Bioaugmentation of pilot-scale slow sand filters can achieve compliant levels for the micropollutant metaldehyde in a real water matrix.

Metaldehyde is a polar, mobile, low molecular weight pesticide that is challenging to remove from drinking water with current adsorption-based micropollutant treatment technologies. Alternative strategies to remove this and compounds with similar properties are necessary to ensure an adequate supply of safe and regulation-compliant drinking water. Biological removal of metaldehyde below the 0.1 µg•L-1 regulatory concentration was attained in pilot-scale slow sand filters (SSFs) subject to bioaugmentation with metaldehyde-degrading bacteria. To achieve this, a library of degraders was first screened in bench-scale assays for removal at micropollutant concentrations in progressively more challenging conditions, including a mixed microbial community with multiple carbon sources. The best performing strains, A. calcoaceticus E1 and Sphingobium CMET-H, showed removal rates of 0.0012 µg•h-1•107 cells-1 and 0.019 µg•h-1•107 cells-1 at this scale. These candidates were then used as inocula for bioaugmentation of pilot-scale SSFs. Here, removal of metaldehyde by A. calcoaceticus E1, was insufficient to achieve compliant water regardless testing increasing cell concentrations. Quantification of metaldehyde-degrading genes indicated that aggregation and inadequate distribution of the inoculum in the filters were the likely causes of this outcome. Conversely, bioaugmentation with Sphingobium CMET-H enabled sufficient metaldehyde removal to achieve compliance, with undetectable levels in treated water for at least 14 d (volumetric removal: 0.57 µg•L-1•h-1). Bioaugmentation did not affect the background SSF microbial community, and filter function was maintained throughout the trial. Here it has been shown for the first time that bioaugmentation is an efficient strategy to remove the adsorption-resistant pesticide metaldehyde from a real water matrix in upscaled systems. Swift contaminant removal after inoculum addition and persistent activity are two remarkable attributes of this approach that would allow it to effectively manage peaks in metaldehyde concentrations (due to precipitation or increased application) in incoming raw water by matching them with high enough degrading populations. This study provides an example of how stepwise screening of a diverse collection of degraders can lead to successful bioaugmentation and can be used as a template for other problematic adsorption-resistant compounds in drinking water purification.

Evaluation of the immunogenicity and protective efficacy of a recombinant CS6-based ETEC vaccine in an Aotus nancymaae CS6 + ETEC challenge model.

Colonization factors or Coli surface antigens (CFs or CS) are important virulence factors of Enterotoxigenic E. coli (ETEC) that mediate intestinal colonization and accordingly are targets of vaccine development efforts. CS6 is a highly prevalent CF associated with symptomatic ETEC infection both in endemic populations and amongst travelers. In this study, we used an Aotus nancymaae non-human primate ETEC challenge model with a CS6 + ETEC strain, B7A, to test the immunogenicity and protective efficacy (PE) of a recombinant CS6-based subunit vaccine. Specifically, we determined the ability of dscCssBA, the donor strand complemented recombinant stabilized fusion of the two subunits of the CS6 fimbriae, CssA and CssB, to elicit protection against CS6 + ETEC mediated diarrhea when given intradermally (ID) with the genetically attenuated double mutant heat-labile enterotoxin LT(R192G/L211A) (dmLT). ID vaccination with dscCssBA + dmLT induced strong serum antibody responses against CS6 and LT. Importantly, vaccination with dscCssBA + dmLT resulted in no observed diarrheal disease (PE = 100%, p = 0.03) following B7A challenge as compared to PBS immunized animals, with an attack rate of 62.5%. These data demonstrate the potential role that CS6 may play in ETEC infection and that recombinant dscCssBA antigen can provide protection against challenge with the homologous CS6 + ETEC strain, B7A, in the Aotus nancymaae diarrheal challenge model. Combined, these data indicate that CS6, and more specifically, a recombinant engineered derivative should be considered for further clinical development.

Extant diversity of bryophytes emerged from successive post-Mesozoic diversification bursts.

Unraveling the macroevolutionary history of bryophytes, which arose soon after the origin of land plants but exhibit substantially lower species richness than the more recently derived angiosperms, has been challenged by the scarce fossil record. Here we demonstrate that overall estimates of net species diversification are approximately half those reported in ferns and ∼30% those described for angiosperms. Nevertheless, statistical rate analyses on time-calibrated large-scale phylogenies reveal that mosses and liverworts underwent bursts of diversification since the mid-Mesozoic. The diversification rates further increase in specific lineages towards the Cenozoic to reach, in the most recently derived lineages, values that are comparable to those reported in angiosperms. This suggests that low diversification rates do not fully account for current patterns of bryophyte species richness, and we hypothesize that, as in gymnosperms, the low extant bryophyte species richness also results from massive extinctions.

Prospective evaluation of reliability of cone-beam computed tomography in detecting different position of vibroplasty middle ear implants.

OBJECTIVES: This study had the following objectives: (i) to determine the accuracy of determination of Vibrant Soundbridge position in the spectrum of typically implanted sites in the middle ear, (ii) to assess interobserver agreement between three observers with different levels of radiology experience and (iii) to determine the suitability of cone-beam computed tomography (CT) to be used as the baseline radiological assessment post implantation, confirm ferromagnetic transducer (FMT) position. DESIGN: Prospective research study. Using four fresh human cadaveric heads, different types of vibroplasty were performed. After each step, cone-beam CT was performed for each of the four cadaveric heads. SETTING: University hospital (ENT and Neuroradiology). PARTICIPANTS: Four fresh cadaveric heads of human donors were operated and analysed by radiological imaging. MAIN OUTCOME MEASURES: There are different ways of coupling an ferromagnetic transducer to the anatomical structures of the middle and inner ear. Possibilities of differentiation between these coupling variants should be presented. RESULTS: The individual reconstruction view was significantly different from a standardised view for each observer (observer 1: P = 0.003; observer 2: P = 0.001; observer 3: P = 0.002) for all coupling variants combined as well as for each individual coupling variant (overall correct diagnosis: 100% versus 60%). Regarding the frequency of correct diagnosis, no significant differences were found between the three observers (P > 0.500) for each individual coupling variant as well as for all coupling variants combined. The worst rates of correct diagnosis were found in the standardised view for incus (42%), stapes (0%) and TORP (17%) vibroplasty. CONCLUSION: Cone-beam CT as a radiological control for Vibrant Soundbridge is safe and adequately sensitive and reliable and is therefore suitable for clinical investigation. The position of the ferromagnetic transducer in the middle ear and the presence or absence of an additional coupler could be determined in this study. Therefore, cone-beam-CT is useful for the assessment of device failure when there has been gross displacement of the ferromagnetic transducer (or smaller displacements in case of a baseline postoperative cone-beam CT). Regarding the quality of imaging, cone-beam CT produced accurate results with different observers with widely varying radiological experience.

[Imaging quality and dosage in ENT--what do we really need?]. / Dosisreduktion unter Betrachtung der erforderlichen Bildqualität--Wie viel Dosis braucht ein Bild?

BACKGROUND: Today, imaging of nose, paranasal sinuses and temporal bone by CT is standard in preoperative diagnostics. The need of reduction of applied dosage leads to the necessity of research in necessary imaging quality. Therefore this paper deals with new developed anatomical checklists and the analysis of imaging quality on anterior and lateral skull base. MATERIAL AND METHODS: With 3 human complete heads over 400 examinations were performed on one cone beam CT device under varying x-ray-tube adjustments. 31 anatomic parameters were evaluated (Excellent, well, poor, not evaluable) for every data set. A summation score was built for every examination. RESULTS: As well for paranasal sinuses as for temporal bone a constant excellent imaging quality could be seen in high dosages. Certainly, in low dosages a reduction of imaging quality was detected. The optimal range (all parameters visualized well as average) could be evaluated for paranasal sinuses between 2,0 and 3,0 mGy and between 3,0 and 4,0 mGy for temporal bone. So, a reduction of 70-80% in comparison to highest adjustments of today is possible and realistic. In comparison to standard protocols, a reduction of about 50% can be reached. CONCLUSION: The possibility of dose reduction by discussion of the necessary imaging quality from clinical point of view could be shown.

Potential of dosage reduction in cone-beam-computed tomography (CBCT) for radiological diagnostics of the paranasal sinuses.

More than 10 years ago, cone-beam-computed tomography (CBCT) was introduced in ENT radiology. Until now, the focus of research was to evaluate clinical limits of this technique. The aim of this work is the evaluation of specific dosages and the identification of potential optimization in the performance of CBCT of the paranasal sinuses. Based on different tube parameters (tube current, tube voltage, and rotation angles), images of the nose and the paranasal sinuses were taken on a phantom head with the Accu-I-tomo F17 (Morita, Kyoto, Japan). The dosages applied to the lens and parotid gland were measured with OSL dosimetry. The imaging quality was evaluated by independent observers. All datasets were reviewed according to a checklist of surgically important anatomic structures. Even for lowest radiation exposure (4 mA, 76 kV, 180°, computed tomography dosage index (CTDI) = 1.8 mGy), the imaging quality was sufficient. Of course a significant reduction of the imaging quality could be seen, so a reliable mean was set for 4 mA, 84 kV, and 180° rotation angle (CTDI = 2.4 mGy). In this combination, a reduction of 92 % in lens-dose and of 77 % of dosage at the parotid gland was observed in comparison to the maximal possible adjustments (8 mA, 90 kV, 360°, CTDI = 10.9 mGy). There is potential for optimization in CBCT. Changing the rotation angle (180° instead of 360°) leads to a dose reduction of 50 %. Furthermore from clinical point of view in case of chronic rhinosinusitis a relevant reduction of dosage is possible. Therefore, it is necessary to intensify the interdisciplinary discussion about the disease specifics required quality of imaging.

Exclusive conservation of mitochondrial group II intron nad4i548 among liverworts and its use for phylogenetic studies in this ancient plant clade.

Liverworts occupy a pivotal position in land plant (embryophyte) phylogeny as the presumed earliest-branching major clade, sister to all other land plants, including the mosses, hornworts, lycophytes, monilophytes and seed plants. Molecular support for this earliest dichotomy in land plant phylogeny comes from strikingly different occurrences of introns in mitochondrial genes distinguishing liverworts from all other embryophytes. Exceptionally, however, the nad5 gene--the mitochondrial locus hitherto used most widely to elucidate early land plant phylogeny--carries a group I type intron that is shared between liverworts and mosses. We here explored whether a group II intron, the other major type of organellar intron, would similarly be conserved in position across the entire diversity of extant liverworts and could be of use for phylogenetic analyses in this supposedly most ancient embryophyte clade. To this end, we investigated the nad4 gene as a candidate locus possibly featuring different introns in liverworts as opposed to the non-liverwort embryophyte (NLE) lineage. We indeed found group II intron nad4i548 universally conserved in a wide phylogenetic sampling of 55 liverwort taxa, confirming clade specificity and surprising evolutionary stability of plant mitochondrial introns. As expected, intron nad4i548g2 carries phylogenetic information in its variable sequences, which confirms and extends previous cladistic insights on liverwort evolution. We integrate the new nad4 data with those of the previously established mitochondrial nad5 and the chloroplast rbcL and rps4 genes and present a phylogeny based on the fused datasets. Notably, the phylogenetic analyses suggest a reconsideration of previous phylogenetic and taxonomic assignments for the genera Calycularia and Mylia and resolve a sister group relationship of Ptilidiales and Porellales.

Identification and characterization of a novel family of pneumococcal proteins that are protective against sepsis.

Four pneumococcal genes (phtA, phtB, phtD, and phtE) encoding a novel family of homologous proteins (32 to 87% identity) were identified from the Streptococcus pneumoniae genomic sequence. These open reading frames were selected as potential vaccine candidates based upon their possession of hydrophobic leader sequences which presumably target these proteins to the bacterial cell surface. Analysis of the deduced amino acid sequences of these gene products revealed the presence of a histidine triad motif (HxxHxH), termed Pht (pneumococcal histidine triad) that is conserved and repeated several times in each of the four proteins. The four pht genes (phtA, phtB, phtD, and a truncated version of phtE) were expressed in Escherichia coli. A flow cytometry-based assay confirmed that PhtA, PhtB, PhtD and, to a lesser extent, PhtE were detectable on the surface of intact bacteria. Recombinant PhtA, PhtB, and PhtD elicited protection against certain pneumococcal capsular types in a mouse model of systemic disease. These novel pneumococcal antigens may serve as effective vaccines against the most prevalent pneumococcal serotypes.

Use of a whole genome approach to identify vaccine molecules affording protection against Streptococcus pneumoniae infection.

Microbial targets for protective humoral immunity are typically surface-localized proteins and contain common sequence motifs related to their secretion or surface binding. Exploiting the whole genome sequence of the human bacterial pathogen Streptococcus pneumoniae, we identified 130 open reading frames encoding proteins with secretion motifs or similarity to predicted virulence factors. Mice were immunized with 108 of these proteins, and 6 conferred protection against disseminated S. pneumoniae infection. Flow cytometry confirmed the surface localization of several of these targets. Each of the six protective antigens showed broad strain distribution and immunogenicity during human infection. Our results validate the use of a genomic approach for the identification of novel microbial targets that elicit a protective immune response. These new antigens may play a role in the development of improved vaccines against S. pneumoniae.

Identification and characterization of SirA, an iron-regulated protein from Staphylococcus aureus.

The acquisition of iron by pathogenic bacteria is often a crucial step in establishing infection. To accomplish this, many bacteria, including Staphylococcus aureus, produce low-molecular-weight iron-chelating siderophores. However, the secretion and transport of these molecules in gram-positive organisms are poorly understood. The sequence, organization, and regulation of genes involved in siderophore transport are conserved among gram-negative bacteria. We used this information to identify a putative siderophore transport locus from an S. aureus genomic sequence database. This locus contains three predicted open reading frames with a high degree of homology to genes involved in siderophore uptake in several bacterial species, in particular the cbr locus of the plant pathogen Erwinia chrysanthemi. The first gene in the locus, which we have designated sir for staphylococcal iron regulated, encodes a putative lipoprotein with a molecular mass of 37 kDa. The open reading frame is preceded by a 19-bp region of dyad symmetry with homology for operator sequences controlling iron-regulated expression of genes in other bacteria. Fur titration experiments indicate that this region of dyad symmetry is sufficient for Fur-dependent regulation in Escherichia coli. The expression of this gene was repressed, in a dose-dependent manner, by the addition of iron to the S. aureus culture medium. sir-encoded proteins may be involved in iron acquisition in vivo and therefore may be targets for antimicrobial agents.

Evaluation of carborane-containing porphyrins as tumour targeting agents for boron neutron capture therapy.

A number of carborane-containing porphyrins were administered to mice bearing subcutaneously transplanted mammary carcinomas. Administration was via serial intraperitoneal (i.p.) injections to assess their relative toxicities and tumour affinities. Three analogues of the natural porphyrin heme and four tetraphenylporphyrins (TPPs) were given at total doses of 78-245 micrograms g-1 body weight. The water-insoluble TPPs were less toxic to mice, and delivered greater amounts of boron to tumour than did the water-soluble TPPS and the heme analogues. One such compound, NiTCP-H, delivered more than 100 micrograms B g-1 to tumour tissue with a tumour:blood boron concentration ratio greater than 500:1 and a tumour: brain boron concentration ratio greater than 50:1, 4 days after the last of six i.p. injections given over 2 days. Another TPP analogue, NiTCP, delivered approximately 50 micrograms B g-1 to tumour with similar boron concentrations in normal tissues. Neither compound was toxic to mice at total doses of approximately 200 micrograms g-1 body weight. In contrast, the heme analogues were toxic and, with the exception of VCDP, delivered less boron to tumour than NiTCP and NiTCP-H. The two porphyrins with the greatest potential for application to boron neutron capture therapy (BNCT), NiTCP and NiTCP-H, yielded higher tumour:blood and tumour:brain boron concentration ratios in mice than could be achieved with p-boronophenylalanine (BPA) and sodium mercaptoundecahydrododecaborate (BSH), the compounds which are currently being used in clinical trials of BNCT in the treatment of glioblastoma. The boron delivered by each of the porphyrins tested remained in tumour tissue longer than did boron delivered by either BPA or BSH. The copper and nickel chelates of these porphyrins behave identically in vivo. The former offer the potential for imaging by 67Cu-mediated single photon emission computed tomography (SPECT) to aid BNCT treatment planning.

sar Genetic determinants necessary for transcription of RNAII and RNAIII in the agr locus of Staphylococcus aureus.

The temporal expression of most virulence factors in Staphylococcus aureus is regulated by pleiotropic loci such as agr and sar. We have previously shown that the sar locus affects hemolysin production because it is required for agr transcription. To delineate the sar genetic determinant required for agr transcription, single copies of fragments from the sar locus, encompassing the individual sar transcripts (sarA, sarC, and sarB), were introduced into a sar mutant via the integration vector pCL84. Although a DNA fragment encompassing the sarA transcript plus a 189-bp upstream region was sufficient for agr expression, complementation analysis revealed that the sarB transcript was the most effective in augmenting agr transcription as determined by RNAII and RNAIII transcription and gel retardation assays with the P2 and P3 promoters of agr. As the region upstream of the sarA transcript encodes a 39-amino-acid open reading frame, ORF3, it is possible that posttranslational cooperation between the sarA gene product and ORF3 may be necessary for optimal agr expression. Deletion studies demonstrated that an intact sarA gene is essential for agr transcription. However, mutagenesis and in vitro translation studies revealed that unlike the agr locus, the required element is the SarA protein and not the RNA molecule. Taken together, these results indicate that the sarA-encoded protein, possibly in conjunction with peptides encoded in the upstream region, regulates hemolysin production by controlling agr P2 and P3 transcription.

Regulation of protein A synthesis by the sar and agr loci of Staphylococcus aureus.

The synthesis of protein A in Staphylococcus aureus is regulated by global regulatory loci such as sar and agr. Phenotypic data indicate that both sar and agr suppress protein A synthesis; like agr, sar also regulates protein A production at the transcriptional level. To determine the genetic requirement of sar in protein A suppression, we transformed shuttle plasmids containing various sar fragments into a sar mutant. Our results indicated that the 560-bp sarA transcript, or, more probably, the SarA protein (13.5 kDa), is sufficient for suppressing protein A gene transcription when introduced on a multicopy plasmid or as a single copy in the chromosome. Immunoblot analysis with a chicken anti-protein A antibody also confirmed the reduction in protein A expression in these sar mutant clones. Complementation studies revealed that the transcription of the protein A gene can be suppressed in a sar mutant background by a plasmid containing RNAIII. Surprisingly, in agr deletion mutant clones and in clones derived from the agr-sar double mutant, protein A gene transcription can also be suppressed by plasmids containing the sarA transcript plus additional upstream sequence but not the sarA transcript alone. These data suggest that the sar locus can down-modulate protein A gene transcription via both RNAIII-dependent and RNAIII-independent pathways. Consistent with the hypothesis of an RNAIII-independent pathway is an additional genetic requirement for protein A suppression in the agr deletion mutant RN6911 as well as the isogenic double sar-agr mutant, whereas in the sar mutant background, the sarA transcript encoding the SarA protein alone is sufficient. These data suggested that both sar and agr are coregulators of protein A synthesis in S. aureus.

Synthesis of a nickel tetracarboranylphenylporphyrin for boron neutron-capture therapy: biodistribution and toxicity in tumor-bearing mice.

Nickel-2,3,7,8,12,13,17,18-octaacetic acid-5,10,15,20-tetra-[3-carboranyl-methoxyphenyl]-porphyrin octamethylester (NiTCP) was given in a Cremophor EL, a polyethoxylated castor oil, and propylene glycol emulsion to BALB/c mice bearing transplanted s.c. KHJJ mammary carcinomas. A total dose of 244 microg NiTCP/gram body weight (gbw) (54 microg B/gbw) was given in 6 i.p. injections over a 32 hr period. Observations of behavior and changes in body weight and chemical and hematological blood tests indicated little or no toxicity from NiTCP over a period of 6-90 hr after injections. Boron concentrations near tumor margins were 160-180 microg B/g at 41-90 hr after the last injection. Tumor:normal brain boron concentration ratios reached approx. 10:1 and tumor:blood ratios reached approx. 250:1 after 4 days. There was no evidence of thrombocytopenia or other potentially important toxicities. Our findings place NiTCP among the leading candidates for pre-clinical experiments aimed toward improvement upon the compounds being tested for boron neutron-capture therapy of glioblastoma multiforme.

The molecular architecture of the sar locus in Staphylococcus aureus.

The global regulator sar in Staphylococcus aureus controls the synthesis of a variety of cell wall and extracellular proteins, many of which are putative virulence factors. The sar locus in strain RN6390 contains a 339-bp open reading frame (sarA) and an 860-bp upstream region. Transcriptional analyses of this locus revealed three different transcripts of 0.58, 0.84, and 1.15 kb (designated sarA, sarC, and sarB, respectively). All three transcripts seemed to be under temporal, growth cycle-dependent regulation, with sarA and sarB being most abundant in early log phase and the sarC concentration being highest toward the late stationary phase. Mapping of the 5' ends of the sar transcripts by primer extension and modified S1 nuclease protection assays demonstrated that transcription is initiated from three separate, widely spaced promoters. The 3' ends of all three sar transcripts are identical, and transcriptional termination occurs upstream of a typical prokaryotic poly(T) termination signal. Northern (RNA) analysis of sar mutant clones containing plasmids that comprised various promoters and the termination signal revealed that individual transcripts can be generated from each of the three promoters, thus suggesting possible activation as independent promoters. The multipromoter system, from which transcription is initiated, bears conserved features for recognition by homologous sigma 70 transcription factors and also by those expressed in the general stress response. Downstream of the two distal promoters (P3 and P2) are two regions potentially encoding short peptides. It is conceivable that posttranslational cooperation between these short peptides and the sarA gene product occurs to modulate sar-related functions. Complementation studies of a sar mutant with a clone expressing all three sar transcripts showed that this clone was able to restore the sar wild-type phenotype to the sar mutant.

Characterization of the sar locus and its interaction with agr in Staphylococcus aureus.

The expression of cell wall and extracellular proteins in Staphylococcus aureus is controlled by global regulatory systems, including sar and agr. We have previously shown that a transposon insertion into the 372-bp sarA gene within the sar locus resulted in decreased expression of several extracellular and cell wall proteins (A. L. Cheung and S. J. Projan, J. Bacteriol. 176:4168-4172, 1994). In this study, Northern (RNA blot) analysis with a 732-bp sarA probe indicated that two major transcripts (0.56 and 1.2 kb) were absent in the sar mutant compared with the parental strain RN6390. Additional transcriptional studies revealed that the sarA gene is encoded within the 0.56-kg transcript. Notably, a plasmid carrying the sarA gene together with a 1.2-kb upstream fragment (1.7 kb total) was able to reestablish the 1.2-kb transcript in the mutant. Although reconstitution of the parental phenotype by the sarA gene was incomplete, the introduction of a plasmid carrying the 1.7-kb fragment to the mutant restored the parental phenotype. Transcription of RNAII and RNAIII, which encode the structural and regulatory genes of agr, respectively, was diminished in the mutant but restored to wild-type levels by complementation with the 1.7-kb fragment. In gel shift assays, cell extracts of this clone were able to retard the mobility of a labeled RNAII promoter probe but not an RNAIII promoter element. These data suggest that sarA and the adjacent upstream DNA are essential to the expression of a DNA-binding protein(s) with specificity for the RNAII promoter, thereby controlling agr-related transcription.