Site-specific Reagents Research Articles

Developing global whole cell standards for the microbiome field

The microbiome field has developed rapidly over the last decade, however there is no effective standardisation of protocols in place, with no accredited or certified reference materials available to the wider community. As part of the NIBSC Microbiome program, we developed whole cell standards for microbiome research which can act as global working standards and will be put forward for consideration as the first whole cell World Health Organization International Reference Reagents for microbiome analysis. The developed reagents consist of common bacteria found in human gut and have been tested using different commercial DNA extraction kits in order to detect biases introduced through different DNA extraction processes. Multiple replicates of DNA extracted the whole cell standard using different DNA extraction kits were assessed using Next Generation Sequencing and evaluated using the number of input cells in the standards, as measured by microscopy and flow cytometry. These NIBSC whole cell standards are aimed to standardise the DNA extraction steps in microbiome analytical pipelines and serve as a tool to more accurately capture the representation of each microbe in the gut microbiome. This could be achieved by setting a specific threshold of accepted error levels by the microbiome community and we aim to set this through a large collaborative study in 2020-21. This work is part of a larger NIBSC microbiome program that is developing site specific DNA, whole cell and matrix spike-in reagents for use in microbiome research.

Developing standards for the microbiome field

BackgroundEffective standardisation of methodologies to analyse the microbiome is essential to the entire microbiome community. Despite the microbiome field being established for over a decade, there are no accredited or certified reference materials available to the wider community. In this study, we describe the development of the first reference reagents produced by the National Institute for Biological Standards and Control (NIBSC) for microbiome analysis by next-generation sequencing. These can act as global working standards and will be evaluated as candidate World Health Organization International Reference Reagents.ResultsWe developed the NIBSC DNA reference reagents Gut-Mix-RR and Gut-HiLo-RR and a four-measure framework for evaluation of bioinformatics tool and pipeline bias. Using these reagents and reporting system, we performed an independent evaluation of a variety of bioinformatics tools by analysing shotgun sequencing and 16S rRNA sequencing data generated from the Gut-Mix-RR and Gut-HiLo-RR. We demonstrate that key measures of microbiome health, such as diversity estimates, are largely inflated by the majority of bioinformatics tools. Across all tested tools, biases were present, with a clear trade-off occurring between sensitivity and the relative abundance of false positives in the final dataset. Using commercially available mock communities, we investigated how the composition of reference reagents may impact benchmarking studies. Reporting measures consistently changed when the same bioinformatics tools were used on different community compositions. This was influenced by both community complexity and taxonomy of species present. Both NIBSC reference reagents, which consisted of gut commensal species, proved to be the most challenging for the majority of bioinformatics tools tested. Going forward, we recommend the field uses site-specific reagents of a high complexity to ensure pipeline benchmarking is fit for purpose.ConclusionsIf a consensus of acceptable levels of error can be agreed on, widespread adoption of these reference reagents will standardise downstream gut microbiome analyses. We propose to do this through a large open-invite collaborative study for multiple laboratories in 2020.93HHEig7BSVpjCFc6M9PenVideo

Using nucleases to stimulate homologous recombination.

In essentially all organisms, double-strand breaks in chromosomal DNA stimulate repair by multiple mechanisms, including homologous recombination. It is possible to use site-specific reagents to produce a break or other recombinagenic damage at a unique site, which makes possible detailed analysis of the repair products. In addition, targeted mutagenesis and gene replacement are stimulated in the immediate vicinity of the break site. To utilize meganucleases with long recognition sequences, it is necessary to introduce the corresponding sequence prior to directed cleavage. The same is typically true of triplex-forming oligonucleotides that target polypurine-polypyrimidine tracts. Zinc-finger nucleases have the potential of being targetable to arbitrarily selected sites, owing to the flexibility of zinc finger recognition of DNA.

Designing and Synthesis of Site-Specific DNA Cleavage Reagents

Designing and Synthesis of Site-Specific DNA Cleavage Reagents

Preparation of avidin-labeled protein nanoparticles as carriers for biotinylated peptide nucleic acid

The possibility of preparing protein nanoparticles followed by covalent linkage of avidin was investigated. Free sulfhydryl groups were introduced onto the surface of protein nanoparticles either by aldehyde quenching with cysteine or reaction of free amino groups with 2-iminothiolane. The number of primary amino groups and sulfhydryl groups on the surface of the resulting particles was quantified with site-specific reagents. Avidin was attached to the surface of the thiolated nanoparticles via a bifunctional spacer which reacted in a first step with amino groups of avidin and in a second step with the sulfyhdryl groups introduced onto the surface of the nanoparticles. Biotinylated peptide nucleic acid (PNA) as a model compound for biotinylated drugs was effectively coupled to the nanoparticles by complex formation with the covalently attached avidin. Since the formation of the interaction between biotin and avidin is very rapid and stable a highly effective drug carrier system for biotinylated compounds such as PNAs was achieved.

Preparation of avidin-labelled gelatin nanoparticles as carriers for biotinylated peptide nucleic acid (PNA)

The possibility of preparing uniform nanoparticles consisting of proteins such as gelatin followed by covalent linkage of avidin was investigated. Gelatin nanoparticles were prepared by two step desolvation. Functional groups at the surface of the particulate system were quantified with site-specific reagents. The surface of the nanoparticles was thiolated and avidin was covalently attached to the nanoparticles via a bifunctional spacer at high levels. Biotinylated peptide nucleic acid (PNA) was effectively complexed by the avidin-conjugated nanoparticles. Avidin-conjugated protein nanoparticles should prove as potential carrier system for biotinylated drug derivatives in antisense therapy.

Involvement of the consensus sequence motif at coil 2b in the assembly and stability of vimentin filaments.

Nearly all intermediate filament (IF) proteins share two sequence motifs located at the N- and the C-terminal ends of their helical rod domain ('coil 1a' and 'coil 2b', respectively). To examine the structural role of the coil 2b motif, we have performed in vitro assembly studies and in vivo microinjection experiments employing two site-specific reagents: (a) a 20-residue synthetic peptide (C-2) representing the conserved motif itself and (b) a monoclonal antibody (anti-IFA) that recognises an epitope within the conserved coil 2b sequence. We demonstrate here that vimentin protofilaments, when induced to assemble in the presence of C-2 or anti-IFA, show a lower propensity to polymerise and yield various abberant structures. The few filaments that are formed under these conditions appear much shorter than normal IFs and are unravelled or aggregated. Furthermore, when preformed vimentin filaments are exposed to C-2 or anti-IFA, most of the normal IFs are converted into shorter filamentous forms that possess an abberant morphology. None of these effects is seen when vimentin subunits are coincubated with control peptides. Microinjection of anti-IFA into the cytoplasm of interphasic 3T3 cells provokes collapse of vimentin IFs into a juxtanuclear mass and formation of numerous amorphous aggregates distributed throughout the cytoplasm. These two effects are not seen when the anti-IFA is microinjected into the cell nucleus. Our results provide experimental evidence supporting previous suggestions for a role for the conserved coil 2b sequence in filament assembly. We propose that this region is interacting with other sites along the vimentin molecule and that these interactions are essential for proper protofilament-protofilament alignment and filament stability.

Esterase-like activity of human serum albumin. VIII. Reaction with amino acid p-nitrophenyl esters.

The reactions of human serum albumin (HSA) with optically active amino acid p-nitrophenyl esters (substrate, S) were examined kinetically at 25 degrees C. The rate data were analyzed in terms of a mechanism involving 1:1 complexing (S.HSA) between S and HSA. The dissociation constant (Ks in M) and the catalytic rate constant (k2 in s-1) of S.HSA were determined. Among ten substrates examined, the reactions with N-carbobenzoxy-D(L)-alanine p-nitrophenyl esters (N-CBZ-D(L)-AlaNP) were most accelerated by HSA. Results of the reaction in the presence of excess N-CBZ-D(L)-AlaNP over HSA indicated the existence of one strong reactive site on HSA. The effects of the reversible binding of the site-specific drug and the chemical modification by site-specific reagents on the HSA activity showed that the reactive site towards N-CBZ-D(L)-AlaNP is the R site located near tyrosine-411 residue of HSA.

Esterase-like Activity of Human Serum Albumin. VII. Reaction with p-Nitrophenyl 4-Guanidinobenzoate.

The reaction of p-nitrophenyl 4-guanidinobenzoate (NPGB) with human serum albumin (HSA) was examined kinetically at various pH's and 25 degrees C. The Michaelis constant (Ks in M) and the catalytic rate constant (k2 in s-1) were determined. The ratio of k2 to k0 (hydrolysis rate constant of NPGB in s-1) at pH 7.4 was 75.6, indicating the esterase-like activity of HSA. The effects of the reversible binding of site-specific drugs and the chemical modification by site-specific reagents on the HSA activity indicated that HSA has multiple reactive sites towards NPGB. Results of the reaction in the presence of excess NPGB over HSA also suggested the existence of multiple active sites. The pH-profile for k2 showed inflection points at about pH 6.0 and pH 10.0, suggesting the involvement of groups with pKa's of 6.0 and 10.0 in HSA.

Heat stable proteinase from Thermomonospora fusca

An extracellular proteinase secreted by the thermophilic bacteria Thermomonospora fusca YX (YX-proteinase) is a serine proteinase as shown by its inactivation by the site specific reagents, phenylmethanesulfonyl fluoride, dansyl fluoride, and carbobenzoxy-L-phenylalanine chloromethyl ketone. This conclusion is further supported by the effect of various proteinase inhibitors on its activity. The activity of the proteinase toward small synthetic ester substrates shows that the enzyme has a primary specificity for the aromatic and hydrophobic amino acids. The amino acid composition and NH2-terminal sequence, as well as its size, suggest that the enzyme is related to the chymotrypsin-like microbial proteinase, alpha-lytic protease from Myxobacter 495 and protease A and B from Streptomyces griseus.

Ecto-ATPase activity in cytolytic T-lymphocytes. Protection from the cytolytic effects of extracellular ATP.

Addition of ATP or ATP analog to the incubation media is shown to result in cell death in experiments with different cultured cell lines as evidenced by the results of several independent assays, both in the absence or presence of extracellular Ca2+. Cytolytic T-lymphocyte (CTL) clone itself was not only resistant to cytolytic effects of ATP, but was able to "rescue" antigen-nonbearing 51Cr-labeled cells from lytic effects of extracellular ATP (but not from lytic effects of adenosine 5'-thiotriphosphate) when present during assay. To test whether the resistance of CTL to ATP is due to a high activity of ecto-ATPase, four independent assays of ATPase activity were utilized to demonstrate the presence and relatively high activity of the ecto-ATPase(s) on CTL surface. Studies of substrate specificity of CTL ecto-ATPase suggest that there is more than one nucleoside 5'-triphosphatase on the surface of CTL. The enzyme(s) activity is Ca2+- and Mg2+-dependent and in this respect is similar to recently described hepatic cells ecto-ATPase. We tested effects of known ATP-binding site-specific reagents fluorescein 5'-isothiocyanate (FITC) and 5'-fluorosulfonylbenzoyladenosine (FSBA) to find covalent modification procedures to be used in studies of functional role of ecto-ATPase. FSBA, but not FITC, inhibits lymphocyte ecto-ATPase but addition of ATP together with FSBA protects ecto-ATPase activity. Inactivation of CTL ecto-ATPase by pretreatment with FSBA makes CTL susceptible to lytic effects of extracellular ATP, as was hypothesized for the functional role of this enzyme in CTL.

Sequential Derivatization and the SIMS Imaging of Coal

Abstract Secondary Ion Mass Spectrometry (SIMS) has been used to examine the low-temperature oxidation of coal, using 18O2 and in conjunction with site-specific reagents in order to identify the surface distribution of oxygen functional groups. This approach leads to some ambiguity in the assignment of functional groups because the inherent complexity of coal chemistry rarely allows any definitive reaction products to be identified. This paper describes results obtained from reaction of coal surfaces with a sequence of reagents designed to remove ambiguous results. This procedure, coupled with SIMS imaging, yields results consistent with an oxidation mechanism which involves the formation of surface peroxides.

The immunodominant site of a synthetic immunogen has a conformational preference in water for a type-II reverse turn.

Many short synthetic peptides have now been shown to induce antibodies reactive with their cognate sequences in the intact folded protein. Aside from the usefulness of such antibodies as site-specific reagents, the frequency with which this recognition occurs has raised several theoretical issues, the central one being that of how an antibody to a short synthetic peptide, which represents one of the most disordered states of a site in a protein, can react with the more ordered version of the same sequence in the folded protein. This apparent paradox can be resolved if the target site on the protein approaches disorder or if the peptide in solution or on a carrier adopts, with significant frequency, a conformation compatible with that of the cognate site in the protein. Various studies already suggest that antigenic sites in proteins correspond to regions of high atomic mobility. We now show, using high-field nuclear magnetic resonance (NMR) spectroscopy, that a nonapeptide selected by several monoclonal antibodies as the immunodominant site of a 36-amino-acid immunogen (residues 75-110 of influenza virus haemagglutinin) adopts a highly populated type-II reverse-turn conformation in water. This suggests that in this case the antibodies have selected a sequence possessing a conformational preference. Apart from helping us to understand immunological recognition, anti-peptide antibodies may provide reagents of sufficient precision for an immunological approach to the problem of protein folding.

Interference of active site specific reagents in plasminogen-streptokinase active site formation.

We have recently observed slow, non-Michaelis-Menten kinetics of activation of native cat plasminogen by catalytic concentrations of streptokinase. In order to understand the reasons for this phenomenon, we undertook to study the formation of the plasminogen-streptokinase activator complex under the same plasminogen activation conditions. The results obtained in this study show that the potential active site in both cat and human plasminogen is capable of binding strongly the specific substrates (S) p-nitrophenyl p-guanidinobenzoate (NPGB) and H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide, through the active site is incapable of hydrolyzing these substrates. Binding studies support these and the following conclusions. Streptokinase binds to this zymogen-substrate complex to create the ternary plasminogen-S-streptokinase complex, which then slowly converts to an acylated plasminogen-streptokinase form. This acylation reaction is 550 times slower than acylation of the preformed plasminogen-streptokinase complex by NPGB. The same reaction also occurs with human plasminogen, though the acylation reaction is 10 times faster than when the cat zymogen is used. NPGB binds specifically to plasminogen but not to streptokinase. These studies proved that inhibition of cat plasminogen activation by streptokinase occurs at the level of activator complex formation. We conclude from our studies that streptokinase binding to both cat and human plasminogen occurs at the potential active site of the zymogen. Consequently, it is probable that plasminogen activation in vivo is inhibited by binding of active site specific inhibitors to plasminogen.

Influence of inactivation of trypsin on immunoreactivity and serum immunoreactive trypsin concentration measured by radioimmunoassay.

The influences of various active site-specific reagents of trypsin and protease inhibitors on the immunoreactivity of trypsin and serum trypsin concentration have been studied by radioimmunoassay (RIA). The RIA using inactivated 125I-trypsin as tracer showed lower Bo/T than the RIA using active 125I-trypsin, but the coefficient of variance of the former was smaller than that of the latter. Normal serum trypsin concentrations were 26.12-36.38 ng/ml with the RIA using inactivated 125I-trypsin as antigen tracer, and 201.15 ng/ml with the RIA using active 125I-trypsin as tracer. The recovery experiment showed that the difference was due to the interaction of serum protease inhibitors and labeled active trypsin.

24] Two-protonic-state electrophiles as probes of enzyme mechanism

A two-protonic-state electrophile is a compound that exhibits different electrophilic reactivities in each of two protonic or ionization states and can be used as reactivity probes with special characteristics that permit the detection and characterization of interaction in molecules in general and in enzyme active centers. In particular, it is used as reporter-group delivery vehicles and in the isolation of thiol-enzymes by covalent chromatography. This chapter presents the theoretical basis for the use as reactivity probes of two-protonic-state electrophiles in general and reviews the applications of pyridyl disulfides in the isolation and study of enzymes. Disulfides containing the 2-pyridyl moiety have been shown to be exceedingly versatile and valuable reagents for the isolation and study by both structural and kinetic methods of thiol-containing molecules. Covalent chromatography is a rapid isolation technique for thiol-enzymes and peptides, and provides considerable economies in both time and materials. It is reported that to draw mechanistic conclusions from kinetic data obtained by using reactivity probes, it is important to take account of the possibility that reactions of enzyme nucleophiles with site-specific reagents that are not formally substrate analogs might well proceed through the intermediacy of adsorptive complexes, albeit characterized by rather large dissociation constants.

Binding of acetaldehyde to rat liver microsomes: Enhancement after chronic alcohol consumption

Summary Rat hepatic microsomal preparations were assessed for their capacity to bind exogenous 14C-acetaldehyde and 14C-acetaldehyde formed endogenously from 14C-ethanol in the microsomal ethanol-oxidizing system (MEOS). Exogenous acetaldehyde was found to bind to rat liver microsomes. Blocking of the free amino groups and thiol groups with site specific reagents (pyridoxal 5′-phosphate and p-hydroxymercuribenzoate) reduced the binding of acetaldehyde. Acetaldehyde formed endogenously from ethanol also bound to hepatic microsomes. This was increased after chronic alcohol consumption, in association with enhanced MEOS activity. The binding of acetaldehyde produced by MEOS was significantly greater than that of an equivalent amount of exogenous acetaldehyde. Thus, acetaldehyde produced in situ may exert local toxic effects on the endoplasmic reticulum.

Human red cell galactose 1-phosphate uridylyltransferase: Effects of site-specific reagents on catalytic activity

Inactivation studies with the group-specific reagents p-chloromercuribenzoate, iodoacetic acid, and diethylpyrocarbonate (ethoxyformic anhydride) have been performed on purified human red cell galactose 1-phosphate Uridylyltransferase. The enzyme is rapidly inactivated by all three reagents at 17 °C. Complementary substrate protection studies using UDP-glucose, UDP-galactose, and galactose 1-phosphate show that none of these substrates exerts any detectable protective effect against inactivation by p-chloromercuribenzoate, but the UDP-hexose substrates specifically shield the enzyme against inactivation by iodoacetic acid and diethylpyrocarbonate. Whereas the protection afforded to the enzyme by treatment with 1 m m amounts of the UDP-hexose substrates is only partial against inactivation by diethylpyrocarbonate, the protection against alkylation by iodoacetic acid is dramatic and complete at a substrate concentration between 25 to 50 μ m. However, the extent of alkylation is dependent upon the presence of dithioerythritol and in the absence of this reducing agent, only about 30% inactivation is possible. Dithioerythritol is also necessary for maximal catalytic activity. It is concluded that the enzyme contains both cysteinyl and histidyl residues within the active center of the enzyme and that these residues are essential for catalytic activity. It is presumed that the requirement for dithioerythritol is to reduce a disulfide bridge at or near the UDP-hexose binding site and expose an essential cysteinyl residue which is required for productive catalysis.

Isolation and characterization of the hatching enzyme from the amphibian, Xenopus laevis

The proteolytic activity released at the time of Xenopus laevis embryo hatching, termed the hatching enzyme, was purified and characterized in terms of its physical and enzymatic properties. Using predominantly isoelectric focusing and preparative ultracentrifugation, the enzyme was purified 2200-fold over the starting crude hatching media. From disc gel electrophoretic experiments, the most highly purified form of the enzyme had two enzymatically active charge isomers present with molecular weights of 62,500. With time, the purified enzyme gave rise to a family of enzymatically active charge isomeric proteins. The enzymatic activity of hatching enzyme toward its 125I-labeled natural substrate, the fertilization envelope, was optimal at pH 7.7 and was ionic strength dependent. The enzyme was inhibited by Zn 2+ and by EDTA. From inhibition by the site-specific reagents diisopropylfluorophosphate and phenylmethylsulfonylfluoride, we concluded that the enzyme was of the serine protease type, although its inhibition by Zn 2+ and EDTA prevents a clear and unequivocal classification of the protease. This enzyme is different from the hatching enzymes reported in fish and echinoderms, on the basis of size, but it is similar to that described in Rana chensinensis on the basis of size and specificity.

The pH-dependence of second-order rate constants of enzyme modification may provide free-reactant pKa values.

1. Reactions of enzymes with site-specific reagents may involve intermediate adsorptive complexes formed by parallel reactions in several protonic states. Accordingly, a profile of the apparent second-order rate constant for the modification reaction (Kobs., the observed rate constant under conditions where the reagent concentration is low enough for the reaction to be first-order in reagent) against pH can, in general, reflect free-reactant-state molecular pKa values only if a quasi-equilibrium condition exists around the reactive protonic state (EHR) of the adsorptive complex. 2. Usually the condition for quasi-equilibrium is expressed in terms of the rate constants around EHR: (formula: see text) i.e. k mod. less than k-2. This often cannot be assessed directly, particularly if it is not possible to determine kmod. 3. It is shown that kmod. must be much less than k-2, however, if kobs. (the pH-independent value of kobs.) less than k+2. 4. Since probable values of k+2 greater than 10(6)M-1.S-1 and since values of kobs. for many modification reactions less than 10(6)M-1.S-1, the equilibrium assumption should be valid, and kinetic study of such reactions should provide reactant-state pKa values. 5. This may not apply to catalyses, because for them the value of kcat./Km may exceed 5 X 10(5)M-1.S-1. 6. The conditions under which the formation of an intermediate complex by parallel pathways may come to quasi-equilibrium are discussed in the Appendix.