Microbial divergence and evolution. The case of anammox bacteria.

Species differentiation and the appearance of novel diversity on Earth is a major issue to understand the past and future of microbial evolution. Herein, we propose the analysis of a singular evolutive example, the case of microorganisms carrying out the process of anammox (anaerobic ammonium oxidation). Anammox represents a singular physiology active on Earth from ancient times and, at present, this group is still represented by a relatively limited number of species carrying out a specific metabolism within the Phylum Planctomycetota. The key enzyme on the anammox pathway is hydrazine dehydrogenase (HDH) which has been used as a model in this study. HDH and rRNA (16S subunit) phylogenies are in agreement suggesting a monophyletic origin. The diversity of this singular phylogenetic group is represented by a few enriched bacterial consortia awaiting to be cultured as monospecific taxa. The apparent evolution of the HDH genes in these anammox bacteria is highly related to the diversification of the anammox clades and their genomes as pointed by phylogenomics, their GC content and codon usage profile. This study represents a clear case where bacterial evolution presents a paralleled genome, gene and species diversification through time from a common ancestor; a scenario that most times is masked by a web-like phylogeny and the huge complexity within the prokaryotes. Besides, this contribution suggests that microbial evolution of the anammox bacteria has followed an ordered, vertical diversification through Earth history and will present a potentially similar speciation fate in the future.

[Epidemic of influenza A(H1N1) 2009 in the French overseas territories of the Americas: epidemiological surveillance set up and main results, April 2009-January 2010]. / L'épidémie de grippe A(H1N1) 2009 dans les territoires français des Amériques: dispositif de surveillance mis en place et principaux résultats, avril 2009-janvier 2010.

Guadeloupe, French Guiana, Martinique, St. Martin and St. Barthelemy were the French territories most exposed to the new influenza A(H1N1)v, and adequate epidemiological surveillance tools were promptly developed in order to detect its emergence. The first stage, "containment phase", consisted in detection and management of individual cases. Then, when an autochthonous A(H1N1)v circulation was confirmed, its evolution has been monitored within the whole population, mainly through data collected from sentinel doctors' networks and virological surveillance. This allowed to detect very early the occurrence of epidemics, and to follow their evolution until they were over. Like all the other Caribbean countries, the five French overseas territories were hit by an outbreak of influenza A(H1N1)v. Although they had globally similar characteristics, each epidemic had its specificity in terms of scale and severity. They started between August and September 2009 in four of the five territories, while the last one, St. Barthelemy, was not affected until the end of the year. Attack rate estimates varied from 28 to 70 per 1000 inhabitants according to the territory, and hospitalisation rate varied from 4.3 to 10.3 per 1000 cases. Severity rate didn't reach 1 per 1000 cases in any of the territories. Compared to metropolitan France, the surveillance system presented several strengths, including the pre-existence of both an active sentinel network and an expert committee on emerging diseases in each territory. On the other hand, specific difficulties appeared, notably linked with logistical aspects of virological surveillance and the co-circulation of dengue virus in Guadeloupe and St. Barthelemy. Despite these difficulties, the different tools allowed early detection of the epidemics and follow-up of their evolution. All of them lead to very concordant results, suggesting that they are completely appropriate to monitor a potential new epidemic wave.

Effects of heat on the centrosomes of Chinese hamster ovary cells.

Chinese hamster ovary cells were heated either at 45.5 degrees for 15 min or at 42 degrees for 1 hr and then were either fixed immediately or allowed to recover at 37 degrees for intervals of up to 12 hr. In addition control cells and cells heated at 45.5 degrees for 15 min were immediately subjected to a cell fractionation procedure that yielded partially purified centrosome preparations. In 100% of the cells fixed and examined immediately after heating, the centrosomes were damaged. The osmiophilic cloud increased in density and became aggregated. The majority of the pericentriolar particles or virus-like particles disappeared, and in some cases the tubules of the wall of the centriole appeared disrupted. These changes were also noted in the much more abundant population of centrosomes in the partially purified cell fraction. Furthermore, in those cells heated at 45.5 degrees for 15 min, no recovery of the centrosomes or return of virus-like particles occurred even after incubation at 37 degrees for 12 hr.

CD4(+)CD45RO(+)CD49d(high) cells are involved in the pathogenesis of relapsing-remitting multiple sclerosis.

In the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis, encephalitogenic T cells differ from the non-encephalitogenic ones in their expression of CD49d. The disease-inducing CD49d(high) and not the CD49d(low) cells enter the brain parenchyma. In this context, we characterized CD4(+)(CD45RO(+))CD49d(high) cells in relapsing-remitting multiple sclerosis (RR-MS) patients. These cells, showing characteristics of activated cells able to produce pro-inflammatory cytokines, were found to be increased in peripheral blood during relapses and present in high numbers in cerebrospinal fluid. These results suggest that the CD4(+)CD45RO(+)CD49d(high) subpopulation in RR-MS patients includes autoreactive cells and may be target for immunotherapy.

Polymeric microballoons as ultrasound contrast agents. Physical and ultrasonic properties compared with sonicated albumin.

Air-filled polymeric microballoons were prepared with number-mean diameters of approximately 3 microns, volume-mean diameters of approximately 5 microns, and narrow particle-size distributions (standard deviation [SD] = 1.2 microns in number and SD = 2.0 microns in volume). More than 99% of the particles were below 8 microns. These particles were found to be highly echogenic for ultrasound, showing backscatter coefficients at 7.5 MHz, similar to the ones obtained with sonicated albumin microspheres. However, at 2.25 MHz, microballoons were less echogenic than albumin microspheres. These results are consistent with ultrasound attenuation measurements, which showed a maximum at 8 to 9 MHz for the microballoons compared with a reported value of 3.5 to 4.5 MHz for albumin microbubbles. Polymeric microballoons were found to be stable in plasma or under applied pressure as evidenced by unchanged particle concentration and echogenicity. Albumin microspheres were particularly unstable to applied pressure (150 mm Hg) and showed a rapid decrease in both particle counts and echogenicity.

BR1: a new ultrasonographic contrast agent based on sulfur hexafluoride-filled microbubbles.

RATIONALE AND OBJECTIVES: The basic characteristics of BR1, a novel echo contrast agent based on stabilized sulfur hexafluoride (SF6) microbubbles have been evaluated. METHODS: The authors determined the physicochemical properties (bubble concentration, bubble size distribution, resistance to pressure, and stability) and the acoustic properties (backscatter and attenuation coefficients) of BR1. The diagnostic value of BR1 was evaluated further in minipigs. Left heart images were recorded before and after injection of different doses of BR1. RESULTS: BR1 is formulated as a lyophilized product, which after addition of saline, provides a suspension containing 2 x 10(8) SF6 microbubbles/mL with a number mean diameter of 2.5 microns. More than 90% of the bubbles are below 8 microns. The use of SF6 rather than air provides an improved resistance to pressure increases such as the ones occuring in the left heart during systole. After reconstitution, the echogenicity and the bubble characteristics are unchanged for more than 8 hours. The high echogenicity remains almost constant over the entire medical frequency range (1-10 MHz). BR1 injections in animals resulted in a homogenous, dose-dependent opacification of the left heart. CONCLUSIONS: Considering its high echogenicity, outstanding stability, and resistance to pressure changes, BR1 is a very promising ultrasound contrast agent.

Application of cell culture toxicity tests to the development of implantable biosensors.

Cell culture toxicity testing methods were modified and applied to the development of implantable glucose microsensors, and positive and negative control materials suitable for the microsensor assessment were established. The location, source and degree of the toxic effect in a multi-component biosensor was spatially visualized with cell monolayers. A freshly prepared sensor showed moderate toxicity, mainly as a result of the presence of glutaraldehyde and the residual solvents in the polymer layers. However, it was possible to reduce the toxicity by removing the leachable toxic substances through extraction in phosphate buffer, and a non-toxic sensor was readily obtained.

Development of the implantation chamber in the pregnant bitch.

Uteri taken from 25 bitches at various times during the early stages of pregnancy were studies cytologically to determine how the implantation chamber developed and how fetal-maternal relations were established. On day 13 after the end of estrus, knobs of trophoblastic syncytium formed and became wedged between cells of the uterine luminal epithelium. The syncytium quickly spread along the uterine lumen and into the mouths of the glands, dislodging and surrounding maternal cells. As invasion continued trophoblastic villi, consisting of cores of cytotrophoblast covered by a continuous layer of syncytium, penetrated deeper into the endometrium. The syncytium spread to surround maternal vessels and decidual cells. By day 26 the trophoblast had extended down to the large lacunae. Here syncytial trophoblast covering tips of the villi degenerated, leaving cytotrophoblast exposed to the necrotic zone. These cells possessed characteristics of absorbing cells. Hematomas were formed by focal necrosis of fetal and endometrial tissue at the poles of the implantation sites. Large pools of extravasated blood accumulated and red blood cells were phagocytized by surrounding trophoblastic cells. Therefore, the endotheliochorial relationship in the canine placenta appeared to be established by syncytial trophoblast invading a cellular endometrium. In the necrotic zone and hematomas, cellular trophoblast may have lost its syncytial covering, but elsewhere maternal vessels and decidual cells in the placenta were in direct contact only with syncytial trophoblast.

Development of the endometrium during the estrous cycle in the bitch.

The endometrium of 40 cycling bitches was studied using cytological, cytochemical, and morphometric techniques. Two principal phases of growth and differentiation can be discerned. Phase one begins at the end of anestrus as serum estrogen levels begin to rise and is completed just prior to estrus. It is characterized by growth of the crypts and differentiation of the glandular epithelial cells into well-developed, mucus-secreting cells. Growth, initially rapid, gradually slows. The second growth phase does not begin until the middle of estrus as serum progestin levels rise and lasts nearly a week. Both hypertrophy and hyperplasia of the glandular epithelium and growth of the basal glands characterize this stage. The gland cells develop many well-defined characteristics of absorptive and secretory cells. Another phase of growth occurs in pregnant animals at the onset of implantation. During the third week of metestrus in non-pregnant bitches, the uterus begins to involute. Acid phosphatase and the number of lysosomes increase dramatically in the epithelial cells particularly in the basal glands. Cells lining the lumen and crypts accumulate numerous large lipid droplets. The data are discussed in relation to the clear separation of two distinct uterine functions: (1) sperm transport and maintenance and (2) production and secretion of nutritive uterine milk. Extended periods of follicular development, breeding, and preimplantation in the bitch probably impose this separation.

Estrogen-induced differentiation of the oviductal epithelium in prepubertal dogs.

PIP: 24 prepubertal 6-8 week old Beagle dogs were injected intramuscularly every other day either with 150 mcg/kg estradiol-beta valerate (14), or progesterone respositol (5), or used as controls (5) to determine what effect estrogen and progesterone had on cytodifferentiation. Light and electron micrographs of the oviductal epithelium of the ampullae of the controls and hormone-treated dogs were studied. The epithelial cells in the controls were uniformly low cuboidal with a high nucleocytoplasmic ratio. After 36 hours of estradiol treatment there was nuclear and nucleolar hypertrophy; an increase in the numbers of cytoplasmic polyribosomes and numerous mitotic figues were apparent. Ciliogenesis began and 2 cell types were distinguishable after 3 days of estradiol treatment. Basal bodies in various stages of formation were randomly distributed throughout the apex of many cells. In others, more mature basal bodies were aligned at the apical border or cilia were evident. 6 days of estradiol resulted in complete ciliation of approximately 60% of all epithelial cells. The remaining cells possessed abundant cisternae of rough endoplasmic reticulum (RER) and appeared to be differentiating into mucus-secreting cells. After 10 days, these cells possessed a large Golgi apparatus, large dilated cisternae of RER, and abundant periodic-acid-Schiff positive granules at their apex. Progesterone treatment alone produced no hypertrophy of the oviductal epithelium. In fact, cytomorphometric measurements indicate that progesterone had an opposite effect, causing atrophy (p less than.05) and increasing the nucleocytoplasmic ratio. It is concluded that estrogen alone was capable of producing complete cytodifferentiation in the oviduct of the prepubertal dog.^ieng

Treatment with methylprednisolone in relapses of multiple sclerosis patients: immunological evidence of immediate and short-term but not long-lasting effects.

Relapses of multiple sclerosis (MS) are treated commonly with high-dose intravenous methylprednisolone (MP) given over a period of 3-5 days. The mechanisms responsible for the beneficial effects of MP in attacks are not clearly established. It is also controversial whether this treatment may have a long-term effect. Here, peripheral blood samples from relapsing--remitting MS patients in acute relapse were analysed by flow cytometry just before steroid treatment and at different time points after initiation of the therapy. We observed an immediate (day 3) decrease in the percentage of CD4+ lymphocytes, with a relative increase in the memory (CD4+CD45R0+) subpopulation. A longer standing effect of MP on IFN-gamma production, CD54, CCR5, CXCR3 and CD95 (Fas) expression was also observed on CD4+ cells after 1 month of treatment initiation. Six months after the therapy, during clinical remission, no changes due to ivMP therapy were detected. These results support that MP treatment of relapses induces immediate post-treatment and short-term effects on the immune system that could partly account for the clinical and radiological improvement observed in MS patients. However, no conclusion can be drawn as to a possible long-term or even intermediate influence of ivMP treatment on the course of the disease.

Amelioration of flulike symptoms at the onset of interferon beta-1b therapy in multiple sclerosis by low-dose oral steroids is related to a decrease in interleukin-6 induction.

Low-dose oral steroid use at the onset of interferon beta-1b (IFNbeta-1b) therapy in relapsing-remitting multiple sclerosis (RR-MS) patients reduces flulike symptoms. To determine the mechanism by which steroid treatment minimizes these side effects, we analyzed the percentage of interleukin-6 (IL-6)-, interferon-gamma (IFN-gamma)-, tumor necrosis factor alpha (TNF-alpha)-, and IL-10-producing cells before and after 3 months of IFNbeta-1b therapy onset. Our results support a relationship between IL-6 induction and fever. Such side effects can be ameliorated by steroids.

Study and development of multilayer needle-type enzyme-based glucose microsensors.

Glucose oxidase (GOD) was covalently coupled to a cellulose acetate (CA) layer, using bovine serum albumin (BSA) and parabenzoquinone (PBQ) linkages. Prior to GOD coupling this CA layer was deposited on the platinum tip of a needle-type sensor and covered with an outer layer of polyurethane (PU). Such microsensors were found to be active, their GOD load reaching 1.6 to 3.0 micrograms mm-2 and their glucose response reaching 1 to 3 microA M-1 mm-2, even when the upper limit of their linear range reached 10-30 mM. Due to the multilayer structure and composition of these microsensors, small anions such as ascorbate were partially discriminated from neutral molecules such as hydrogen peroxide. When implanted subcutaneously in anaesthetized rats, sensor responses correlated correctly with blood glucose concentration but presented sensitivity coefficients significantly different to those determined in vitro: a 2 point calibration procedure was found necessary for in vivo experiments.