10 (100) | 2.8-4.3 | 3.4 | 0/10 | 0 | 0 |
W5 | 10/10 (100) | 2-2,420 | 687 | 10/10 (100) | 5.5-6.9 | 6.3 | 7/10 (70) | 0-3.2 | 1.7 | 5/10 (50) | 0-3.1 | 1.2 | 0/10 | 0 | 0 |
W6 | 10/10 (100) | 3-1,990 | 389 | 10/10 (100) | 5.5-7 | 6.3 | 9/10 (90) | 0-4.3 | 2.8 | 6/10 (60) | 0-5.1 | 2 | 1/10 | 0-3.4 | 1.3 |
W7 | 7/7 (100) | 5-2,420 | 445 | 7/7 (100) | 5.7-7.8 | 7 | 6/7 (86) | 0-3.8 | 2.6 | 5/7 (71) | 0-4.4 | 2.4 | 2/7 | 0-5.1 | 2.8 |
W8 | 10/10 (100) | 17-980 | 160 | 10/10 (100) | 6.3-8.6 | 7.1 | 8/10 (80) | 0-4.6 | 2.8 | 7/10 (70) | 0-4.4 | 2.3 | 0/10 | 0 | 0 |