DNA sequence analysis of point mutations in traA, the F pilin gene, reveal two domains involved in F-specific bacteriophage attachment

Summary Six missense point mutations in traA (WPFL43,44,45,46,47 and 51), the gene encoding F pilin in the transfer region of the F plasmid, have been characterized for their effect on the transfer ability, bacteriophage (R17, QB and fl) sensitivity and levels of piliation expressed by the plasmid. The sequence analysis of the first five of these mutations revealed two domains in the F pilin subunit exposed on the surface of the F pilus which mediate phage attachment. These two domains include residues 14–17 (approximately) and the last few residues at the carboxy-terminus of the pilin protein. One of these mutants had a pleiotropic affect on pilus function and was thought to have affected pilus assembly. The sixthe point mutant (WPFL51), previously thought to be in traA, was complemented by chimeric plasmids carrying the traG gene of the F transfer region, which may be involved in the acetylation of the pilin subunit. A traA nonsense mutant (JCFL1) carried an amber mutation near the amino-terminus which is well suppressed in SuI⁺ (supD) and SuIII⁺ (supF) strains. Neither the antigenicity of the pilin nor the efficiency of plating of F-specific bacteriophages were affected when this plasmid was harbored by either suppressor strain. A second amber mutant (JCFL25) which is not suppressible, carried its mutation in the codon for the single tryptophan in F pilin, suggesting that this residue is important in subunit interactions during pilus assembly. Two other point mutants (JCFL32 and 44) carried missense mutations in the leader sequence (positions 9 and 13) which affected the number of pili per cell presumably by altering the processing of propilin to pilin.     

Bacterial feeding by the rotifer Brachionus calyciflorus: Clearance and ingestion rates,behavior and population dynamics

Summary The rotifer Brachionus calyciflorus is capable of collecting and ingesting cells or short chains of a laboratory-grown bacterium Aerobacter aerogenes. Clearance rate, the volume of water effectively processed animal ^-1h^-1, does not vary systematically with bacterial density between 0.01 and 100 g dry weight ml^-1. Consequently, ingestion rates are strongly density-dependent, reaching maximal values at the highest food densities tested. Bacterial feeding rates are consistently lower than those determined with larger food types, except in very dense cell suspensions. A. aerogenes in high concentration (100 g ml^-1) induces Brachionus to orient their pseudotrochal cirri to form screens over the buccal funnel; this behavior is at least four times less frequently observed at low (10 g ml^-1) food density. Despite its occurrence, pseudotrochal screening appears ineffective in regulating bacterial ingestion rate. B. calyciflorus can be cultured xenically for greater than 40 generations fed A. aerogenes alone, with no diminution in net reproductive rate or intrinsic rate of natural increase, and no lengthening in cohort generation time.     

Automatic cell identification and enrichment in lung cancer. I. Light scatter and fluorescence parameters.

Two physical parameters were investigated to automatically recognize cells in sputum from human squamous cell carcinoma of the lung and to separate them for preparation by the Papanicolaou methods, for human interactive identification and for automated high resolution image analysis. The two parameters, 0.5-15.0 degrees forward argon-ion laser light scatter to estimate total cell size and 546 nm Acridine orange fluorescence to approximate total cell DNA content, were measured in a flow-through fluorescence activated cell sorting system. Enrichment for neoplastic cells in three cases of squamous cell carcinoma of the lung averaged 7.8-fold over the original sputum when only green fluorescence was used and 10.5-fold using green fluorescence and forward light scatter. The average enrichment for neoplastic cells was 65.6-fold relative to polymorphonuclear deenrichment.     

Stoichiometric and growth responses of a freshwater filamentous green alga to varying nutrient supplies: slow and steady wins the race

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A quantitative method for the detection and localization of quantum-limited events from radionuclides in cells and tissue sections by computer-enhanced video microscopy

Cellular dynamics often involve extremely low concentrations of biologically active substances, which can be radiolabeled and detected, localized and quantitated by autoradiography. The latter may require exposures from a few days to many months. The objective of this research was to demonstrate the feasibility of reducing this long period of data collection by one to two orders of magnitude, while maintaining or improving the spatial resolution and localization in tissues and the quantitative characteristics inherent in autoradiography. A mathematical model describing the complete system was generated using energy partition calculations to estimate photon production via scintillant per H3 beta particle emission and to estimate the subsequent photon capture based upon imaging system parameters and microscope geometry. Calculations showed that, typically, a single tritium beta particle produces a maximum of 5.8 X 10(3) photons. A photon-limited camera and microscope imaging system were selected and optimized in conjunction with a specially developed physical scintillation model. Results showed that the number of detected photoevents increases monotonically with both signal integration time and, independently, with the concentration of the radionuclide. Consequently, this work demonstrates that video microscopy imaging methods can spatially and temporally quantify very low concentrations of radiolabeled substances and can reduce data acquisition times.     

Effects of exogenous guanosine on chromatophore differentiation in the axolotl

Guanosine is shown to dramatically alter the pigment phenotype of axolotls by suppressing melanization and enhancing the biosynthesis and deposition of purine-derived pigments. Phenotypic changes caused by guanosine are manifested by altered chromatophore differentiation patterns such that few black pigment cells (melanophores) differentiate (and those that do are punctate and necrotic in appearance), whereas the development of yellow (xanthophore) and reflecting (iridophore) pigment cells is enhanced. Mechanisms for changes in chromatophore differentiation, and thus pattern formation, are discussed, including the possibility that pigment cells may undergo transdifferentiation in vivo.     

A Tetrachrome Stain for Fresh, Mineralized Bone Sections, Useful in the Diagnosis of Bone Diseases

Fresh, unprocessed bone is ground to sections 75-100 μ thick, stained in an aqueous solution composed of fast green FCF, 0.1 gm; orange G, 2.0 gm; distilled water, 100.0 ml; and adjusted to pH 6.65, then in a mixture of 1 part alcoholic solution of 0.25% celestine blue B and 9 parts of alcoholic solution of 0.1% basic fuchsin. Surface stain is removed by grinding sections to 50 μ and washing them in 1% invert soap (Zephiran) to remove adherent debris. (Commercial detergents and alkaline soaps may interfere with chromophore groups of the dyes.) Wash in tap water; rinse in distilled water and differentiate in 1% acetic alcohol. Dehydrate in ascending alcohols, clear in xylene and mount permanently in a neutral, synthetic resin. Active osteoid seams stain dark to light green; resting osteoid seams, red to bright orange red; transitional osteoid seams, geenish-yellow, orange red to red; older, partly mineralized matrix, orange; new, partly mineralized matrix, red; osteocyte nuclei, red; osteoblasts and osteoclasts, greenish-blue to dark purple nuclei and green or light green cytoplasm. Hyper-trophic and differentiating cartilage cells are stained light pink and dark red respectively. The staining reactions are consistent; the solutions are stable.     

Epigenetic signature of birth weight discordance in adult twins

Background

A low birth weight has been extensively related to poor adult health outcomes. Birth weight can be seen as a proxy for environmental conditions during prenatal development. Identical twin pairs discordant for birth weight provide an extraordinary model for investigating the association between birth weight and adult life health while controlling for not only genetics but also postnatal rearing environment. We performed an epigenome-wide profiling on blood samples from 150 pairs of adult monozygotic twins discordant for birth weight to look for molecular evidence of epigenetic signatures in association with birth weight discordance.

Results

Our association analysis revealed no CpG site with genome-wide statistical significance (FDR < 0.05) for either qualitative (larger or smaller) or quantitative discordance in birth weight. Even with selected samples of extremely birth weight discordant twin pairs, no significant site was found except for 3 CpGs that displayed age-dependent intra-pair differential methylation with FDRs 0.014 (cg26856578, p = 3.42e-08), 0.0256 (cg15122603, p = 1.25e-07) and 0.0258 (cg16636641, p = 2.05e-07). Among the three sites, intra-pair differential methylation increased with age for cg26856578 but decreased with age for cg15122603 and cg16636641. There was no genome-wide statistical significance for sex-dependent effects on intra-pair differential methylation in either the whole samples or the extremely discordant twins.

Conclusions

Genome-wide DNA methylation profiling did not reveal epigenetic signatures of birth weight discordance although some sites displayed age-dependent intra-pair differential methylation in the extremely discordant twin pairs.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1062) contains supplementary material, which is available to authorized users.     

Fermentable carbohydrate alters hypothalamic neuronal activity and protects against the obesogenic environment

Obesity has become a major global health problem. Recently, attention has focused on the benefits of fermentable carbohydrates on modulating metabolism. Here, we take a system approach to investigate the physiological effects of supplementation with oligofructose-enriched inulin (In). We hypothesize that supplementation with this fermentable carbohydrate will not only lead to changes in body weight and composition, but also to modulation in neuronal activation in the hypothalamus. Male C57BL/6 mice were maintained on a normal chow diet (control) or a high fat (HF) diet supplemented with either oligofructose-enriched In or corn starch (Cs) for 9 weeks. Compared to HF+Cs diet, In supplementation led to significant reduction in average daily weight gain (mean ± s.e.m.: 0.19 ± 0.01 g vs. 0.26 ± 0.02 g, P < 0.01), total body adiposity (24.9 ± 1.2% vs. 30.7 ± 1.4%, P < 0.01), and lowered liver fat content (11.7 ± 1.7% vs. 23.8 ± 3.4%, P < 0.01). Significant changes were also observed in fecal bacterial distribution, with increases in both Bifidobacteria and Lactobacillius and a significant increase in short chain fatty acids (SCFA). Using manganese-enhanced MRI (MEMRI), we observed a significant increase in neuronal activation within the arcuate nucleus (ARC) of animals that received In supplementation compared to those fed HF+Cs diet. In conclusion, we have demonstrated for the first time, in the same animal, a wide range of beneficial metabolic effects following supplementation of a HF diet with oligofructose-enriched In, as well as significant changes in hypothalamic neuronal activity.     

Simple epidemiological dynamics explain phylogenetic clustering of HIV from patients with recent infection

Phylogenies of highly genetically variable viruses such as HIV-1 are potentially informative of epidemiological dynamics. Several studies have demonstrated the presence of clusters of highly related HIV-1 sequences, particularly among recently HIV-infected individuals, which have been used to argue for a high transmission rate during acute infection. Using a large set of HIV-1 subtype B pol sequences collected from men who have sex with men, we demonstrate that virus from recent infections tend to be phylogenetically clustered at a greater rate than virus from patients with chronic infection ('excess clustering') and also tend to cluster with other recent HIV infections rather than chronic, established infections ('excess co-clustering'), consistent with previous reports. To determine the role that a higher infectivity during acute infection may play in excess clustering and co-clustering, we developed a simple model of HIV infection that incorporates an early period of intensified transmission, and explicitly considers the dynamics of phylogenetic clusters alongside the dynamics of acute and chronic infected cases. We explored the potential for clustering statistics to be used for inference of acute stage transmission rates and found that no single statistic explains very much variance in parameters controlling acute stage transmission rates. We demonstrate that high transmission rates during the acute stage is not the main cause of excess clustering of virus from patients with early/acute infection compared to chronic infection, which may simply reflect the shorter time since transmission in acute infection. Higher transmission during acute infection can result in excess co-clustering of sequences, while the extent of clustering observed is most sensitive to the fraction of infections sampled.