Cinemicrographic Study of the Development of Subsurface Colonies of Staphylococcus aureus in Soft Agar

Cinemicrographic studies revealed that the development of elongated subsurface colonies of Staphylococcus aureus in soft agar (<0.2% agar) originated with a colony-forming unit of about 10 to 20 cells. It was then observed that small clusters of 3 to 12 cells broke off from the main colony unit and drifted away under the combined influence of gravity and Brownian motion. Once the downward or slightly sideward motion of the small clusters ceased, the clusters would continue to increase in size; at the same time, additional small clusters broke off, and the cycle was repeated until the entire colony was formed. Displacement and velocity measurements were made on the drifting small clusters. When compared with the dimensional growth rate and geometry of the subsurface colony, these showed that a correlation existed between the movement and velocity of the small clusters and the subsequent colony development. A relationship between the role of gravity reported in these results and the development of spherical colonies after rotation on a clinostat is suggested.     

Species delimitation and hybridization history of a hazel species complex

Background and AimsHybridization increases species adaptation and biodiversity but also obscures species boundaries. In this study, species delimitation and hybridization history were examined within one Chinese hazel species complex (Corylus chinensis–Corylus fargesii). Two species including four varieties have already been described for this complex, with overlapping distributions.MethodsA total of 322 trees from 44 populations of these four varieties across their ranges were sampled for morphological and molecular analyses. Climatic datasets based on 108 geographical locations were used to evaluate their niche differentiations. Flowering phenology was also observed for two co-occurring species or varieties.Key ResultsFour statistically different phenotypic clusters were revealed, but these clusters were highly inconsistent with the traditional taxonomic groups. All the clusters showed statistically distinct niches, with complete or partial geographical isolation. Only two clusters displayed a distributional overlap, but they had distinct flowering phenologies at the site where they co-occurred. Population-level evidence based on the genotypes of ten simple sequence repeat loci supported four phenotypic clusters. In addition, one cluster was shown to have an admixed genetic composition derived from the other three clusters through repeated historical hybridizations.ConclusionsBased on our new evidence, it is better to treat the four clusters identified here as four independent species. One of them was shown to have an admixed genetic composition derived from the other three through repeated historical hybridizations. This study highlights the importance of applying integrative and statistical methods to infer species delimitations and hybridization history. Such a protocol should be adopted widely for future taxonomic studies.     

Sample Size Considerations for GEE Analyses of Three‐Level Cluster Randomized Trials

Summary Cluster randomized trials in health care may involve three instead of two levels, for instance, in trials where different interventions to improve quality of care are compared. In such trials, the intervention is implemented in health care units (“clusters”) and aims at changing the behavior of health care professionals working in this unit (“subjects”), while the effects are measured at the patient level (“evaluations”). Within the generalized estimating equations approach, we derive a sample size formula that accounts for two levels of clustering: that of subjects within clusters and that of evaluations within subjects. The formula reveals that sample size is inflated, relative to a design with completely independent evaluations, by a multiplicative term that can be expressed as a product of two variance inflation factors, one that quantifies the impact of within‐subject correlation of evaluations on the variance of subject‐level means and the other that quantifies the impact of the correlation between subject‐level means on the variance of the cluster means. Power levels as predicted by the sample size formula agreed well with the simulated power for more than 10 clusters in total, when data were analyzed using bias‐corrected estimating equations for the correlation parameters in combination with the model‐based covariance estimator or the sandwich estimator with a finite sample correction.     

Clusters of repeated sequences of chicken DNA are extensively methylated but contain specific undermethylated regions

In the chicken genome there are middle repetitive DNA sequences with a clustered organization. Each cluster is composed of members of different families of repeated DNA sequences and usually contains only one member of each family. Many clusters have the same assortment of repeated sequences but they are in scrambled order from cluster to cluster. These clusters usually exceed 20 × 10³ bases in length and comprise at least 10% of the repeated DNA of the chicken. The repeated sequences that are cluster components are extensively methylated. Methylation was detected by comparing HpaII and MspI digests of total DNA, where the occurrence of the sequence C-m⁵C-G-G is indicated when HpaII (cleaves C-C-G-G) fragments are larger than those generated by MspI (cleaves C-m⁵C-G-G or C-C-G-G). In hybridization experiments with Southern (1975) blots of total DNA digested with either HpaII or MspI, the cloned probes representing clustered repeated sequences showed a dramatic difference in the lengths of restriction fragments detected in the two digests. Many of the sequences that comprise these clusters are methylated in most of their genomic occurrences. There are patterns of methylation that are reproduced faithfully from copy to copy. The overall distribution of methylation within clusters seems to be regional, with long methylated DNA segments interrupted by specific undermethylated regions.     

Molecular and cytologic analysis of clusters of moderate repeats of bird genomes

Pigeon genome long sequences containing clusters of moderately repeating elements have been cloned. Molecular analysis has shown a dispersed distribution of the repeats in both pigeon and chicken genomes. Within a single cluster, a scrambled distribution of elements belonging to different families of repeats has been shown. Similar repeated sequences have been revealed within clusters. The analysed clusters of repeats are characterized by a limited structural variability in the genomes. In situ hybridization revealed the localization of sequences complementary to the cloned clusters in pigeon and chicken macrochromosomes. Preferential localization has been demonstrated in telomeric and centromeric chromosome regions as well as in the region of R-bands.     

Nonrandom localization of recombination events in human alpha satellite repeat unit variants: implications for higher-order structural characteristics within centromeric heterochromatin.

Tandemly repeated DNA families appear to undergo concerted evolution, such that repeat units within a species have a higher degree of sequence similarity than repeat units from even closely related species. While intraspecies homogenization of repeat units can be explained satisfactorily by repeated rounds of genetic exchange processes such as unequal crossing over and/or gene conversion, the parameters controlling these processes remain largely unknown. Alpha satellite DNA is a noncoding tandemly repeated DNA family found at the centromeres of all human and primate chromosomes. We have used sequence analysis to investigate the molecular basis of 13 variant alpha satellite repeat units, allowing comparison of multiple independent recombination events in closely related DNA sequences. The distribution of these events within the 171-bp monomer is nonrandom and clusters in a distinct 20- to 25-bp region, suggesting possible effects of primary sequence and/or chromatin structure. The position of these recombination events may be associated with the location within the higher-order repeat unit of the binding site for the centromere-specific protein CENP-B. These studies have implications for the molecular nature of genetic recombination, mechanisms of concerted evolution, and higher-order structure of centromeric heterochromatin.     

High symbiont diversity in the bone-eating worm Osedax mucofloris from shallow whale-falls in the North Atlantic

Osedax worms are whale-fall specialists that infiltrate whale bones with their root tissues. These are filled with endosymbiotic bacteria hypothesized to provide their hosts with nutrition by extracting organic compounds from the whale bones. We investigated the diversity and distribution of symbiotic bacteria in Osedax mucofloris from shallow-water whale-falls in the North Atlantic using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization (FISH). We observed a higher diversity of endosymbionts than previously described from other Osedax species. Endosymbiont sequences fell into eight phylogenetically distinct clusters (with 91.4-98.9% similarity between clusters), and considerable microdiversity within clusters (99.5-99.7% similarity) was observed. Statistical tests revealed a highly significant effect of the host individual on endosymbiont diversity and distribution, with 68% of the variability between clusters and 40% of the variability within clusters explained by this effect. FISH analyses showed that most host individuals were dominated by endosymbionts from a single cluster, with endosymbionts from less abundant clusters generally confined to peripheral root tissues. The observed diversity and distribution patterns indicate that the endosymbionts are transmitted horizontally from the environment with repeated infection events occurring as the host root tissues grow into the whale bones.     

A comparison of lymphocyte antigen specificities in Norwegian and Swiss goats

Summary. In all, 363 alloantireagents were tested in Berne, Switzerland and in Oslo, Norway against lymphocytes from 1679 goats of different breeds. The same lymphocytotoxicity test was used at both laboratories. The test data were pooled, and correlation coefficients for pairs of sera were used to group the sera in clusters. Twelve clusters were accepted as defining lymphocyte antigen specificities believed to be coded by genes within the major histocompatibility complex (MHC). The specificities defined by these clusters were designated Eu 1-Eu12. Two clusters defined specificities which were not coded from loci within the MHC. These were designated GLY-1.1 and GLY-2.1, and the loci GLY-1 and GLY-2, respectively. GLY-1.1 was also located on erythrocytes.     

Advances in the structure and chemistry of metallothioneins

A low molecular weight (6-7 kDa) class of metalloproteins, designated as metallothioneins (MTs), exhibit repeated sequence motifs of either CxC or CxxC through which mono or divalent d(10) metal ions are bound in polymetallic-thiolate clusters. The preservation of metal-thiolate clusters in an increasing number of three-dimensional structures of these proteins signifies the importance of this structural motif. This review focuses on the recent developments regarding the versatile and striking chemical reactivity of MTs as well as on the existence of conformational/configurational dynamics within their structure. Both properties and their interplay are likely to be essential for the still elusive biological function of these proteins.     

Prediction of operon-like gene clusters in the Arabidopsis thaliana genome based on co-expression analysis of neighboring genes

Operon-like arrangements of genes occur in eukaryotes ranging from yeasts and filamentous fungi to nematodes, plants, and mammals. In plants, several examples of operon-like gene clusters involved in metabolic pathways have recently been characterized, e.g. the cyclic hydroxamic acid pathways in maize, the avenacin biosynthesis gene clusters in oat, the thalianol pathway in Arabidopsis thaliana, and the diterpenoid momilactone cluster in rice. Such operon-like gene clusters are defined by their co-regulation or neighboring positions within immediate vicinity of chromosomal regions. A comprehensive analysis of the expression of neighboring genes therefore accounts a crucial step to reveal the complete set of operon-like gene clusters within a genome. Genome-wide prediction of operon-like gene clusters should contribute to functional annotation efforts and provide novel insight into evolutionary aspects acquiring certain biological functions as well. We predicted co-expressed gene clusters by comparing the Pearson correlation coefficient of neighboring genes and randomly selected gene pairs, based on a statistical method that takes false discovery rate (FDR) into consideration for 1469 microarray gene expression datasets of A. thaliana. We estimated that A. thaliana contains 100 operon-like gene clusters in total. We predicted 34 statistically significant gene clusters consisting of 3 to 22 genes each, based on a stringent FDR threshold of 0.1. Functional relationships among genes in individual clusters were estimated by sequence similarity and functional annotation of genes. Duplicated gene pairs (determined based on BLAST with a cutoff of E<10(-5)) are included in 27 clusters. Five clusters are associated with metabolism, containing P450 genes restricted to the Brassica family and predicted to be involved in secondary metabolism. Operon-like clusters tend to include genes encoding bio-machinery associated with ribosomes, the ubiquitin/proteasome system, secondary metabolic pathways, lipid and fatty-acid metabolism, and the lipid transfer system.     

Large-scale periodicity of nucleosome positioning signal in pericentric regions of chromosomes (Drosophila melanogaster)

Nucleosome positioning signal (NPS) in heterochromatin is not uniform. We suggest the analysis of its heterogeneity by correlation with periodic function (analog of Furrier analysis). It was established the periodical repetition of the nucleosome clusters of large size in pericentric regions in a discontinuous manner. In the 3L pericentric region, it was revealed the domination of 78–85?kbp wavelength in the correlation coefficient profile and also strong presentation of 50?kbp signal. In further to centromere position, the 69?kbp value strongly dominates as well as the 50?kbp value in the closest proximity. In addition to the long wavelength signals, there are plenty of short wavelengths signals especially in the closest vicinity to centromere. In some positions throughout pericentric region of 2L chromosome, there are two sizes of repeated intermingled correlation signals (50, and 75?kbp) with dominating value of 75?kbp in proximity and 50?kbp distantly to centromere, the situation for 2R is analogous. Some genes with long introns support these quantitative characteristics of NPSs and to some extent their dominating character in each region. The characteristic repeat periods for 3L pericentric region coincide with the distances between heterochromatin epigenetic mark clusters and their distribution throughout this region for fly embryos, larvae, and some cell lines.     

An approach to the analysis of repeated measurements

A linear model for repeated measurements is proposed in which the correlation structure within each time sequence of measurements includes parameters for measurement error, variation between experimental units, and serial correlation within units. An approach to data analysis is presented which involves preliminary analysis by ordinary least squares, use of the empirical semi-variogram of residuals to suggest a suitable correlation structure, and formal inference using likelihood-based methods. Applications to two biological data sets are described.     

Conserved and clustered RNA recognition sequences are a critical feature of signals directing RNA localization in Xenopus oocytes

Although it is widely regarded that the targeting of RNA molecules to subcellular destinations depends upon the recognition of cis-elements found within their 3' untranslated regions (UTR), relatively little is known about the specific features of these cis-sequences that underlie their function. Interaction between specific repeated motifs within the 3' UTR and RNA-binding proteins has been proposed as a critical step in the localization of Vg1 RNA to the vegetal pole of Xenopus oocytes. To understand the relative contributions of repeated localization element (LE) sequences, we used comparative functional analysis of Vg1 LEs from two frog species, Xenopus laevis and Xenopus borealis. We show that clusters of repeated VM1 and E2 motifs are required for efficient localization. However, groups of either site alone are not sufficient for localization. In addition, we present evidence that the X. borealis Vg1 LE is recognized by the same set of RNA-binding proteins as the X. laevis Vg1 LE and is capable of productive interactions with the X. laevis transport machinery as it is sufficient to direct vegetal localization in X. laevis oocytes. These results suggest that clustered sets of cis-acting sites within the LE direct vegetal transport through specific interactions with the localization machinery.     

Matrix resistance stress: A key parameter for immobilized cell growth regulation

Microenvironmentally restricted yeast cell growth within Ca-alginate beads with and without entrapped gas bubbles was considered based on experimental data. Cell growth dynamics was described by (1) the dimensionless cell number density as a function of the cell growth time and (2) the cell distribution per bead cross sections. One of the key control parameters for bioprocess optimization is the matrix resistance stress generated during immobilized cell expansion. The dynamics of the increase in matrix stress was described theoretically based on a multi-scale mathematical model. In order to estimate and reduce the accumulation of matrix stress we considered repeated stress relaxation cycles in separate rheological experiments without immobilized cells.The results revealed that the increase in resistance stress within the Ca-alginate matrix was significant (∼7 kPa) after 10 repeated cycles, even under a low compression strain of 2% per cycle. The stress could be reduced by using the Ca-alginate matrix with entrapped gas bubbles. The final cell concentration within the beads with entrapped bubbles was 3.3 times higher in comparison with the beads without bubbles. The bubbles could locally amortize the compression effects within the surrounding cell clusters.     

Whole miRNome-wide Differential Co-expression of MicroRNAs

Co-regulation of genes has been extensively analyzed, however, rather limited knowledge is available on co-regulations within the miRNome. We investigated differential co-expression of microRNAs (miRNAs) based on miRNome profiles of whole blood from 540 individuals. These include patients suffering from different cancer and non-cancer diseases, and unaffected controls. Using hierarchi-cal clustering, we found 9 significant clusters of co-expressed miRNAs containing 2-36 individual miRNAs. Through analyzing multiple sequencing alignments in the clusters, we found that co-expression of miRNAs is associated with both sequence similarity and genomic co-localization. We calculated correlations for all 371,953 pairs of miRNAs for all 540 individuals and identified 184 pairs of miRNAs with high correlation values. Out of these 184 pairs of miRNAs, 16 pairs (8.7%) were differentially co-expressed in unaffected controls, cancer patients and patients with non-cancer diseases. By computing correlated and anti-correlated miRNA pairs, we constructed a network with 184 putative co-regulations as edges and 100 miRNAs as nodes. Thereby, we detected specific clusters of miRNAs with high and low correlation values. Our approach represents the most comprehensive co-regulation analysis based on whole miRNome-wide expression profiling. Our findings further decrypt the interactions of miRNAs in normal and human pathological processes.     

Character process model for semen volume in AI rams: evaluation of correlation structures for long and short-term environmental effects

The objective of this study was to build a character process model taking into account serial correlations for the analysis of repeated measurements of semen volume in AI rams. For each ram, measurements were repeated within and across years. Therefore, we considered a model including three environmental effects: the long-term environmental effect, which is a random year* subject effect, the short-term environmental effect, which is a random within year subject* collection effect, and the classical measurement error. We used a four-step approach to build the model. The first step explored graphically the serial correlations. The second step compared four models with different correlation structures for the short-term environmental effect. We selected fixed effects in the third step. In the fourth step, we compared four correlation structures for the long-term environmental effect. The model, which fitted best the data, used a spatial power correlation structure for the short-term environmental effect and a first order autoregressive process for the long-term environmental effect. The heritability estimate was 0.27 (0.04), the within year repeatability decreased from 0.56 to 0.44 and the repeatability across years decreased from 0.43 to 0.37.     

Imputation strategies for missing continuous outcomes in cluster randomized trials

In cluster randomized trials, intact social units such as schools, worksites or medical practices - rather than individuals themselves - are randomly allocated to intervention and control conditions, while the outcomes of interest are then observed on individuals within each cluster. Such trials are becoming increasingly common in the fields of health promotion and health services research. Attrition is a common occurrence in randomized trials, and a standard approach for dealing with the resulting missing values is imputation. We consider imputation strategies for missing continuous outcomes, focusing on trials with a completely randomized design in which fixed cohorts from each cluster are enrolled prior to random assignment. We compare five different imputation strategies with respect to Type I and Type II error rates of the adjusted two-sample t -test for the intervention effect. Cluster mean imputation is compared with multiple imputation, using either within-cluster data or data pooled across clusters in each intervention group. In the case of pooling across clusters, we distinguish between standard multiple imputation procedures which do not account for intracluster correlation and a specialized procedure which does account for intracluster correlation but is not yet available in standard statistical software packages. A simulation study is used to evaluate the influence of cluster size, number of clusters, degree of intracluster correlation, and variability among cluster follow-up rates. We show that cluster mean imputation yields valid inferences and given its simplicity, may be an attractive option in some large community intervention trials which are subject to individual-level attrition only; however, it may yield less powerful inferences than alternative procedures which pool across clusters especially when the cluster sizes are small and cluster follow-up rates are highly variable. When pooling across clusters, the imputation procedure should generally take intracluster correlation into account to obtain valid inferences; however, as long as the intracluster correlation coefficient is small, we show that standard multiple imputation procedures may yield acceptable type I error rates; moreover, these procedures may yield more powerful inferences than a specialized procedure, especially when the number of available clusters is small. Within-cluster multiple imputation is shown to be the least powerful among the procedures considered.     

A comparison of lymphocyte antigen specificities in Norwegian and Swiss goats

In all, 363 alloantireagents were tested in Berne, Switzerland and in Oslo, Norway against lymphocytes from 1679 goats of different breeds. The same lymphocytotoxicity test was used at both laboratories. The test data were pooled, and correlation coefficients for pairs of sera were used to group the sera in clusters. Twelve clusters were accepted as defining lymphocyte antigen specificities believed to be coded by genes within the major histocompatibility complex (MHC). The specificities defined by these clusters were designated Eu1-Eu12. Two clusters defined specificities which were not coded from loci within the MHC. These were designated GLY-1.1 and GLY-2.1, and the loci GLY-1 and GLY-2, respectively. GLY-1.1 was also located on erythrocytes.