Electrolyzed Saline Targets Biofilm Periodontal Pathogens In Vitro.

Preventing the development and recurrence of periodontal diseases often includes antimicrobial mouthrinses to control the growth of the periodontal pathogens. Most antimicrobials are nonselective, targeting the symbiotic oral species as well as the dysbiosis-inducing ones. This affects the overall microbial composition and metabolic activity and consequently the host-microbe interactions, which can be detrimental (associated with inflammation) or beneficial (health-associated). Consequently, guiding the antimicrobial effect for modulating the microbial composition to a health-associated one should be considered. For such an approach, this study investigated electrolyzed saline as a novel rinse. Electrolyzed saline was prepared from sterile saline using a portable electrolysis device. Multispecies oral homeostatic and dysbiotic biofilms were grown on hydroxyapatite discs and rinsed daily with electrolyzed saline (EOS). Corresponding positive (NaOCl) and negative (phosphate-buffered saline) controls were included. After 3 rinses, biofilms were analyzed with viability quantitative polymerase chain reaction and scanning electron microscopy. Supernatants of rinsed biofilms were used for metabolic activity analysis (high-performance liquid chromatography) through measuring organic acid content. In addition, human oral keratinocytes (HOKs) were exposed to EOS to test biocompatibility (cytotoxicity and inflammation induction) and also to rinsed biofilms to assess their immunogenicity after rinsing. Rinsing the dysbiotic biofilms with EOS could reduce the counts of the pathobionts (>3 log10 Geq/mm2 reduction) and avert biofilm dysbiosis (≤1% pathobiont abundance), leading to the dominance of commensal species (≥99%), which altered both biofilm metabolism and interleukin 8 (IL-8) induction in HOKs. EOS had no harmful effects on homeostatic biofilms. The scanning electron micrographs confirmed the same. In addition, tested concentrations of EOS did not have any cytotoxic effects and did not induce IL-8 production in HOKs. EOS showed promising results for diverting dysbiosis in in vitro rinsed biofilms and controlling key periopathogens, with no toxic effects on commensal species or human cells. This novel rinsing should be considered for clinical applications.

Independent phasing of rephasing and non-rephasing 2D electronic spectra.

Assigning absolute phase to two-dimensional (2D) third-order nonlinear optical signals generally requires acquiring both the rephasing and the non-rephasing signals and comparing the sum of the two to spectrally resolved pump-probe spectra. To date, however, Gradient Assisted Photon Echo Spectroscopy (GRAPES) has only been able to acquire rephasing spectra. Such a constraint requires a new phasing protocol. Here, we analytically prove that the rephasing and non-rephasing spectra can be phased independently using pump-probe signal. We verify this result holds even for finite duration pulses by simulation. This relationship holds for all 2D spectroscopies, not only GRAPES. In addition, we present improvements to GRAPES that enable acquisition of rephasing and non-rephasing signals in different phase-matched directions. We employ our phasing protocol to phase the data for laser dye IR-144, leading to reconstruction of purely absorptive 2D spectrum.

Short communication: serum immunoglobulin G and total protein concentrations in dairy calves fed a colostrum-replacement product.

Neonatal calf health is largely dependent on the ingestion and absorption of maternally derived antibodies via colostrum administration. The objective of this study was to evaluate the efficacy of a commercially available plasma-derived colostrum-replacement (CR) product as compared with bovine colostrum. Holstein calves were removed from the dam immediately after birth and randomly allocated to 1 of 3 groups. Group 1 calves (n=22) were fed 1 package of the CR product; group 2 calves (n=22) were fed 2 packages of the CR product; and group 3 calves (n=22) were fed 3 L of bovine colostrum. Blood samples were collected from all calves 24h after colostrum or CR feeding and analyzed for serum IgG and total protein concentrations. Calves fed bovine colostrum had significantly higher serum IgG and total protein concentration than calves in either group fed the CR product. Group 1 calves (1 package of CR product) had a significantly higher incidence of failure of transfer of passive immunity than calves in groups 2 or 3. The results of this study indicated that 2 packages of this CR product achieved adequate IgG concentrations in calves. However, calves fed 1 package of this CR product consistently had failure of transfer of passive immunity.

Insulin-like growth factor II and WT1 transcript localization in human fetal kidney and Wilms' tumor.

In situ hybridization was used to examine, in parallel, the localization of insulin-like growth factor II (Igf2) and WT1 transcripts in normal fetal kidney and Wilms' tumor. The expression of Igf2 and WT1 transcripts in the fetal kidney is almost complementary in both the epithelial and stromal cell lineages derived from the undifferentiated metanephrogenic blastema. The patterns of transcription of Igf2 in three Wilms' tumors appeared to be perturbed as compared to the normal fetal kidney. In these tumors Igf2 transcripts were detected in structures that are developmentally equivalent to the renal vesicle, which in the normal kidney do not contain Igf2 transcripts. These results suggest that Wilms' tumors arise from an alteration in the regulation of Igf2 mRNA synthesis.

Expression of the FcRn receptor (alpha and beta) gene homologues in the intestine of suckling brushtail possum (Trichosurus vulpecula) pouch young.

The neonatal IgG transporter FcRn consists of two chains, FcRn alpha and beta (also known as beta(2) microglobulin), and is involved in transferring IgG molecules across both mammary and intestinal epithelial cells. Developmental changes in FcRn IgG alpha and beta chain mRNA levels were investigated in the gut of brushtail possum (Trichosurus vulpecula) pouch young (PY) using Northern hybridisation. FcRn alpha transcripts were detected in the PY proximal intestine at all times examined, between days 1 and 195 of post-natal life, with increased levels detected from around day 110. The beta(2) microglobulin transcript levels in the PY proximal intestine were low to undetectable until day 110 of post-natal life and then increased dramatically after day 159. Both the FcRn alpha and beta gene transcripts were detected in a wide range of tissues in the adult possum (>365 days). Genomic sequences located 5' to the start of transcription of the FcRn alpha and beta(2) microglobulin genes were cloned and analysed for predicted cis-acting transcription control elements. Both the FcRn alpha and beta(2) microglobulin genomic sequences contained STAT5 binding motifs consistent with the transcription of both genes being modulated by prolactin. Using in situ hybridisation, the FcRn alpha and beta(2) microglobulin transcripts were localised to the epithelial cells of the PY intestine. However, no prolactin receptor transcripts were detected in the same epithelial cells suggesting that the observed changes in FcRn alpha and beta(2) microglobulin gene expression in the proximal intestine are not modulated directly by prolactin. The results are consistent with the hypothesis that changes in FcRn alpha and beta(2) microglobulin gene expression take place in the possum PY intestine to accommodate changes in maternal milk composition to meet the changing immunological demands of the PY.

Expression and secretion of a biologically active glycoprotein hormone, ovine follicle stimulating hormone, by Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, has been used to co-express recombinant genes formed by fusion of the mating factor-alpha (MFalpha) leader and ovine follicle stimulating hormone (oFSH) alpha and beta subunit coding sequences. Pichia strains carrying single copies of the two fusion genes secreted recombinant oFSH (roFSH) to concentrations of approximately 51.0 ng/ml and 17.5 ng/ml, measured by RIA or in vitro bioassay respectively, whereas a strain with two copies of the alpha and one copy of the beta subunit fusion genes secreted roFSH to concentrations of 61 ng/ml (RIA) and 22 ng/ml (bioassay). It appears that the Pichia-derived roFSH had about one-third the in vitro bioactivity of native oFSH or, alternatively, only one-third of the roFSH is bioactive. Measurements of secreted roFSH alpha and beta subunit concentrations indicated less than 10% of alpha and 25-33% of beta subunits were stably dimerized. The receptor binding properties of the roFSH resemble those of native oFSH. In summary this paper reports the production, by P. pastoris, of a heterodimeric glycoprotein hormone (roFSH) that has in vitro biological activity.

The Australian brushtail possum (Trichosurus vulpecula) gonadotrophin alpha-subunit: analysis of cDNA sequence and pattern of expression.

A cDNA sequence from the gonadotrophin alpha-subunit mRNA of Australian brushtail possum (Trichosurus vulpecula) has been determined and analysed. Comparison with seven eutherian mammalian gonadotrophin alpha-subunit gene sequences revealed an average of 82.6% homology between the coding region nucleotide sequences and 88.8% identity between the predicted amino acid sequences. The predicted possum gonadotrophin alpha-subunit protein has ten evolutionarily conserved cysteine residues, two potential N-linked glycosylation sites and a putative enzyme recognition sequence which it has been suggested is required for sulphation of carbohydrate moieties. Comparison of the possum gonadotrophin alpha-subunit 3' untranslated region (UTR) sequence with the 3' UTRs of eutherian alpha-subunit transcripts revealed sequence homology. In particular, an 18 nucleotide imperfect palindromic sequence present in the possum 3' UTR, with the potential to form a hairpin loop, was found to be evolutionarily conserved and present in five out of seven eutherian alpha-subunit 3' UTR sequences. In situ hybridization localized the transcripts to a sub-population of anterior pituitary cells presumed to be gonadotrophs and thyrotrophs. In summary, these results indicate considerable conservation of the structure and function of the gonadotrophin alpha-subunit protein since the divergence of the marsupial and eutherian mammalian lineages.

cDNA sequence analysis, gene expression and protein localisation of the inhibin alpha-subunit of Australian brushtail possum (Trichosurus vulpecula).

An inhibin alpha-subunit cDNA sequence from the Australian brushtail possum (Trichosurus vulpecula) has been identified and analysed. The cDNA includes an open reading frame encoding a predicted precursor protein of 361 amino acids. The predicted protein sequence includes four possible proteolytic cleavage sites, 12 evolutionarily conserved cysteine residues and three potential N-linked glycosylation sites. The mature alpha-subunit is the carboxyl terminal fragment (alphaC) consisting of 131 amino acids. The full-length precursor protein shows a mean identity with eutherian homologues of 69.8%. The homology is not evenly distributed, with the putative alphaC fragment showing the highest level (79.7%). Using Northern hybridisation, an alpha-subunit transcript of approximately 1.6 kb was detected in adult possum ovary. Using in situ hybridisation and immunocytochemistry, inhibin alpha-subunit was localised exclusively to the granulosa cell layers of follicles. Hybridisation and immunostaining for the inhibin alpha-subunit were first observed in granulosa cells of primary follicles and the expression continued throughout all stages of follicular growth. Inhibin alpha-subunit mRNA and protein were also detected in cells of the corpus luteum. In summary, results indicate considerable conservation of the structure and possible function of the inhibin alpha-subunit protein since the divergence of the marsupial and eutherian mammalian lineages. The expression data suggest that, in the adult possum, inhibin may have a role in ovarian follicular growth from the primary stage of development.

Production of biologically active tethered ovine FSHbetaalpha by the methylotrophic yeast Pichia pastoris.

The pituitary-derived glycoprotein hormone FSH plays a central role in controlling vertebrate gonadal function. In female mammals the maturation of ovarian follicles is critically dependent upon stimulation by FSH. Moreover, injection of exogenous FSH is used extensively to stimulate increased numbers of follicles to ovulate. Structurally FSH is a heterodimeric glycoprotein composed of two non-covalently associated polypeptide subunits. The tertiary structures of both the alpha- and beta-subunits are constrained by intramolecular disulphide bonds and are post-translationally modified with two N-linked carbohydrate moieties, the structure of which appears to modulate in vivo biological activity. Here we report the expression of ovine FSH (oFSH) as a biologically active single-chain polypeptide using the methylotrophic yeast Pichia pastoris. Sequences encoding the mature oFSH alpha- and beta-proteins were fused to form a gene encoding a fusion protein with the C-terminus of the beta-chain joined to the N-terminus of the alpha-chain, with the chains separated by a two amino acid linker sequence. This fusion gene was itself fused to two alternative Pichia leader sequences (mating factor alpha and acid phosphatase) and transformed into the Pichia strains GS115 and SMD1168. The recombinant fusion protein (oFSHbetaalpha) was expressed at approximately 0.1 microg/ml in 'shake-flask' cultures. The Pichia-expressed tethered protein was biologically active in an in vitro bioassay, had a molecular mass of 28 kDa, as determined by SDS-PAGE, and bound the bovine FSH receptor with a binding profile similar to that of native oFSH.

Growth and paracrine factors regulating follicular formation and cellular function.

The purpose of this paper is to review, using fetal sheep as the animal model, aspects of ovarian development related to follicular formation and to report on the identity of growth and paracrine factors which might be involved in this process. Before follicular formation there is a massive and sustained colonisation of the fetal ovary by mesonephric cells, which become a precursor source of follicular cells. From within the ovarian medulla, somatic 'cell-streams' branch into the cortex around nests of oogonia and oocytes. These 'cell-streams', which contain elongated cells with either flattened or cuboidal shaped nuclei, express steroidogenic factor-1 (SF-1), steroid acute regulatory protein (StAR), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450(scc), and P450(aromatase) mRNA and/or protein. Follicles form from the association of an oocyte with the 'cell-stream' with either a single layer of flattened cells (i.e. type 1 follicle) or with a mixture of flattened and cuboidal cells (i.e. type 1a follicle). These newly-formed follicles have between 3 and 57 somatic cells (i.e. granulosa cells) and contain oocytes which vary in diameter between 23 and 52 microm. Newly formed and early growing follicles have been identified with growth factors or growth factor receptors in either the oocytes or granulosa cells. Many of the growth factors are from the TGFbeta superfamily and are expressed in a cell- and stage-specific manner.

Field test of a rapid assessment technique against a probabilistic community survey: operational implications for neonatal tetanus elimination.

Neonatal tetanus (NNT) is a leading cause of neonatal mortality in developing countries and is frequently called 'the silent epidemic'. It is endemic in over 90 countries throughout the world. Incidence is often not known, obstructing cost-effective resource management for control measures. In many developing countries NNT is responsible for one-half of the neonatal mortality and up to one-quarter of infant mortality. Case-fatality rates (CFR) can, even with treatment, reach 80-90%. Operational tools for the rapid identification of NNT risk areas need to be developed for WHO's programme which calls for the elimination of NNT by 1995. Results of a rapid assessment technique, carried out in 1990, were compared with those found in a household survey, which was independently carried out in Jalisco, Mexico, in 1988. One approach used random sample survey techniques in rural communities, which in previous years had reported NNT. Of 40 neonatal deaths, 8 (20%) were attributable to NNT. The annual incidence rate was 4.6/1000 livebirths. Using this as the 'gold standard', a rapid assessment technique was evaluated. The NNT cases seen at health services were randomly matched with other neonatal illnesses obtained from health records and then mapped. Age-specific catchment areas for hospitals under investigation and risk areas for NNT were obtained. Areas without NNT cases but with other neonatal illnesses have been operationally considered to be at low risk for NNT. The use of health services by neonates with other pathologies supports the hypothesis that an NNT case, if it occurred within the same time period and area under investigation, would most probably have been admitted.(ABSTRACT TRUNCATED AT 250 WORDS)

Residual neurologic deficits 30 years after occupational exposure to elemental mercury.

A battery of tests of peripheral and central nervous system function was administered to 205 former workers of a large heavy industrial plant, 104 of whom were previously exposed to inorganic mercury. The mean age of those examined was 71 years. Exposed subjects had participated in a urine-mercury exposure monitoring program during the time of operation of a process that required the use of mercury and its subsequent clean-up. Mercury exposure had been high (mean peak urine mercury concentration was >600 microg/l) and had ended 30 years or more prior to the investigation. Peripheral nerve function outcomes that were statistically significantly associated with cumulative mercury exposure after controlling for covariates included classification as having peripheral neuropathy, peroneal motor nerve conduction velocity, ulnar motor nerve conduction velocity, and peroneal motor nerve F-wave latency. Quantitative assessment of resting tremor was nearly significantly associated with cumulative mercury exposure (p=0.07). Among tests of central nervous system function, results of the Handeye Coordination test were significantly associated with cumulative mercury exposure after controlling for covariates. Cumulative mercury exposure was not observed to be associated with a quantitative measure of dementia or with a number of cognitive neurobehavioral test outcomes. The statistically significant associations with mercury exposure were observed in spite of greater mortality among the exposed group than the unexposed group. These results suggest that substantial occupational mercury exposure can have long-term adverse effects on the peripheral nervous system detectable decades after cessation of exposure. Such long-term adverse effects were not observed for a measure of dementia or other measures of cognitive function.

The follicle-stimulating hormone beta-subunit gene of the common brushtail possum (Trichosurus vulpecula): analysis of cDNA sequence and expression.

Reverse transcription-PCR has been used to obtain a cDNA sequence from the follicle-stimulating hormone (FSH) beta-subunit gene of the Australian brushtail possum (Trichosurus vulpecula). Comparisons of the possum FSHbeta-mRNA coding region nucleotide sequence with that of six eutherian mammal homologues reveals a mean percent identity of 77.3% and 76.8% at the nucleotide and predicted amino acid-sequence levels respectively. Furthermore, the predicted amino acid sequence of the possum FSHbeta mature protein shows evolutionary conservation of twelve cysteine residues and two potential N-linked glycosylation sites. The protein lacks the CAGY motif present in most reported glycoprotein beta-subunit sequences. The translation termination codon and consensus polyadenylation sequence overlap, a feature observed in other mammalian FSHbeta genes. Northern hybridization of total RNA from adult female possum pituitary revealed three hybridizing transcripts of approximately 2.8, 1.2 and 0.5 kb which may arise from utilizing alternative polyadenylation signals. In situ hybridization localized the FSHbeta transcripts to a sub-population of anterior pituitary cells interpreted as being gonadotropes. In summary the results indicate considerable evolutionary conservation of the structure of the FSH beta-subunit gene between the marsupial and eutherian mammalian lineages.

Screening the foods of an endangered parrot, the kakapo (Strigops habroptilus), for oestrogenic activity using a recombinant yeast bioassay.

In recent years the possibility of environmental oestrogens affecting the reproduction of vertebrates has become an issue of both public and scientific interest. Although the significance of such chemicals remains controversial there is clear evidence that, in some contexts, environmental oestrogens can influence the fertility of vertebrates. Highly endangered species represent a situation in which even modest reductions in the fertility of key individuals may have implications for the survival of the entire species. This paper reports the screening of both natural and supplementary foods of the kakapo (Strigops habroptilus), a critically endangered New Zealand nocturnal parrot, for oestrogenic activity using a recombinant yeast based bioassay. Low levels of oestrogenic activity were detected in one of the 'chick-raising' foods, but no oestrogenic activity was detected in the adult supplementary foods. The oestrogenicity of a range of phytochemicals possibly associated with the kakapo natural diet was also examined. Two such phytochemicals, podocarpic acid and its reduced derivative podocarpinol, showed weak oestrogenic activity (approximately 10(-6) and 10(-4) of the activity of 17-beta-oestradiol, respectively).

Infertility: from a personal to a public health problem.

The inability to conceive a child is most often viewed as a private matter, but public health perspectives and skills can contribute greatly to our knowledge about infertility, and the development of effective and rational public policy for prevention, access to health care, and regulation of new technologies. We offer a primer of public health aspects of infertility in an effort to encourage the broad spectrum of public health professionals to become more knowledgeable about these topics and join in the national debate about preventive strategies, cost-benefit assessment, resource allocation, and ethics.

Influence of developer exhaustion on accuracy of quantitative digital subtraction radiography: an in vitro study.

OBJECTIVE: The aim of the study was to evaluate the influence of developer exhaustion on accuracy of quantitative digital subtraction radiography. STUDY DESIGN: Six objects, each incorporating a section of dry human mandible, were radiographed with 4 exposure times. Baseline films were processed in fresh solutions, whereas follow-up films were processed in fresh and in increasingly exhausted solutions (ie, 1, 2, and 3 weeks old). Bone loss and bone gain were computer simulated in 17 regions of interest on baseline radiographs. Area and volume of changes in mineralization were measured in subtracted images, obtained by subtraction of baseline from their corresponding follow-up radiographs. Friedman's 2-way analysis of variance by ranks and Wilcoxon signed-rank test were used for statistical analysis. RESULTS: Because of exhausted developer, bone loss was relatively underestimated from 6.6% to 16.5% (P <.05), whereas bone gain was relatively overestimated from 9.7% to 16.7% (P <.05). CONCLUSIONS: This in vitro study demonstrates that films for quantitative digital subtraction radiography should be processed in fresh developer or error might be introduced.

The effect of image content on detail preservation and file size reduction in lossy compression.

OBJECTIVES: To demonstrate the effect of image content on image detail preservation and file size reduction. METHODS: The first set, containing 16 in vitro images with variable projection geometry, exposure time, bone level and number of teeth, was compressed with three compression modes: JPEG quality factor (JPQF), JPEG2000 quality factor (J2QF) and JPEG2000 compression ratio (J2CR). Image detail degradation was evaluated by local mean square error (MSE) on a standardized region of interest (ROI), containing bone. The second set, containing 105 clinical bitewings, was compressed with the same compression modes at 3 quality factors/compression ratios and local MSEs were calculated on two ROIs, containing bone and crown. RESULTS: For the first image set, nearly constant MSE was found for the JPQF and J2QF compression modes, while file size depended on projection geometry, exposure time, bone level and the number of teeth. In contrast, file size reduction was nearly constant for the J2CR compression mode, while MSE depended on the abovementioned factors. Similarly, for the second image set, nearly constant MSE and variation of file size reduction were found for JPQF and J2QF but not for the J2CR compression mode. All of these results were consistent for all three quality factors/compression ratios. CONCLUSIONS: Constant image detail preservation, crucial for diagnostic accuracy in radiology, can only be assured in QF compression mode in which the file size of the compressed image depends on the original image content. CR compression mode assures constant file size reduction, but image detail preservation depends on image content.

Avian Clock gene polymorphism: evidence for a latitudinal cline in allele frequencies.

In comparison with most animal behaviours, circadian rhythms have a well-characterized molecular genetic basis. Detailed studies of circadian clock genes in 'model' organisms provide a foundation for interpreting the functional and evolutionary significance of polymorphic circadian clock genes found within free-living animal populations. Here, we describe allelic variation in a region of the avian Clock orthologue which encodes a functionally significant polyglutamine repeat (ClkpolyQcds), within free-living populations of two passerine birds, the migratory bluethroat (Luscinia svecica) and the predominantly nonmigratory blue tit (Cyanistes caeruleus). Multiple ClkpolyQcds alleles were found within populations of both species (bluethroat: 12 populations, 7 alleles; blue tit: 14 populations, 9 alleles). Some populations of both species were differentiated at the ClkpolyQcds locus as measured by F(ST) and R(ST) values. Among the blue tit, but not bluethroat populations, we found evidence of latitudinal clines in (i) mean ClkpolyQcds repeat length, and (ii) the proportions of three ClkpolyQcds genotype groupings. Parallel analyses of microsatellite allele frequencies, which are considered to reflect selectively neutral processes, indicate that interpopulation allele frequency variation at the ClkpolyQcds and microsatellite loci does not reflect the same underlying demographic processes. The possibility that the observed interpopulation ClkpolyQcds allele frequency variation is, at least in part, maintained by selection for microevolutionary adaptation to photoperiodic parameters correlated with latitude warrants further study.

Lossy JPEG compression: easy to compress, hard to compare.

OBJECTIVES: To review the literature on lossy compression in dental radiography and to discuss the importance and suitability of the methodology used for evaluation of image compression. METHODS: A search of Medline (from 1966 to October 2004) was undertaken with the search expression "(Radiography, dental) and compression". Inclusion criterion was that the reference should be evaluating the effect of lossy image compression on diagnostic accuracy. For all included studies, information in relation to mode of image acquisition, image content, image compression, image display, and method of image evaluation was extracted. RESULTS: 12 out of 32 papers were included in the review. The design of these 12 studies was found to vary considerably. Parameters used to express the degree of information loss (DIL) were either or both compression ratio (CR) and compression level (CL). The highest acceptable CR reported in the studies ranged from 3.6% to 15.4%. Furthermore, different CR values were proposed even for the same diagnostic task, for example, for caries diagnosis CR ranged from 6.2% to 11.1%. CONCLUSION: Lossy image compression can be used in clinical radiology if it does not conflict with national law. However, the acceptable DIL is difficult to express and standardize. CR is probably not suitable to express DIL, because it is image content dependent. CL is also probably not suitable to express DIL because of the lack of compression software standardization.