Process for the integrated extraction, identification and quantification of metabolites, proteins and RNA to reveal their co-regulation in biochemical networks

A novel extraction protocol is described with which metabolites, proteins and RNA are sequentially extracted from the same sample, thereby providing a convenient procedure for the analysis of replicates as well as exploiting the inherent biological variation of independent samples for multivariate data analysis. A detection of 652 metabolites, 297 proteins and clear RNA bands in a single Arabidopsis thaliana leaf sample was validated by analysis with gas chromatography coupled to a time of flight mass spectrometer for metabolites, two-dimensional liquid chromatography coupled to mass spectrometry for proteins, and Northern blot analysis for RNA. A subset of the most abundant proteins and metabolites from replicate analysis of different Arabidopsis accessions was merged to form an integrative dataset allowing both classification of different genotypes and the unbiased analysis of the hierarchical organization of proteins and metabolites within a real biochemical network.     

Taxonomic continuum of Alchemilla (Rosaceae) in Estonia

23 widespread apomictic Alchemilla microspecies occurring in Estonia are analyzed to investigate whether the species and higher rank taxa are distinct, how variable these taxa are and which characters distinguish them better. Cluster analysis, discriminant analysis, analysis of variance, principal component analysis and continuum analysis are used for data processing. The characters form four correlative groups, describing (i) vegetative and (ii) generative parts of the plant body, (iii) hairiness characters and, (iv) leaf teeth measurements. The best characters according to analysis of variance for disünguishing species are hairiness characters, but often they distinguish only few species very clearly and cannot be used for the remaining ones. Hence the other characters cannot be excluded. From the studied species only A. plicata, A. semilunaris and A. lindbergiana are significantly distinct from all others. The remaining ones form a complicated network of mutually indistinct pairs. Higher rank taxa — sections and series according to Rothmaler, Fröhner and Yuzepchuk are better separated, containing very few mutually indistinct pairs. Results from species centroids' clustering are most congruent with Fröhner's system, but still some changes seem to be necessary and a corrected system is proposed here. Section Plicatae is split into two series: Pubescentes and Barbulatae , sections Alchemilla, Ultravulgares and Decumbentes are joined as three series of section Hirsutae , and A. filicaulis is moved from section Plicatae to section Coriaceae. Coriaceae should also be split into three series: Exuentes (A. filicaulis), Glabricaules (A. glabricaulis) and Coriaceae (other species).     

Crystallographic study of endogenous alpha-amylase inhibitor from wheat

Endogenous alpha-amylase inhibitor from wheat has been crystallized by a microdialysis method. There are two forms of monoclinic crystal in a microdialysis cell with a space group of P2(1). The unit cell dimensions are a = 43.5 A, b = 64.8 A, c = 32.2 A, beta = 113 degrees for the rod-like crystal, and a = 42.5 A, b = 65.2 A, c = 32.2 A, beta = 112 degrees for the plate-like crystal. The former is suitable for structure analysis because it gives the sharp diffraction beyond 2.0 A resolution, and the latter tends to form a twin crystal. A heavy-atom derivative has been successfully prepared with the heavy-atom reagent K2PtCl4, and structure analysis is in progress.     

Survival analysis using auxiliary variables via multiple imputation, with application to AIDS clinical trial data

We develop an approach, based on multiple imputation, to using auxiliary variables to recover information from censored observations in survival analysis. We apply the approach to data from an AIDS clinical trial comparing ZDV and placebo, in which CD4 count is the time-dependent auxiliary variable. To facilitate imputation, a joint model is developed for the data, which includes a hierarchical change-point model for CD4 counts and a time-dependent proportional hazards model for the time to AIDS. Markov chain Monte Carlo methods are used to multiply impute event times for censored cases. The augmented data are then analyzed and the results combined using standard multiple-imputation techniques. A comparison of our multiple-imputation approach to simply analyzing the observed data indicates that multiple imputation leads to a small change in the estimated effect of ZDV and smaller estimated standard errors. A sensitivity analysis suggests that the qualitative findings are reproducible under a variety of imputation models. A simulation study indicates that improved efficiency over standard analyses and partial corrections for dependent censoring can result. An issue that arises with our approach, however, is whether the analysis of primary interest and the imputation model are compatible.     

Mass spectrometry-assisted study reveals that lysine residues 1967 and 1968 have opposite contribution to stability of activated factor VIII

The A2 domain rapidly dissociates from activated factor VIII (FVIIIa) resulting in a dampening of the activity of the activated factor X-generating complex. The amino acid residues that affect A2 domain dissociation are therefore critical for FVIII cofactor function. We have now employed chemical footprinting in conjunction with mass spectrometry to identify lysine residues that contribute to the stability of activated FVIII. We hypothesized that lysine residues, which are buried in FVIII and surface-exposed in dissociated activated FVIII (dis-FVIIIa), may contribute to interdomain interactions. Mass spectrometry analysis revealed that residues Lys(1967) and Lys(1968) of region Thr(1964)-Tyr(1971) are buried in FVIII and exposed to the surface in dis-FVIIIa. This result, combined with the observation that the FVIII variant K1967I is associated with hemophilia A, suggests that these residues contribute to the stability of activated FVIII. Kinetic analysis revealed that the FVIII variants K1967A and K1967I exhibit an almost normal cofactor activity. However, these variants also showed an increased loss in cofactor activity over time compared with that of FVIII WT. Remarkably, the cofactor activity of a K1968A variant was enhanced and sustained for a prolonged time relative to that of FVIII WT. Surface plasmon resonance analysis demonstrated that A2 domain dissociation from activated FVIII was reduced for K1968A and enhanced for K1967A. In conclusion, mass spectrometry analysis combined with site-directed mutagenesis studies revealed that the lysine couple Lys(1967)-Lys(1968) within region Thr(1964)-Tyr(1971) has an opposite contribution to the stability of FVIIIa.     

Capillary electrophoresis.

The past year has seen major advances in capillary electrophoresis in terms of broadening applicability. A variety of successful approaches to peptide/protein and DNA separation and analysis are now available, and techniques for saccharide analysis are developing rapidly. Capillary electrophoresis--mass spectrometry continues to demonstrate its potential as a tool for high-resolution structure analysis.     

Feature identification in circadian rhythms of mice strains using in vivo information

The objective of this work was to identify strain-specific characteristics from real-time measurements of circadian rhythms of two inbred mouse strains. In particular, heart rate, temperature, and activity data collected from A/J and C57BL/6J (B6) mice using telemetry are analyzed. The influence of activity on heart rate and temperature is minimized by correlation analysis followed by regression analysis. The correlation analysis is used to determine the length of the activity data filter that results in the best correlation between activity data and heart rate or temperature. After the activity data are filtered, they are used in regression analysis. The temperature and heart rate rhythms obtained as the intercepts of the regression analysis are interpreted as the zero-activity rhythms and consequently are good estimates of the circadian rhythms. The circadian temperature rhythms for the B6 mice follow a smoother cosine-like time waveform, whereas those for the A/J mice follow a more square-wave-like waveform. To quantify the difference between these two temperature rhythms, a feature based on Fourier analysis of the time-series data is used. Detrended fluctuation analysis is used to identify features in the heart rate rhythms. The results of this work show that the features for the circadian temperature and heart rate rhythms can be used as distinguishing characteristics of the A/J and B6 strains. This work provides the foundation for future studies directed at investigating the influence of chromosomal substitutions on the regulation of circadian rhythms in these two strains.     

Recommended Practice Regarding Selection of Sensitivity Analysis Methods Applied to Microbial Food Safety Process Risk Models

A guideline is presented for selection of sensitivity analysis methods applied to microbial food safety process risk (MFSPR) models. The guideline provides useful boundaries and principles for selecting sensitivity analysis methods for MSFPR models. Although the guideline is predicated on a specific branch of risk assessment models related to food-borne diseases, the principles and recommendations provided are typically generally applicable to other types of risk models. Applicable situations include: prioritizing potential critical control points; identifying key sources of variability and uncertainty; and refinement, verification, and validation of a model. Based on the objective of the analysis, characteristics of the model under study, amount of detail expected from sensitivity analysis, and characteristics of the sensitivity analysis method, recommendations for selection of sensitivity analysis methods are provided. A decision framework for method selection is introduced. The decision framework can substantially facilitate the process of selecting a sensitivity analysis method.     

Cumulant analysis in fluorescence fluctuation spectroscopy

A novel technique for the analysis of fluorescence fluctuation experiments is introduced. Fluorescence cumulant analysis (FCA) exploits the factorial cumulants of the photon counts and resolves heterogeneous samples based on differences in brightness. A simple analytical model connects the cumulants of the photon counts with the brightness epsilon and the number of molecules N in the optical observation volume for each fluorescent species. To provide the tools for a rigorous error analysis of FCA, expressions for the variance of factorial cumulants are developed and tested. We compare theory with experiment by analyzing dye mixtures and simple fluorophore solutions with FCA. A comparison of FCA with photon-counting histogram (PCH) analysis, a related technique, shows that both methods give identical results within experimental uncertainty. Both FCA and PCH are restricted to data sampling times that are short compared to the diffusion time of molecules through the observation volume of the instrument. But FCA theory, in contrast to PCH, can be extended to treat arbitrary sampling times. Here, we derive analytical expressions for the second factorial cumulant as a function of the sampling time and demonstrate that the theory successfully models fluorescence fluctuation data.     

Quantification of Orofacial Phenotypes in Xenopus

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.     

Genetic manipulation of Aspergillus nidulans: meiotic progeny for genetic analysis and strain construction

The multicellular microbial eukaryote Aspergillus nidulans is an excellent model for the study of a wide array of biological processes. Studies in this system contribute significantly to understanding fundamental biological principles and are relevant for biotechnology and industrial applications, as well as human, animal and plant fungal pathogenesis. A. nidulans is easily manipulated using classical and molecular genetics. Here, we describe the storage and handling of A. nidulans and procedures for genetic crossing, progeny analysis and growth testing. These procedures are used for Mendelian analysis of segregation of alleles to show whether a mutant phenotype segregates as a single gene and independent assortment of genes to determine the linkage relationship between genes. Meiotic crossing is used for construction of multiple mutant strains for genetic analysis. Genetic crossing and analysis of progeny can be undertaken in 2-3 weeks and growth testing takes 2-3 days.     

A collection of programs for one‐dimensional Ising analysis of linear repeat proteins with point substitutions

A collection of programs is presented to analyze the thermodynamics of folding of linear repeat proteins using a 1D Ising model to determine intrinsic folding and interfacial coupling free energies. Expressions for folding transitions are generated for a series of constructs with different repeat numbers and are globally fitted to transitions for these constructs. These programs are designed to analyze Ising parameters for capped homopolymeric consensus repeat constructs as well as heteropolymeric constructs that contain point substitutions, providing a rigorous framework for analysis of the effects of mutation on intrinsic and directional (i.e., N‐ vs. C‐terminal) interfacial coupling free‐energies. A bootstrap analysis is provided to estimate parameter uncertainty as well as correlations among fitted parameters. Rigorous statistical analysis is essential for interpreting fits using the complex models required for Ising analysis of repeat proteins, especially heteropolymeric repeat proteins. Programs described here are available at https://github.com/barricklab-at-jhu/Ising_programs .     

Dimensional analysis in mathematical biology I. General discussion

Dimensional analysis is discussed from the viewpoint of its basic group properties and shown to be an algebraic Abelian group that is useful for analysis of physical measurements. The application of the method to various types of equations and the formulation of previously unclassified dimensions are discussed. Functional dimensional analysis is applied to the problems of cell size and biomass proliferation; future applications are also noted. A number of dimensionless terms have been formulated for cellular physiochemical phenomena. They apparently represent the first systematic study of biological dimensionless numbers recorded in the literature. A dimensionless proliferation law is suggested. A brief analysis of the physical dimensionality associated with information measures is carried out. Entropy and “information” are shown to be completely different in their dimensional meaning; other informational measures of possible interest in biology are proposed. The dimensional coding and computor analysis of biomathematical equations is suggested.     

Positive association of CC2D1A and CC2D2A gene haplotypes with mental retardation in a Han Chinese population

The CC2D1A and CC2D2A genes are involved in Ca(2+)-regulated signaling pathways and have recently been implicated in the etiology of mental retardation (MR). The aim of this study was to investigate whether CC2D1A and CC2D2A polymorphisms are associated with susceptibility to MR in a Han Chinese population using a family based association approach. The sample included 172 trios (parents and offspring), and all subjects were genotyped for several single-nucleotide polymorphisms covering CC2D1A and CC2D2A. Linkage disequilibrium (LD) analysis revealed that the rs6511901 and rs10410239 polymorphisms of CC2D1A were in strong LD (D'=0.865), and haplotype analysis showed evidence for over-transmission from parents to MR offspring (p=0.0009). The LD analysis also revealed that CC2D2A single-nucleotide polymorphisms rs10025837, rs13116304, and rs7661102 were in strong LD (D'=0.848), and haplotype analysis showed significant transmission disequilibrium (p=0.0004). The results suggest the involvement of CC2D1A and CC2D2A in MR in the Han Chinese population, and some specific haplotypes may be susceptible or protective.     

A rapid, small-scale procedure for the structural characterization of lipid A applied to Citrobacter and Bordetella strains: discovery of a new structural element

Endotoxins [lipopolysaccharides (LPSs)] are part of the outer cell membrane of Gram-negative bacteria. Their biological activities are associated mainly with the lipid component (lipid A) and even more specifically with discrete aspects of their fine structure. The need for a rapid and small-scale analysis of lipid A motivated us to develop a procedure that combines direct isolation of lipids A from bacterial cells with sequential release of their ester-linked fatty acids by a mild alkali treatment followed by MALDI-MS analysis. This method avoids the multiple-step LPS extraction procedure and lipid A isolation. The whole process can be performed in a working day and applied to lyophilized bacterial samples as small as 1 mg. We illustrate the method by applying it to the analysis of lipids A of three species of Citrobacter that were found to be identical. On the other hand, when applied to two batches of Bordetella bronchiseptica strain 4650, it highlighted the presence, in one of them, of hitherto unreported hexosamine residues substituting the lipid A phosphate groups, possibly a new camouflage opportunity to escape a host defense system.     

Crystallographic data for Streptomyces avidinii streptavidin

Crystallization conditions are reported for Streptomyces avidinii streptavidin with and without bound biotin. X-ray examination of the free and bound crystal forms shows the streptavidin-biotin complex crystals to be most suitable for high resolution structure analysis. A complete x-ray data set to 2.6 A resolution was collected for the streptavidin-biotin crystals using a two-dimensional area detector. Reduction and analysis of the x-ray diffraction pattern show that the complex crystallizes in the tetragonal space group I4(1)22 (a = b = 98.4 A, c = 125.8 A), with half of the streptavidin tetramer in the crystallographic asymmetric unit.     

Sensitivity analysis for matched case-control studies

A sensitivity analysis in an observational study indicates the degree to which conclusions would be altered by hidden biases of various magnitudes. A method of sensitivity analysis previously proposed for cohort studies is extended for use in matched case-control studies with multiple controls, where slightly different derivations and calculations are required. Also discussed is a sensitivity analysis for case-control studies that have two distinct types of controls, say hospital and neighborhood controls, where the two types may be affected by different biases. For illustration, the method is applied to five case-control studies, including a study of herniated lumbar disc in which there are three types of cases, and a study of breast cancer with two types of controls.     

Molecular dynamics simulation in vacuo and in solution of [Aib5,6-D-Ala8] cyclolinopeptide A: a conformational and comparative study.

The conformation of a Cyclolinopeptide A analogue, c-(Pro-Pro-Phe-Phe-Aib-Aib-Ile-D-Ala-Val), has been investigated by means of molecular dynamics simulations, in various molecular environments. The molecular dynamics results are compared with that obtained for Cyclolinopeptide A and a detailed analysis of the different behaviour for the two compounds is reported. A complete analysis of hydrogen bonds is presented.