Assessment of biochemical methods to detect enzymatic depolymerization of polysaccharides

Biochemical methods for detection of depolymerisation were compared. Currently used methods such as reducing sugars assays, double bond monitoring or molecular weight determination were tested to follow the kinetic of depolymerization with different enzyme/polysaccharide combinations. The range of concentrations of different enzymes allowed us to identify the most sensitive and appropriate method to detect polysaccharide degradation. Reducing sugars assays are quantitative, sensitive and usable with all kind of polysaccharide but some compounds may interfere with them. When the polysaccharide is charged, agarose gel electrophoresis, although being a qualitative assay is as sensitive as high performance size exclusion chromatography analysis, easy to handle, high-throughput and this is preferred.     

The relative merits of foregut and hindgut fermentation

Mammalian herbivores cannot break down cellulose except by fermentation, and may have Termentation chambers at either end of the gut: ruminants have their principal fermentation chamber in the stomach but horses ferment only in the hindgut. A mathematical model (Alexander, 1991) predicted that foregut fermenters should do better than hindgut fermenters on poor foods, and the reverse on richer. less fibrous foods. Further, the optimum gut for poor foods would have the hindgut fermentation chamber only a little smaller than the foregut chamber. However. it has been claimed that horses do better than ruminants on poor food, and the hindgut of ruminants is much smaller than the rumen.
In this paper, the basic model is modified in ways designed to make it more realistic and the effects are investigated. None of the modifications alters the conclusion that the optimum gut for poor food has a large foregut fermentation chamber. However, the optimum proportions of fore-to hindgut, for poor diets, become more like those of real ruminants when account is taken of the diminishing volume of the food passing through the gut, and of incomplete mixing in the rumen.     

Chemical methods to detect S-nitrosation

Nitric oxide (NO) is a cell-signaling molecule involved in a number of physiological and pathophysiological processes. Modification of cysteine residues by NO (or NO metabolites), that is S-nitrosation, changes the function of a broad spectrum of proteins. This reaction represents an important post-translational modification that transduces NO-dependent signals. However, the detection and quantification of S-nitrosation in biological samples remain a challenge mainly because of the lability of S-nitrosation products: S-nitrosothiols (SNO). In this review we summarize recent developments of the methods to detect S-nitrosation. Our focus is on the methods which can be used to directly conjugate the site(s) of S-nitrosation.     

Multiple hypothesis testing to detect lineages under positive selection that affects only a few sites

Detection of positive Darwinian selection has become ever more important with the rapid growth of genomic data sets. Recent branch-site models of codon substitution account for variation of selective pressure over branches on the tree and across sites in the sequence and provide a means to detect short episodes of molecular adaptation affecting just a few sites. In likelihood ratio tests based on such models, the branches to be tested for positive selection have to be specified a priori. In the absence of a biological hypothesis to designate so-called foreground branches, one may test many branches, but a correction for multiple testing becomes necessary. In this paper, we employ computer simulation to evaluate the performance of 6 multiple test correction procedures when the branch-site models are used to test every branch on the phylogeny for positive selection. Four of the methods control the familywise error rates (FWERs), whereas the other 2 control the false discovery rate (FDR). We found that all correction procedures achieved acceptable FWER except for extremely divergent sequences and serious model violations, when the test may become unreliable. The power of the test to detect positive selection is influenced by the strength of selection and the sequence divergence, with the highest power observed at intermediate divergences. The 4 correction procedures that control the FWER had similar power. We recommend Rom's procedure for its slightly higher power, but the simple Bonferroni correction is useable as well. The 2 correction procedures that control the FDR had slightly more power and also higher FWER. We demonstrate the multiple test procedures by analyzing gene sequences from the extracellular domain of the cluster of differentiation 2 (CD2) gene from 10 mammalian species. Both our simulation and real data analysis suggest that the multiple test procedures are useful when multiple branches have to be tested on the same data set.     

Real-time molecular methods to detect infectious viruses

Waterborne transmitted viruses pose a public health threat due to their stability in aquatic environment and the easy transmission with high morbidity rates at low infectious doses. Two major challenge of virus analysis include a lack of adequate information in infectivity and the inability to cultivate certain epidemiologically important viruses in vitro. The use of fluorescent probes in conjunction with fluorescence microscopy allows us to reveal dynamic interactions of the viruses with different cellular structures in living cells that are impossible to detect by immunological or PCR-based experiments. Real-time viral detection in vivo provides sufficient information regarding multiple steps in infection process at molecular level, which will be valuable for the prevention and control of viral infection.     

Improved methods to detect GTP-binding proteins from plants

Improved methods are described for the detection of G1P-binding proteins (G-proteins) in the protonema of mossFunaria hygrometrica and coleoptiles of corn(Zea mays) and sorghum(Sorghum vulgare). We optimized conditions for the transfer of proteins to nitrocellulose, production of high titer polyclonal anti-Gα (common) antibodies and finally the detection of G-proteins by amplification. In addition to the α-subunit of heterotrimeric G-proteins (M _r 41–43 kDa), a small molecular weight class (< 30 kDa) was also detected by anti-Gα (common) antibodies. An easy, reliable and efficient filter assay is also described to quantify the toxin catalyzed ADP-ribosylation. The apparentK _m of the NAD has been determined to be approximately 1.5μM for the microsomal fraction of moss. Inclusion of G1P stimulated ADP-ribosylation by 2–27-fold. One to three polypeptides representing the α-subunit of heterotrimeric G-proteins of (M_r 37–43 kDa) were ADP-ribosylated in all three plants. The anti-Gβ (C-terminus) antibody cross-reacted strongly with 39 and 34 kDa polypeptide in moss and corn respectively. By employing improved methods two classes of G-proteins have been shown to be present in three plant species.     

Morphometric evolutionary trends in the dental complex ofPongo

This paper examines whether there has been an overall reduction in the likely size ofPongo from the Pleistocene to the present day. It is concluded that the original suggestion byHooijer (1948) that the evolution ofPongo over the last half-million years was characterized by a reduction in dental size, corresponding to a reduction in overall body size, is likely. There were, however, fluctuations in dental size during the Pleistocene. Overall, in the Sundaic region a slight reduction in overall dental size continued from Late Pleistocene to the present day.     

Exploring large vegetation databases to detect temporal trends in species occurrences

Question: Can vegetation relevé databases be used to analyse species losses and gains in specific vegetation types in Germany over time? Does the type of response (increase or decline in relative frequency) conform to observed large‐scale environmental trends in the last decades? Location: Germany. Exploring the German Vegetation Reference Database Halle (GVRD) that was established for forest and grassland vegetation within the framework of German Biodiversity Exploratories. Methods: Use of generalized linear models (GLMs) for testing changes in temporal frequency of plant taxa in a semi‐dry grassland data set (Mesobromion) and a beech forest data set (Fagion). Data were either aggregated by year, decade or by a balanced re‐sampling approach. Interpretation of the observed changes was based on species traits. Results: In both data sets significant temporal changes were observed, although the frequency of the majority of species remained unchanged. In both data sets, species with a temporal increase in frequency had higher Ellenberg N and F indicator values, compared to species that decreased, thus indicating effects of widespread atmospheric nitrogen deposition. In the forest data set, the observed increase in recruitment of deciduous trees pointed to a change in management, while trends in the grassland data set suggested use abandonment, as seen in an increased frequency of woody species. Conclusion: We demonstrate that vegetation databases represent very valuable resources for analysis of temporal changes in species frequencies. GLMs proved their value in detecting these trends, as also shown by the interpretability of model results with species traits. In contrast, the method of aggregation or re‐sampling had little influence on the general outcome of analyses.     

The relative merits of open- and closed-path analysers for measurement of eddy fluxes

Theoretical and practical aspects of measuring eddy fluxes of trace gases using open-and closed-path analysers are presented. Trace gas fluxes measured with an open-path analyser require the concurrent measurement of sensible and latent heat fluxes to correct for density fluctuations in trace gas concentration caused by these fluxes. A closed-path analyser eliminates the corrections due to sensible heat flux, but not for water vapour, provided temperature fluctuations are completely removed without significantly reducing fluctuations in the trace gas mixing ratio. Theory for the design of heat exchangers and for the attenuation of concentration fluctuations during air flow through tubes is used to provide design criteria for closed-path systems. Spectral transfer functions are used to estimate flux losses caused by flow through the sampling tube and gas analyser. Other factors considered include cross-sensitivity of infrared CO₂ analysers to water vapour, and deterioration of system performance caused by contaminants on the walls of sampling tubes. Of two open-path, infrared CO₂ analysers tested, one showed a strong interaction between CO₂ and water vapour, while the other showed little sensitivity to the presence of water vapour, other than caused by dilution. A commercial closed-path CO₂ analyser also showed little cross-sensitivity to water vapour. Compared to results for a clean sampling tube, the spectral bandwidth for water vapour fluctuations decreased significantly after several weeks of sampling. No such deterioration in bandwidth was observed for CO₂. These findings are attributed to differential adsorption/desorption of water vapour by dust or salt on the tubing walls. Rain and dust must be removed from open-path analysers to obtain satisfactory measurements. Careful system design and maintenance is required for both open- and closed-path systems to ensure satisfactory long-term measurement of trace gas fluxes. With these precautions, both approaches will provide satisfactory flux measurements.     

Chemiluminescent-based methods to detect subpicomole levels of c-type cytochromes

A variety of luminol-based substrates and either an autoradiographic film or a charge-coupled device (CCD) imaging system were used for chemiluminescence detection of c-type cytochromes. The Pierce Femto peroxidase substrate was at least 10 times more sensitive when using film than the highly cited 3,3('),5,5(')-tetramethylbenzidine (benzidine derivative) staining method and 50 times more sensitive when using a CCD imaging system. Film or CCD imaging result in highly sensitive and quantitative signals. The quantitative detection of c-type cytochromes from single colonies or from less than 1ml of a bacterial culture is possible.     

Comparison of methods to detect resistance of Penicillium expansum to thiabendazole

Aims:  Because of the lack of a standard method, the aim of this work is to evaluate the suitability of the broth microdilution method CLSI M38-A in determining the resistance level of some Penicillium expansum isolates to thiabendazole (TBZ). The ability of the isolates to produce patulin (PAT) and citrinin (CIT) has been also assessed.
Methods and Results:  Penicillium expansum isolates (128) were assayed (apples, pears, grapes and five reference strains). It was observed that 69·4% of the strains isolated from apples and pears were resistant to TBZ. Sensitive isolates were inhibited at 0·25–0·5 μg ml⁻¹ whilst resistant isolates still grew at 512 μg ml⁻¹. PAT was produced by all P. expansum isolates. CIT was detected in 98·8% of TBZ-resistant isolates and in 89·1% of the TBZ-sensitive isolates.
Conclusions:  The preliminary screening method combined with the adaptation of the method CLSI M38-A, can be a good strategy to be used in assessing the in vitro activity of TBZ against a large number of isolates.
Significance and Impact of the Study:  The proposed methodology can be a contribution to the standardization of susceptibility tests to fungicides against P. expansum.     

Screening methods to detect toxigenic fungi in liquid medium

Rapid and sensitive methods for identifying toxin production by toxigenic fungi cultivated in liquid medium were developed. The production of aflatoxins B₁, B₂, G₁, G₂, sterigmatocystin, ochratoxin A, patulin, penicillic acid, citrinin and zearalenone was detected employing thin layer chromatography and high performance liquid chromatography detecion with or without extraction and purification procedures. No significant variation was found and toxins could be detected after 2–4 days incubation. The sensitivity of the methods and recovery after extraction have been estimated.     

Improved immunomatrix methods to detect protein:protein interactions

Immunoprecipitation (IP) and coimmunoprecipitation (co-IP) are key techniques for studying protein-protein interactions. These methods utilize immobilized Protein A or Protein G to isolate antibody-bound target antigens. The main disadvantage of traditional IP and co-IP is that the conditions used to elute the precipitated antigen also release the antibody thus contaminating the antigen and destroying the antibody support. To overcome these problems, we describe two methods to generate a reusable antibody support by cross-linking the antibody to immobilized Protein A or Protein G, or by coupling it directly to the resin (see Scheme 1). Antibody cross-linking can be done in 1 h while antibody coupling requires 4 h. IP or co-IP is accomplished by incubating the antibody resin with the protein sample. Washes and elutions are carried out in a spin column to reduce resin loss and decrease assay time. Target proteins are eluted with 0.1 M glycine (pH 2.8) and the resin-bound antibody is re-equilibrated in phosphate-buffered saline (PBS) for reuse. Our studies have demonstrated that the immobilization efficiency for the antibody coupling method was similar for several species of antibody. Furthermore, we illustrate that using both methods of antibody immobilization yield IP and co-IP results similar to traditional protocols but eliminate the antibody heavy and light chain contamination.     

Integrative tracking methods elucidate the evolutionary dynamics of a migratory divide

Migratory divides, the boundary between adjacent bird populations that migrate in different directions, are of considerable interest to evolutionary biologists because of their alleged role in speciation of migratory birds. However, the small size of many passerines has traditionally limited the tools available to track populations and as a result, restricted our ability to study how reproductive isolation might occur across a divide. Here, we integrate multiple approaches by using genetic, geolocator, and morphological data to investigate a migratory divide in hermit thrushes (Catharus guttatus). First, high genetic divergence between migratory groups indicates the divide is a region of secondary contact between historically isolated populations. Second, despite low sample sizes, geolocators reveal dramatic differences in overwintering locations and migratory distance of individuals from either side of the divide. Third, a diagnostic genetic marker that proved useful for tracking a key population suggests a likely intermediate nonbreeding location of birds from the hybrid zone. This finding, combined with lower return rates from this region, is consistent with comparatively lower fitness of hybrids, which is possibly due to this intermediate migration pattern. We discuss our results in the context of reproductive isolating mechanisms associated with migration patterns that have long been hypothesized to promote divergence across migratory divides.     

Assessment of a rigorous transitive profile based search method to detect remotely similar proteins

Profile-based sequence search procedures are commonly employed to detect remote relationships between proteins. We provide an assessment of a Cascade PSI-BLAST protocol that rigorously employs intermediate sequences in detecting remote relationships between proteins. In this approach we detect using PSI-BLAST, which involves multiple rounds of iteration, an initial set of homologues for a protein in a 'first generation' search by querying a database. We propagate a 'second generation' search in the database, involving multiple runs of PSI-BLAST using each of the homologues identified in the previous generation as queries to recognize homologues not detected earlier. This non-directed search process can be viewed as an iteration of iterations that is continued to detect further homologues until no new hits are detectable. We present an assessment of the coverage of this 'cascaded' intermediate sequence search on diverse folds and find that searches for up to three generations detect most known homologues of a query. Our assessments show that this approach appears to perform better than the traditional use of PSI-BLAST by detecting 15% more relationships within a family and 35% more relationships within a superfamily. We show that such searches can be performed on generalized sequence databases and non-trivial relationships between proteins can be detected effectively. Such a propagation of searches maximizes the chances of detecting distant homologies by effectively scanning protein "fold space".     

不同方法检测血清HBV DNA水平的比较

目的:研究不同方法检测血清HBVDNA水平的比较.方法:采用151份标本分别采用COBAS Amplicor、实时定量PCR(LightCycler及Mx3000p)方法进行平行检测.结果:①检测灵敏度:COBAS Amplicor方法灵敏度高于实时PCR方法(P=0.005、0.014),而Mx3000p灵敏度高于LightCycler方法(P=0.042).②相关性:实时定量PCR(LightCycler及Mx3000p)与COBAS Amplicor方法所测HBV DNA结果均呈线性相关(r=0.842,P＜0.01;r=0.946,P＜0.01).结论:实时定量PCR方法是较为经济且准确的HBV DNA检测方法,而Mx3000p在准确性和灵敏度上可能优于LightCycler方法.     

Power to detect trends in ecological indicators in the East Usambara Mountains, Tanzania

We evaluated the statistical power of monitoring protocols to detect, over a 10‐year period, trends in indices of abundance of primates, hornbills and forest interior dung beetles and growth rates of epiphytic ferns in the Amani Nature Reserve, in the East Usambara Mountains, Tanzania. Local technicians are responsible for the day‐to‐day gathering of data. The existing monitoring protocols for blue monkey, silver‐cheeked hornbills, trumpeter hornbills, forest interior dung beetles and Asplenium nidus have sufficient statistical power (>0.80) to detect trends of 65% or less over a 10‐year period. Monitoring protocols for black and white colobus and Asplenium holstii have lower statistical power (<0.80). We therefore conclude that the majority of monitoring protocols of the East Usambara Ecological Monitoring Project have the capacity both logistically and statistically to detect long‐term trends in important functional groups in the East Usambara Mountains.     

Comparison of methods to detect Pasteurella multocida in carrier waterfowl

We conducted laboratory challenge trials using mallard ducks (Anas platyrhynchos) to compare methods for detecting carriers of Pasteurella multocida, the bacterium that causes avian cholera, in wild birds. Birds that survived the initial infection were euthanized at 2-4 wk intervals up to 14 wk post challenge. Isolates of P. multocida were obtained at necropsy from 23% of the birds that survived initial infection. We found that swab samples (oral, cloacal, nasal, eye, and leg joint) were most effective for detecting carrier birds up to 14 wk post infection. No detectable differences in isolation were observed for samples stored in either 10% dimethysulfoxide or brain heart infusion broth. The frequency of detecting carriers in our challenge trials appeared to be related to mortality rates observed during the trial, but was not related to a number of other factors including time after challenge, time delays in collecting tissues postmortem, and route of infection. In our trials, there was little association between antibody levels and carrier status. We concluded that swabs samples collected from recently dead birds, stored in liquid nitrogen, and processed using selective broth provide a feasible field method for detecting P. multocida carriers in wild waterfowl.