T-RFLP analysis of bacterial communities in cyclodextrin-amended bioreactors developed for biodegradation of polychlorinated biphenyls

In this study, T-RFLP analysis was used to determine the structure and spatial distribution of the indigenous bacterial community of an actual-site PCB-contaminated soil treated in aerobic packed-bed loop reactors (PBLRs) in the absence or in the presence of a mixture of randomly methylated beta-cyclodextrins (RAMEB) at 0.5 or 1% w/w. RAMEB was found to significantly enhance the aerobic bioremediation of soil with effects that increased proportionally with the concentration at which it was applied. At the end of treatment (180 days), T-RFLP analysis of the soil samples collected from the top and bottom regions of the PBLRs showed a series of 50 single T-RFs. Remarkably, the number of T-RFs was significantly lower (13-22) in samples collected from different sections of the RAMEB-amended bioreactors with respect to equivalent samples collected from the RAMEB-free reactor. Cluster analysis based on the presence or the absence of T-RFs peaks revealed high similarity, inside each reactor, between the top and bottom parts of its soil bed. Soil samples collected at the top and bottom regions of the two bioreactors amended with RAMEB, clustered together while the equivalent samples of the bioreactor without RAMEB formed a separate cluster which was distantly related to the soil samples obtained from the parallel amended bioreactor. Notably, T-RFLP analyses combined with extensive sequencing of 16S rDNA allowed us to tentatively allocate a series of bacterial species corresponding to specific peaks of the T-RFLP profiles and to determine their phylogenetic affiliation.     

Parasite communities of freshwater fish under flood conditions

The parasite communities of the freshwater fish species roach ( Rutilus rutilus), bitterling ( Rhodeus sericeus amarus) and perch ( Perca fluviatilis) were investigated during a major flood. Differences in parasite community structure due to changes in the host environment were expected. We therefore tested for differences in parasite species abundance and diversity as a consequence of the flood. Potential changes in parasite community structure due to seasonal influences were also considered. We found differences in the composition of parasite species and the proportion of ecto- and endoparasites in the three host species. The parasite community of roach was richer in species than those of perch and bitterling. Roach harboured more ectoparasite than endoparasite species. Parasite communities of both perch and bitterling were richer in endoparasite species and had a lower number of ectoparasite species. Parasite communities of the three different host species responded in different ways to environmental change. Considering all parasite species, no effect of either season or flood was found on the species diversity in either perch or bitterling. The flood caused an increase in the parasite species diversity in roach. For roach and perch, higher parasite abundance were found before the flood compared to after flood. However, no difference was found between the different seasons after the flood. By contrast, bitterling had the highest parasite abundance in the season after the flood. When analysing ecto- and endoparasites separately, a decrease in ectoparasite abundance was found in roach and perch, probably related to the flood, and due mainly to a decrease in monogenean species. A significant increase in the abundance of endoparasites was found in perch. In bitterling, the post-flood increase in ectoparasites was due to a high abundance of Gyrodactylus spp. We conclude that the parasite communities of the three fish species responded in different ways to the flood. This may be due to changes in the host life history strategy or in the immune response in conditions of stress.     

In vitro differentiation of Trypanosoma cruzi under chemically defined conditions

Metacyclic trypomastigotes of Trypanosoma cruzi have been obtained in chemically defined axenic culture. The differentiating medium, composed of artificial triatomine urine supplemented with proline, allows high yields of metacyclic trypomastigotes after 72-h incubation of T. cruzi cells at 27 degrees C. Morphological differentiation of the parasites is gradual under these chemically defined conditions and is preceded by the expression of stage-specific polypeptides. The yield of in vitro-induced metacyclic trypomastigotes depends upon the age of the epimastigote culture, the size of the inoculum and the depth of the medium. Metacyclic trypomastigotes differentiated in vitro from the Dm 28c clone of T. cruzi are both resistant to complement lysis and to macrophage digestion. They are able to infect mice with an efficiency similar to that obtained for natural metacyclic trypomastigotes obtained from triatomine excreta.     

Structural plasticity of developing optic synapses under different lighting conditions

Four-month-old male hooded rats were reared from birth under constant light and darkness conditions. Changes in the amount of postsynaptic density material in the optic synapses in the suprachiasmatic nucleus of these rats were compared with animals maintained under routine light-dark (12 h) cycles. The thickness of postsynaptic density material was found to be significantly greater in dark-reared rats relative to light-reared animals. The plasticity of this structure may have functional implications in the sensitivity of postsynaptic response. There was no significant difference in the lengths of the synaptic apposition and the size of boutons.     

Proliferation and differentiation sequence of osteoblast histogenesis under physiological conditions in rat periodontal ligament

To define the mechanism of osteoblast histogenesis, nuclear morphometry was utilized as a marker for precursor cell differentiation. One hour after ³H-thymidine injection, groups of 7-week-old rats were killed at hourly intervals over one complete 24-hr photoperiod (LD 12:12). S-phase and mitosis were assessed in autoradiographs of 3-μm sections of molar periodontal ligament (PDL) adjacent to a physiological bone-forming surface. Labeled nuclei were divided into four categories according to morphometry of nuclear size: A (40–79 μm³), B (80–119 μm³), C (120–169 μm³), and D (≥ 170 μm³) cells. C and D cells synthesize DNA during the light and divide in the following dark phase; the rhythm for A cells is the opposite. B cells demonstrated no preference and were subsequently determined to be nonosteogenic. Compared to A cells the S-phase photoperiod of C and D cells (combined) is approximately a one-to-one reciprocal relationship, suggesting two proliferating progenitors in series. Based on arrest points in the histogenesis sequence, five compartments are defined: (1) A cells, less differentiated, self-perpetuating precursors; (2) A′ cells, committed osteoprogenitors; (3) C cells, G₁ stage preosteoblasts; (4) D cells, G₂ stage preosteoblasts; and (5) Ob cells, morphologically distinct osteoblasts. Minimal elapsed time for the A →A′ → C → D → Ob sequence is about 60 hr (five alternating dark/light cycles). A stress/strain-mediated increase in nuclear volume (A′ → C) is an important, rate-limiting step in osteoblast differentiation.     

Structural variations and growth potential of Yersinia enterocolitica under different culture conditions

The growth potential and the polypeptide composition of Yersinia enterocolitica serotype 0:3 isolated from patients with uncomplicated diarrhoea, reactive arthritis or septicemia were evaluated under different culture conditions. The expression of polypeptides varied with presence of the virulence-associated 40-48 Mdal plasmid, growth medium, growth temperature and gas composition of the culture (air, carbon dioxide, oxygen). Also the initial growth medium at 26 degrees C, before temperature shift to 37 degrees C, influenced the subsequent growth potential and expression of polypeptides. The plasmid encoded at least 7 polypeptides. This plasmid also inhibited the multiplication of bacteria under defined culture conditions. The dominating plasmid-encoded polypeptides were optimally expressed in air or oxygen-supplemented growth medium. The majority of the chromosomally encoded polypeptides were expressed independently of presence of the plasmid, whereas the expression of at least 8 were repressed by the plasmid. Five chromosomally encoded polypeptides were expressed only in carbon dioxide and five only in oxygen environment. These results indicate that Y. enterocolitica may express different molecules in different environments in vivo. This may be of importance for host-parasite relationship and immune response.     

On certain changes in the degree of corneal tissue differentiation under experimental conditions

Summary Circular burn of the corneal surface reduces the differentiation and induces proliferative growth in the central area in the form of a polyp. Transplants from the cornea with incomplete development, mature rapidly, after being placed between the superficial layers of the corneal stroma. Nerve branches growing into the transplant are visible in the preparations impregnated according to Campos. In the same transplants placed between the deep corneal layers, where the nervous apparatus is poor developed, the degree of differentiation is decreased and follows the type of the tissue culture in vivo. It is suggested that the degree of the tissue differentiation and its functional adequacy are maintained by the nervous apparatus, whereas denervation provokes tissue autonomism, i.e., reduction of differentiation and acceleration of tissue growth.(Presented by Active Member of the Akad. Med. Nauk SSSR N. A. Kraevskii) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 56, No. 7, pp. 99–103, July, 1963     

Isolation and characterization of Chlamydomonas temperature-sensitive mutants affecting gametic differentiation under nitrogen-starved conditions

Summary Two conditional mutants of Chlamydomonas reinhardtii, dif-1 and dif-2, affecting gametic differentiation under conditions of nitrogen (N)-starvation, have been isolated. These mutant cells remain vegetative at the restrictive temperature (35°C) in — N medium, as defined by assays of cell body-agglutinin and cell wall lytic enzyme activities in the soluble fractions of cell homogenates. Moreover, the mutants fail to form mating structures at the restrictive temperature, but do so at the permissive temperature (25°C). Temperature-shift experiments show that mutant cells which have differentiated into gametes at 25°C dedifferentiate into vegetative cells under N-starvation conditions after transfer to 35°C, but differentiate again into gametes at 25°C. Genetic analyses indicate that the dif-1 and dif-2 genes are recessive and unlinked to each other or to the matingtype locus; the dif-1 phenotype cosegregates with a conditional flagellales phenotype expressed in both +N and-N medium at the restrictive temperature.     

Formation and structure of mixed bacterial communities.

Mixed bacterial communities are formed by unrelated bacteria on solid media. Mixed bacterial communities on solid media are similar to "classical" colonies and are formed after the growth of a large number of unrelated bacteria simultaneously plated onto a limited area of agar. The morphology of the mixed bacterial communities was similar for different combinations of bacteria and did not change when the bacteria were plated on different media. Different bacterial strains form zones of individual and mixed growth in the structure of mixed bacterial communities. The results of electron microscopic examination indicate that mixed bacterial communities are isolated from their external environment by a surface film. The basic part of this film is formed by an elementary membrane. The membrane of the surface film of mixed bacterial communities is a stable structure occupying a large surface area. The results of this investigation seem to indicate the existence of a special type of co-operation between different species of bacteria. This type of co-operation may be very important in the regulation of interactions between different bacteria and between bacteria and the environment.     

Inhibition of bacterial and leukocyte adhesion under shear stress conditions by material surface chemistry

Biomaterial-centered infections, initiated by bacterial adhesion, persist due to a compromised host immune response. Altering implant materials with surface modifying endgroups (SMEs) may enhance their biocompatibility by reducing bacterial and inflammatory cell adhesion. A rotating disc model, which generates shear stress within physiological ranges, was used to characterize adhesion of leukocytes and Staphylococcus epidermidis on polycarbonate-urethanes and polyetherurethanes modified with SMEs (polyethylene oxide, fluorocarbon and dimethylsiloxane) under dynamic flow conditions. Bacterial adhesion in the absence of serum was found to be mediated by shear stress and surface chemistry, with reduced adhesion exhibited on materials modified with polydimethylsiloxane and polyethylene oxide SMEs. In contrast, bacterial adhesion was enhanced on materials modified with fluorocarbon SMEs. In the presence of serum, bacterial adhesion was primarily neither material nor shear dependent. However, bacterial adhesion in serum was significantly reduced to < or = 10% compared to adhesion in serum-free media. Leukocyte adhesion in serum exhibited a shear dependency with increased adhesion occurring in regions exposed to lower shear-stress levels of < or = 7 dyne/cm2. Additionally, polydimethylsiloxane and polyethylene oxide SMEs reduced leukocyte adhesion on polyether-urethanes. In conclusion, these results suggest that surface chemistry and shear stress can mediate bacterial and cellular adhesion. Furthermore, materials modified with polyethylene oxide SMEs are capable of inhibiting bacterial adhesion, consequently minimizing the probability of biomaterial-centered infections.     

Inhibition of bacterial and leukocyte adhesion under shear stress conditions by material surface chemistry

Biomaterial-centered infections, initiated by bacterial adhesion, persist due to a compromised host immune response. Altering implant materials with surface modifying endgroups (SMEs) may enhance their biocompatibility by reducing bacterial and inflammatory cell adhesion. A rotating disc model, which generates shear stress within physiological ranges, was used to characterize adhesion of leukocytes and Staphylococcus epidermidis on polycarbonate-urethanes and polyetherurethanes modified with SMEs (polyethylene oxide, fluorocarbon and dimethylsiloxane) under dynamic flow conditions. Bacterial adhesion in the absence of serum was found to be mediated by shear stress and surface chemistry, with reduced adhesion exhibited on materials modified with polydimethylsiloxane and polyethylene oxide SMEs. In contrast, bacterial adhesion was enhanced on materials modified with fluorocarbon SMEs. In the presence of serum, bacterial adhesion was primarily neither material nor shear dependent. However, bacterial adhesion in serum was significantly reduced to 10% compared to adhesion in serum-free media. Leukocyte adhesion in serum exhibited a shear dependency with increased adhesion occurring in regions exposed to lower shear-stress levels of 7 dyne/cm². Additionally, polydimethylsiloxane and polyethylene oxide SMEs reduced leukocyte adhesion on polyether-urethanes. In conclusion, these results suggest that surface chemistry and shear stress can mediate bacterial and cellular adhesion. Furthermore, materials modified with polyethylene oxide SMEs are capable of inhibiting bacterial adhesion, consequently minimizing the probability of biomaterial-centered infections.     

Perception of blindness and blinding eye conditions in rural communities

PURPOSE: The purpose of this qualitative study was to explore the causes and management of blindness and blinding eye conditions as perceived by rural dwellers of two Yoruba communities in Oyo State, Nigeria. METHODS: Four focus group discussions were conducted among residents of Iddo and Isale Oyo, two rural Yoruba communities in Oyo State, Nigeria. Participants consisted of sighted, those who were partially or totally blind and community leaders. Ten patent medicine sellers and 12 traditional healers were also interviewed on their perception of the causes and management of blindness in their communities. FINDINGS: Blindness was perceived as an increasing problem among the communities. Multiple factors were perceived to cause blindness, including germs, onchocerciasis and supernatural forces. Traditional healers believed that blindness could be cured, with many claiming that they had previously cured blindness in the past. However, all agreed that patience was an important requirement for the cure of blindness. The patent medicine sellers' reports were similar to those of the traditional healers. The barriers to use of orthodox medicine were mainly fear, misconception and perceived high costs of care. There was a consensus of opinion among group discussants and informants that there are severe social and economic consequences of blindness, including not been able to see and assess the quality of what the sufferer eats, perpetual sadness, loss of sleep and dependence on other persons for daily activities. CONCLUSION: Local beliefs associated with causation, symptoms and management of blindness and blinding eye conditions among rural Yoruba communities identified have provided a bridge for understanding local perspectives and basis for implementing appropriate primary eye care programs.     

The bacterial mutagenicity of synthetic all-trans fecapentaene-12 changes when assayed under anaerobic conditions

Fecapentaenes are potent mutagens produced in the anaerobicenvironment of the human colon. The aim of this study was todetermine the effect of anaerobic conditions on the bacterialmutagenicity of all-trans fecapentaene-12, a synthetic fecapen-taene.Fecapentaene-12 was tested aerobkally and anaer-obkaUy at dosesfrom 0.25 to 4 µg/plate in agar-overlay assays with Salmonellatyphimurium TA98 and TA100 and Escherichia coli WP2urnA(pKM101),and 0.01 to 2 µg/ml in fluctuation test with TA100. Inagar-overlay tests, fecapentaene-12 was less mutagenic to theframeshift mutant TA98 under aerobic conditions than under anaerobicconditions (average slopes of 3.8 and 31.6 revertants/µgrespectively). Aerobic assays using TA100 and E.coli WP2uvrA-(pKM101)gave respective slopes of 62.9 and 167.6. Anaerobic assays withthese base-substitution mutants gave negative results underconditions in which positive controls were mutagenic. However,the numbers of spontaneous revertants in these anaerobic assayswere substantially lower than normal. Microtitre fluctuationtests, known to perform equally well under both aerobic andanaerobic conditions, were conducted with TA100 to confirm thatthe activity of fecapentaene-12 as a base-substitution mutagenwas attenuated under anaerobic conditions. Replicate aerobicassays gave an average slope (revertants/well/µg) of 0.41,compared with 0.056 for anaerobic assays—a > 7-folddifference. There was no significant difference in slope betweenaerobic and anaerobic positive controls. Thus, fecapentaene-12may have two distinct modes of action, acting as a base-substitutionmutagen and as a frameshift mutagen. Anaerobic conditions suppressthe base-substitution activity but not the frameshift activity.These findings suggest that reactive but labile compounds suchas fecapentaenes formed in the faecal stream are unreactivein the anaerobic environment of the lumen of the large bowelbut could become reactive if they reached the oxygenated intestinalmucosa where expression of their genotoxicity could initiateneoplasia. ¹To whom correspondence should be addressed     

The role of von Hippel-Lindau protein in the differentiation of neural progenitor cells under normoxic and anoxic conditions

Von Hippel-Lindau protein (pVHL) normally functions to cause ubiquitin-mediated degradation of hypoxia-inducible factor-1alpha (HIF-1alpha) under normoxic but not under hypoxic conditions, and induces neuronal differentiation of neural progenitor cells. However, the role of pVHL in the differentiation of neural progenitor cells under either condition has not been fully elucidated. Herein, we show that under the anoxic condition the expression of pVHL and neuronal markers in neural progenitor cells was inhibited, while HIF-1alpha was induced. In addition, neural progenitor cells expressing pVHL following gene transfer showed distinct neuronal differentiation and no induction of HIF-1alpha under the normoxic condition but not under the anoxic condition. In conclusion, neuronal differentiation induced by pVHL is associated with degradation of HIF-1alpha and occurs normally under the normoxic condition but not under the anoxic condition. Differentiation of neuronal progenitor cells may thus depend on oxygen density.     

Influence of cultivation conditions on bacterial DNA content and bacterial membrane permeability

The study included measurement of intracellular and extracellular DNA content and evaluation of bacterial membrane condition during plankton growth and in isolated communities. This was performed at different stages of bacterial development and after heating at the temperature of heat shock system activation. The content of intracellular bacterial DNA in the community decreased after 72 hours of cultivation growth more prominently than during plankton growth. Change of intracellular bacterial DNA content in the community overtook the increase of membrane permeability, which may indicate the onset of apoptosis. Heating at 420' decreased the permeability of bacterial membranes in the community, but did not affect this parameter in the bacteria during plankton growth. Extracellular DNA was found in the matrix of the communities after 24 hours of growth. Appearance of this DNA after 48-74 hours of cultivation may be associated with apoptotic process.     

Expression of neuronal and inducible nitric oxide synthases in the thymus under normal conditions and after administration of bacterial endotoxin

The topography of thymocytes expressing neuronal and inducible nitric oxide synthases and changes in the content of luminescent immunoreactive products in these cells after intraperitoneal injection of bacterial lipopolysaccharide were studied by double immunohistochemical labeling. Under normal conditions neuronal nitric oxide synthase-immunopositive cells formed a wide network in thymus medulla (except for perivascular regions). Inducible nitric oxide synthase was expressed in single cells at the corticomedullary boundary. Lipopolysaccharide markedly increased the intensity of luminescence and number of inducible nitric oxide synthase-immunoreactive cells. However, this agent sharply decreased the intensity of luminescence in neuronal nitric oxide synthase-immunopositive cells of the stroma. Our results indicate that neuronal and inducible nitric oxide synthases are synthesized in various stromal cells of the thymus. Expression of these enzyme isoforms undergoes opposite changes during inflammation.     

Spatial and temporal variability of bacterial communities in high alpine water spring sediments

Water springs are complex, fragile and taxa-rich environments, especially in highly dynamic ecosystems such as glacier forefields experiencing glacier retreat. Bacterial communities are important actors in alpine water body metabolism, and have shown both high seasonal and spatial variations. Seven springs from a high alpine valley (Matsch Valley, South Tyrol, Italy) were examined via a multidisciplinary approach using both hydrochemical and microbiological techniques. Amplified ribosomal intergenic spacer analysis (ARISA) and electric conductivity (EC) measurements, as well as elemental composition and water stable isotopic analyses, were performed. Our target was to elucidate whether and how bacterial community structure is influenced by water chemistry, and to determine the origin and extent of variation in space and time. There existed variations in both space and time for all variables measured. Diversity values more markedly differed at the beginning of summer and then at the end; the extent of variation in space was prevalent over the time scale. Bacterial community structural variation responded to hydrochemical parameter changes; moreover, the stability of the hydrochemical parameters played an important role in shaping distinctive bacterial communities.     

Changes in gut bacterial communities in canaries infected by Macrorhabdus ornithogaster

Macrorhabdus ornithogaster is an opportunistic yeast that colonizes the gastric mucosa of many avian species. Until now, no studies have focused on the influence of a gastric infection on the balance of the intestinal microbiota of birds. In this study, 44 faecal samples from individual canaries, with and without M. ornithogaster infection, were analysed. The detection of the yeast was evaluated by 18S rRNA PCR. In order to evaluate the impact of the Macrorhabdus infection on the bacterial communities, culture-independent methods, by the use of amplicon-based sequencing as well as 16S rRNA-DGGE, were adopted. The different health status of animals affected the relative abundance of the main OTUs, with a greater diversification of the gut microbiota in healthy animals compared to the infected. In particular, Lactococcus, Pseudomonas, Acinetobacter, Lachnospiraceae, Propionibacterium and Weissella were found to be characteristic of uninfected animals (FDR?<?0.05), while Lactobacillus and Candidatus Arthromitus were characteristic of infected animals (FDR?<?0.05). Both these taxa have been reported as immunostimulatory, involved in immunological disorders. In infected animals the inferred metagenome assessed by PICRUST clearly showed a positive correlation between the presence of M. ornithogaster and KEGG genes related to ether lipid metabolism, already reported to be immunostimulatory by activation of macrophages and to play a pathophysiological role in several immunological disorders. Finally, our results show an interaction between infection of the digestive tract and intestinal microbiota of pet birds and provide insight into the changing of the complex enteric bacterial community.

• HIGHLIGHTS

• Macrorabdus ornithogaster is a gastric yeast that colonizes a wide range of birds.

• Differences were found between infected and healthy animals in gut microbiota.

• Candidatus Arthromitus was closely associated with infected birds.

• M. ornithogaster can affect intestinal microbiota composition of canaries.