Homology among nearly all plasmids infecting three Bacillus species.

We have surveyed naturally occurring plasmids in strains of Bacillus subtilis and the closely related species B. mojavensis and B. licheniformis. Previous studies have failed to find host-benefitting functions for plasmids of these species, suggesting that these plasmids are nonmutualistic. Only one type of plasmid was found in each plasmid-bearing strain, suggesting that most of the plasmids infecting these Bacillus species are in the same incompatibility group. A sample of 18 plasmids from these species ranged in size from 6.9 to 16 kb, with all but 6 plasmids falling into three size groups. These groups differed in the sizes of their host ranges and geographical ranges. All but 1 of the 18 plasmids from these three host species are homologous with one another. The cryptic plasmids from these three species are far less diverse than are plasmids (from other species) that are known to benefit their bacterial hosts. The low-level diversity among these cryptic plasmids is consistent with the hypothesis that host-benefitting adaptations play an important role in fostering the coexistence of plasmid populations, but other explanations for the low-level plasmid diversity are possible. Comparison of the phylogenies of the plasmids with those of their hosts suggests that Bacillus plasmids are horizontally transferred in nature at a low rate similar to that found for the colicin plasmids of Escherichia coli.     

Effects of molecular oxygen, oxidation-reduction potential, and antioxidants upon in vitro replication of Treponema pallidum subsp. pallidum.

The effects of various concentrations of dithiothreitol, molecular oxygen, and several antioxidants upon the in vitro replication of Treponema pallidum were studied. The optimal dithiothreitol concentration was between 0.65 and 1.62 mM, and the optimum oxygen concentration was 3.0% +/- 0.5% in both the presence and absence of additional antioxidants. It was discovered that the reduced sulfhydryl concentration and the oxidation-reduction potential of the medium were stabilized after 5 days. The water-soluble antioxidants cobalt chloride, cocarboxylase, mannitol, and histidine were individually tested for their ability to increase treponemal growth in vitro. The optimum concentrations for these antioxidants were 21 nM, 4.3 nM, 0.55 mM, and 0.23 mM, respectively. When combined at these concentrations, the mixture of antioxidants stimulated the in vitro replication of T. pallidum. The number of treponemes in cultures with the antioxidants averaged a 59-fold increase, compared with a 43-fold increase in cultures lacking the antioxidants. It was further demonstrated that histidine and mannitol were the most critical components of this mixture. Catalase and superoxide dismutase were investigated for their ability to promote the growth and maintain viability of T. pallidum in tissue culture. The optimum concentrations for these enzymes were 10,000 U/liter and 25,000 U/liter, respectively. When these enzymes and the above antioxidants were combined and added to a chemically reduced modified Eagle medium, the treponemes increased an average of 70-fold, compared with an average of 35-fold in cultures lacking them. Furthermore, this medium, T. pallidum culture medium, supported the replication of T. pallidum at oxygen concentrations from 5 to 7% with little loss in yield or viability. The lipid-soluble antioxidants vitamin A and vitamin E acetate were also shown to enhance the in vitro growth of T. pallidum in this medium.     

Pathogenic Rickettsia species acquire vitronectin from human serum to promote resistance to complement‐mediated killing

Bacteria of the genus Rickettsia are transmitted from arthropod vectors and primarily infect cells of the mammalian endothelial system. Throughout this infectious cycle, the bacteria are exposed to the deleterious effects of serum complement. Using Rickettsia conorii, the etiologic agent of Mediterranean spotted fever (MSF), as a model rickettsial species, we have previously demonstrated that this class of pathogen interacts with human factor H to mediate partial survival in human serum. Herein, we demonstrate that R. conorii also interacts with the terminal complement complex inhibitor vitronectin (Vn). We further demonstrate that an evolutionarily conserved rickettsial antigen, Adr1/RC1281, interacts with human vitronectin and is sufficient to mediate resistance to serum killing when expressed at the outer‐membrane of serum sensitive Escherichia coli. Adr1 is an integral outer‐membrane protein whose structure is predicted to contain eight membrane‐embedded β‐strands and four ‘loop’ regions that are exposed to extracellular milieu. Site‐directed mutagenesis of Adr1 revealed that at least two predicted ‘loop’ regions are required to mediate resistance to complement‐mediatedkilling and vitronectin acquisition. These results demonstrate that rickettsial species have evolved multiple mechanisms to evade complement deposition and that evasion of killing in serum is an evolutionarily conserved virulence attribute for this genus of obligate intracellular pathogens.     

3D imaging provides a high-resolution,volumetric approach for analyzing biofouling

A volumetric approach for determining the fouling burden on surfaces is presented, consisting of a 3D camera imaging system with fine (5?μm) resolution. Panels immersed in an estuary on the southwest coast of Florida, USA were imaged and the data were used to quantify seasonal changes in the biofouling community. Test panels, which were submerged in seawater for up to one?year, were analyzed before and after gentle scrubbing to quantify the biovolume of the total fouling community (ie soft and hard organisms) and the hard fouling community. Total biofouling ranged from 0.01 to 1.16?cm³ cm^?2 throughout the immersion period; soft fouling constituted 22–87% of the total biovolume. In the future, this approach may be used to inform numerical models of fluid–surface interfaces and to evaluate, with high resolution, the morphology of fouling organisms in response to antifouling technologies.     

Propulsive adaptation to changing gait speed

Understanding propulsion and adaptation to speed requirements is important in determining appropriate therapies for gait disorders. We hypothesize that adaptations for changing speed requirements occur primarily at the hip. The slow, normal and fast gait of 24 healthy young subjects was analyzed. The linear power was analyzed at the hip joint. The anterior-posterior and vertical induced accelerations of the hip were also determined. Linear power and anterior-posterior-induced acceleration (IA) analyses of the hip reveal that the lower limb joint's moments contribute to body forward propulsion primarily during late swing and early stance. Propulsive adaptations to speed changes occur primarily at the hip and secondarily at the ankle. These analyses show that hip muscles, particularly the hip extensors, are critical to propulsion. They also show that ankle function is primarily for support, but is important to propulsion, especially at slow speeds.     

Quantitation of daunorubicin and its metabolites by high-performance liquid chromatography with electrochemical detection

A selective and sensitive high-performance liquid chromatographic method was developed for the separation and quantitation of daunorubicin and its metabolites in serum, plasma, and other biological fluids. Daunorubicin and metabolites in human plasma were injected directly into the high-performance liquid chromatography system via a loop-column to pre-extract the drugs from the plasma, and quantitated against a multilevel calibration curve with adriamycin as the internal standard. The column effluent was monitored with an electrochemical detector at an applied oxidative potential of 0.65 V and by fluorescence. Daunorubicin and four metabolites were separted and characterized by this method. In a blinded evaluation of accuracy and precision, the mean coefficients of variation were 3.8, 3.6 and 9.8% at concentrations of 150, 75 and 15 ng/ml, respectively, and blank samples gave negligible readings. The amperometric sensitivity was greater than achieved by fluorescence detection, and offers an alternative method for quantitation of these compounds. The new method has a limit of detection of less than 2 ng on column, allowing quantitation of < 10 ng/ml in plasma samples without organic extraction prior to chromatographic analysis.     

The automatic pilot of honeybees

Using scanning harmonic radar, we make visible for the first time the complete trajectories of "goal-vector" flights in honeybees. We demonstrate that bees captured at an established feeding station, and released elsewhere, nevertheless embark on the previously learned vector flight that would have taken them directly home from the station, had they not been artificially displaced. Almost all of the bees maintained accurate compensation for lateral wind drift, and many completed the full length of the vector flight before starting to search for their hive. Our results showed that bees tend to disregard landscape cues during these vector flights, at least initially, and rely on the "optic flow" of the ground beneath them, and their sun compass, to judge both direction and distance.     

Conditions that influence the response to Fgf during otic placode induction

More than a century ago, several embryologists described sites of hematopoietic activity in the vascular wall of mid-gestation vertebrate embryos, and postulated the transient existence of a blood generating endothelium during ontogeny. This hypothesis gained significant attention in the 1970s when orthotopic transplantation experiments between quail and chick embryos revealed specific vascular areas as the site of the origin of definitive hematopoiesis. However, the vascular origin of hematopoietic precursors remained elusive and controversial for decades. Only recently, multiple experimental approaches have clearly documented that during vertebrate development definitive hematopoietic precursors arise from a subset of vascular endothelial cells. Interestingly, this differentiation is promoted by the intravascular fluid mechanical forces generated by the establishment of blood flow upon the initiation of heartbeat, and it is therefore connected with cardiovascular development in several critical aspects. In this review we present our current understanding of the relationship between vascular and definitive hematopoietic development through an historical analysis of the scientific evidence produced in this area of investigation.     

Free mycolic acid accumulation in the cell wall of the mce1 operon mutant strain of Mycobacterium tuberculosis

The lipid-rich cell wall of Mycobacterium tuberculosis, the agent of tuberculosis, serves as an effective barrier against many chemotherapeutic agents and toxic host cell effector molecules, and it may contribute to the mechanism of persistence. Mycobacterium tuberculosis strains mutated in a 13-gene operon called mce1, which encodes a putative ABC lipid transporter, induce aberrant granulomatous response in mouse lungs. Because of the postulated role of the mce1 operon in lipid importation, we compared the cell wall lipid composition of wild type and mce1 operon mutant M. tuberculosis H37Rv strains. High resolution mass spectrometric analyses of the mce1 mutant lipid extracts showed unbound mycolic acids to accumulate in the cell wall. Quantitative analysis revealed a 10.7 fold greater amount of free mycolates in the mutant compared to that of the wild type strain. The free mycolates were comprised of alpha, methoxy and keto mycolates in the ratio 1:0.9:0.6, respectively. Since the mce1 operon is regulated in vivo, the free mycolates that accumulate during infection may serve as a barrier for M. tuberculosis against toxic products and contribute to the pathogen’s persistence.     

Expanded divalent metal-ion tolerance of evolved ligase ribozymes

Class I ligases are artificial ribozymes that catalyze the joining of two single-stranded RNAs. These ribozymes are between 120 and 160 nucleotides in length, making them intermediate in size for catalytic RNAs. Previous characterization of the b1-207 ribozyme suggests that it behaves similar to larger ribozymes in terms of divalent metal-ion dependence. This molecule displays a strong preference for magnesium for catalysis, and is inactive in any other metal except manganese, which actually inhibits its operation in magnesium. Here, we sought to examine the metal-ion usages of two ligases that were obtained through continuous evolution in vitro from the b1-207 sequence framework. We found an expanded catalytic range for the E(100)(#3) and B(16)(#19) ribozymes, as they are both catalytically active in calcium and strontium, and less inhibited by manganese. Though not selected for activity in these salts, the evolved ribozymes exhibit several adaptations to in vitro catalysis, and their ability to accommodate metals other than magnesium can be viewed as an example of a molecular exaptation.