Systematic update to the mammalian relative potency estimate database and development of best estimate toxic equivalency factors for dioxin-like compounds.

The World Health Organization (WHO) assesses potential health risks of dioxin-like compounds using Toxic Equivalency Factors (TEFs). This study systematically updated the relative potency (REP) database underlying the 2005 WHO TEFs and applied advanced methods for quantitative integration of study quality and dose-response. Data obtained from fifty-one publications more than doubled the size of the previous REP database (∼1300 datasets). REP quality and relevance for these data was assessed via application of a consensus-based weighting framework. Using Bayesian dose-response modeling, available data were modeled to produce standardized dose/concentration-response Hill curves. Study quality and REP data were synthesized via Bayesian meta-analysis to integrate dose/concentration-response data, author-calculated REPs and benchmark ratios. The output is a prediction of the most likely relationship between each congener and its reference as model-predicted TEF uncertainty distributions, or the 'best estimate TEF' (BE-TEF). The resulting weighted BE-TEFs were similar to the 2005 TEFs, though provide more information to inform selection of TEF values as well as to provide risk assessors and managers with information needed to quantitatively characterize uncertainty around TEF values. Collectively, these efforts produce an updated REP database and an objective, reproducible approach to support development of TEF values based on all available data.

Assessing children's exposure to manganese in drinking water using a PBPK model.

A previously published human PBPK model for manganese (Mn) in infants and children has been updated with Mn in drinking water as an additional exposure source. Built upon the ability to capture differences in Mn source-specific regulation of intestinal uptake in nursing infants who are breast-fed and formula-fed, the updated model now describes the bioavailability of Mn from drinking water in children of ages 0-18. The age-related features, including the recommended age-specific Mn dietary intake, age-specific water consumption rates, and age-specific homeostasis of Mn, are based on the available human data and knowledge of the biology of essential-metal homeostasis. Model simulations suggest that the impact of adding drinking-water exposure to daily Mn exposure via dietary intake and ambient air inhalation in children is not greater than the impacts in adults, even at a drinking-water concentration that is 2 times higher than the USEPA's lifetime health advisory value. This conclusion was also valid for formula-fed infants who are considered at the highest potential exposure to Mn from drinking water compared to all other age groups. Our multi-route, multi-source Mn PBPK model for infants and children provides insights about the potential for Mn-related health effects on growing children and will thereby improve the level of confidence in properly interpreting Mn exposure-health effects relationships in children in human epidemiological studies.

Feature analysis of ultrasound elastography image for quantitative assessment of cutaneous carcinoma.

BACKGROUND: To evaluate the feasibility of using quantitative texture features computed from high frequency ultrasound and ultrasound elastography (USE) images in the discrimination of benign from malignant skin lesions. METHODS: A commercial ultrasound system with a 14 MHz transducer was used to visualize skin lesions requiring biopsy on clinical evaluation. Patients were enrolled over a 6-month period and imaged prospectively by operators blind to the histopathologic diagnosis. Anatomic ultrasound and USE imaging of the skin lesions was performed using a 2-4 mm gel standoff pad before biopsy and histopathologic evaluation. The ElastoAnalysis software developed for the texture analysis of USE images was provided by Hitachi. The software computes thirteen texture features within a region of interest (ROI), which have demonstrated promise in diagnostic characterization of liver fibrosis staging and in quantitative elastography of breast cancer. This approach has not yet been studied in the quantitative assessment of skin cancer. Results were retrospectively compared to the histopathologic diagnosis and a diagnostic criteria with the goal of maximizing sensitivity was evaluated for each textural feature. RESULTS: Of the 37 lesions included, among 30 patients who participated, 12 lesions were malignant and 25 were benign. Eleven out of thirteen textural metrics computed by the software were useful in differentiating benign from malignant lesions with 100% sensitivity and specificities ranging from 28% to 85%. CONCLUSIONS: This feasibility study demonstrated that feature analysis of USE may be useful in quantitatively differentiating cancerous from benign primary solitary skin lesions prior to biopsy.

Prospective analysis of visual outcomes using apodized, diffractive multifocal intraocular lenses following phacoemulsification for cataract or clear lens extraction.

BACKGROUND: To evaluate efficacy, safety and predictability of apodized, diffractive multifocal intraocular lenses. DESIGN: Prospective, observational study. PARTICIPANTS OR SAMPLE: Two hundred three patients (363 eyes) underwent surgery for cataract (54.8%) or clear lens extraction (45.2%). METHODS: A study of consecutive patients undergoing lens extraction with insertion of the AcrySof Natural ReSTOR intraocular lens (SN60D3). Assessment included: refraction, corneal topography, biometry, monocular and binocular uncorrected and best spectacle corrected distance and near visual acuity. MAIN OUTCOME MEASURES: Visual acuity, spectacle dependence. RESULTS: Mean preoperative best corrected visual acuity was 6/12 (cataract) and 6/6 (clear lens extraction). One hundred sixty-one (98.2%) of the clear lens extraction group were hyperopic and presbyopic (mean spherical equivalent +4.25±3.5D). Postoperatively uncorrected visual acuity was ≥6/12 in 96.5% of eyes (51.9% ≥6/6) and ≥ N5 in 95% of eyes. At 6 months, 182 eyes (91.4%) (cataract) and 137 eyes (83.5%) (clear lens extraction) were ≤0.50 D of target spherical equivalent. Spectacle independence was reported in 81.9% (n=168). Minor halos or other visual phenomena were reported in 10.2%. No subjects required intraocular lens exchange. Safety index at 6 months was 2.5 and 1.17 for cataract and clear lens extraction, respectively. CONCLUSIONS: The apodized, diffractive multifocal intraocular lens predictably provided excellent near uncorrected visual acuity and good distance uncorrected visual acuity following cataract and clear lens extraction surgery with a high rate of spectacle independence. A minority of subjects note halos and other visual phenomena that may be related to intraocular lens design.

Do early stages of lumbar intervertebral disc degeneration really cause instability? Evaluation of an in vitro database.

Early stages of intervertebral disc degeneration are postulated to cause instability. In the literature, however, some authors report the opposite. These contradictory positions are probably supported by the mostly small number of segments which are investigated. The aim of this project therefore was to investigate the influence of intervertebral disc degeneration on lumbar spine rotational stability using a large data set. The flexibility data from all spine specimens tested in our institute so far were collected in a large in vitro database. From this database, all lumbar spine specimens were selected, which had been tested for flexibility under pure moment loads of ±7.5 N m and for which radiographs were accessible. 203 segments met these criteria. Their radiographic degree of disc degeneration was determined on a scale from 0 (no degeneration) to 3 (severe degeneration) and their influence on the respective range of motion and neutral zone was examined. The different lumbar levels differ in flexibility, which increases the variability of the data if pooled together. To minimise this effect a statistical model was fitted. The model-based mean estimates showed a decrease of the range of motion from grade 0 to 3 in flexion/extension (by 3.1°, p < 0.05) and lateral bending (by 3.4°, p < 0.05). In contrast, in axial rotation the range of motion tended to increase; however, not only from grade 0 to 1 but also towards grade 3 (by 0.2°) (p > 0.05). The neutral zone was affected in a similar way but to a smaller degree (p > 0.05). In conclusion, the results indicated that early stages of intervertebral disc degeneration do not necessarily cause rotational instability. In contrast, stability increased in flexion/extension and lateral bending. Only in axial rotation stability tended to decrease.

Measuring interoception: The phase adjustment task.

Interoception, perception of one's bodily state, has been associated with mental health and socio-emotional processes. However, several interoception tasks are of questionable validity, meaning associations between interoception and other variables require confirmation with new measures. Here we describe the novel, smartphone-based Phase Adjustment Task (PAT). Tones are presented at the participant's heart rate, but out of phase with heartbeats. Participants adjust the phase relationship between tones and heartbeats until they are synchronous. Data from 124 participants indicates variance in performance across participants which is not affected by physiological or strategic confounds. Associations between interoception and anxiety, depression and stress were not significant. Weak associations between interoception and mental health variables may be a consequence of testing a non-clinical sample. A second study revealed PAT performance to be moderately stable over one week, consistent with state effects on interoception.

Transgene expression and effective gene silencing in vagal afferent neurons in vivo using recombinant adeno-associated virus vectors.

Vagal afferent fibres innervating thoracic structures such as the respiratory tract and oesophagus are diverse, comprising several subtypes of functionally distinct C-fibres and A-fibres. Both morphological and functional studies of these nerve subtypes would be advanced by selective, effective and long-term transduction of vagal afferent neurons with viral vectors. Here we addressed the hypothesis that vagal sensory neurons can be transduced with adeno-associated virus (AAV) vectors in vivo, in a manner that would be useful for morphological assessment of nerve terminals, using enhanced green fluorescent protein (eGFP), as well as for the selective knock-down of specific genes of interest in a tissue-selective manner. We found that a direct microinjection of AAV vectors into the vagal nodose ganglia in vivo leads to selective, effective and long-lasting transduction of the vast majority of primary sensory vagal neurons without transduction of parasympathetic efferent neurons. The transduction of vagal neurons by pseudoserotype AAV2/8 vectors in vivo is sufficiently efficient such that it can be used to functionally silence TRPV1 gene expression using short hairpin RNA (shRNA). The eGFP encoded by AAV vectors is robustly transported to both the central and peripheral terminals of transduced vagal afferent neurons allowing for bright imaging of the nerve endings in living tissues and suitable for structure-function studies of vagal afferent nerve endings. Finally, the AAV2/8 vectors are efficiently taken up by the vagal nerve terminals in the visceral tissue and retrogradely transported to the cell body, allowing for tissue-specific transduction.

The opposite and antagonistic effects of the closely related POU family transcription factors Brn-3a and Brn-3b on the activity of a target promoter are dependent on differences in the POU domain.

The Brn-3a, Brn-3b, and Brn-3c POU family transcription factors are closely related to one another and are members of the group IV subfamily of POU factors. Here we show that despite this close relationship, the factors have different effects on the activity of a target promoter: Brn-3a and Brn-3c stimulate the promoter whereas Brn-3b represses it. Moreover, Brn-3b can antagonize the stimulatory effect of Brn-3a on promoter activity and can also inhibit promoter activation by the Oct-2.1 POU factor. The difference in the transactivation activities of Brn-3a and Brn-3b is dependent upon the C-terminal region containing the POU domain of the two proteins, since exchange of this domain between the two factors converts Brn-3a into a repressor and Brn-3b into an activator.

Detection of an intrinsic marker in hypoxic cells.

An autoradiographic method is presented for detecting, within a cell population, those cells which have been subjected to chronic hypoxia. No radioisotope is administered; rather the photographic emulsion is chemically reduced by intrinsic constituents of the cells. Hypoxic regions in the sandwich system, a multicellular in vitro tumor model, were detected in this manner. These regions were then compared with hypoxic sandwich regions as demonstrated by [3H]misonidazole labeling. Auxiliary studies, including studies on hypoxic monolayers, were consistent with the sandwich results. In all cases, the intracellular distribution of the chemographic grains was found to be cytosolic. Often the grains were clustered near the nucleus, perhaps in the region of the endoplasmic reticulum and the Golgi. We conclude that cells in a state of hypoxia and nutrient deprivation similar to that found in solid tumors retain a detectably altered biology for a significant period after reoxygenation. Therefore systematic methods of detecting previous hypoxia in histological tumor sections are feasible.

Characterization of a Plasmodium vivax cysteine proteinase gene identifies uniquely conserved amino acids that may mediate the substrate specificity of malarial hemoglobinases.

The gene encoding a cysteine proteinase of the human malaria parasite Plasmodium vivax has been identified and characterized. The sequence predicted by the proteinase gene shares several unique features with the sequences of two recently characterized cysteine proteinases of other malarial species. These features include the conservation of a number of amino acids that are predicted, based on a recently devised model for the related Plasmodium falciparum cystine proteinase, to be located near the enzyme's active site. We hypothesize that these residues have been conserved to maintain optimal proteolytic specificity in the hydrolysis of globin by malaria parasites.

Taxonomy and conformational analysis of loops in proteins.

We propose a general classification scheme for loops, aperiodic segments of protein structure. In an effort to avoid the geometric complexity created by non-repeating phi psi angles, a morphologic definition that focuses upon the linearity and planarity of loops is utilized. Out of 432 loops (4 to 20 residues in length) extracted from 67 proteins, 205 are classified as linear (straps), 133 as non-linear and planar (omegas), and 86 as non-linear and non-planar (zetas). The remaining 8 are classified as compound loops because they contain a combination of strap, omega, and zeta morphologies. We introduce a structural alphabet as a shorthand notation for describing local conformation. The symbols of this alphabet are based on the virtual dihedral angle joining four consecutive alpha carbons. The notation is used to provide a compact description of loop motifs in phosphate binding and calcium binding proteins. Since similar loop conformations form similar "words", the structural sequence facilitates the search for common structural motifs in a family of loops. Contrary to the view of loops as "random coils", we find loops to have positional preferences for amino acid residues analogous to those previously described for beta-turns.

Anti-malarial drug development using models of enzyme structure.

BACKGROUND: The trophozoite stage of the malaria parasite infects red blood cells. During this phase of their life-cycle, the parasites use hemoglobin as their principal source of amino acids, using a cysteine protease to degrade it. We have previously reported a three-dimensional model of this cysteine protease, based on the structures of homologous proteases, and the use of the program DOCK to identify a ligand for the malaria protease. RESULTS: Here we describe the design of improved ligands starting from this lead. Ligand design was based on the predicted configuration of the lead compound docked to the model three-dimensional structure of the protease. The lead compound has an IC50 of 6 microM, and our design/synthesis strategy has resulted in increasingly potent derivatives that block the ability of the parasites to infect and/or mature in red blood cells. The two best derivatives to date have IC50(s) of 450 nM and 150 nM. CONCLUSIONS: A new class of anti-malarial chemotherapeutics has resulted from a computational search that was based on a model of the target protease. Despite the lack of a detailed experimental structure of the target enzyme or the enzyme-inhibitor complex, we have been able to identify compounds with increased potency. These compounds approach the activity of chloroquine (IC50 = 20 nM), but have a distinct mechanism of action. This series of compounds could thus lead to new therapies for chloroquine-resistant malaria.

3H-misonidazole labeling and viability of hypoxic cells in the sandwich system, an in vitro tumor analogue.

3H-misonidazole was used as a marker of hypoxic cells in an in vitro tumor analogue, the sandwich system. MISO binding was assessed in situ, using autoradiography. Binding profiles indicate that there are regions of radiobiological hypoxia surrounding the necrotic center in sandwiches of the V79 cell line and in sandwiches of the 9L cell line. Grains per cell were counted and detailed statistics on the variation of intrinsic binding among cells in the same microenvironment are presented. There is a systematic decrease in the standard deviation of grains per cell as one examines populations of cells further and further from the nutrient and oxygen source. Kinetic studies show that the growth fraction of the cell population also decreases with distance from the nutrient source. These findings taken together suggest that MISO binding is proportional to cell size and cells in the inner noncycling portion of the sandwich are more nearly uniform in size. Sandwich cells which exhibit heavy MISO binding, and are presumably radiobiologically hypoxic, were shown to be still viable if restored to good nutrient and oxygen conditions.

Comparison of 3H-misonidazole binding between CHO and 9L cells using the sandwich system.

3H-misonidazole binding of 9L cells was compared with that of CHO cells using an in vitro tumor analog, the sandwich system. In sandwiches there is a gradient of microenvironments, with cells adjacent to the necrotic center subjected to low concentrations of oxygen and glucose and to high concentrations of metabolites. Mixed sandwiches, having 9L and CHO cells interspersed, were used along with sandwiches of each individual cell line. MISO binding was assessed in situ, using autoradiography. Grains per cell were counted and detailed statistics were obtained on the variation in MISO binding among cells located in the same microenvironment. In all cases binding in the regions near the necrotic center was more than 50 times the binding found at the sandwich edge and found in control monolayers, indicating radiobiological hypoxia near the necrotic center. 9L cells began to significantly increase binding of MISO metabolites at a somewhat higher oxygen concentration than did CHO cells. At all oxygen tensions, average per cell binding of the 9L cells was 3 times or more that of the CHO cells, a factor greater than can be explained by the ratio of cell volumes alone. Statistical analyses of the variation in binding among cells in a given microenvironment give some evidence that in the mixed CHO/9L sandwiches there are interactions between the cells of the two different lines which affect the growth patterns of the cells. No preferred binding of misonidazole in the nucleus or cytoplasm was noted within the cells.

Adenovirus-mediated gene delivery to the corneal endothelium.

Genetic manipulation of donor cornea prior to transplantation has the potential to modulate the allogeneic response, as well as the endothelial cell function. This study examined the feasibility of gene transfer to corneal endothelial cells using replication-defective recombinant adenoviral vectors. Adult rabbits corneas were infected with recombinant adenovirus RAd35, containing the Escherichia coli beta-galactosidase (lacZ) gene. Localization of gene transfer was assessed by histochemical staining for beta-galactosidase and recombinant protein production was quantified by a soluble assay. In initial experiments, the efficiency of gene transfer and kinetics of expression were studied ex vivo, using organ culture of transfected corneas. Following coculture of whole corneal fragments with RAd35, high levels of gene expression were evident on days 107, diminishing after that time. Gene transfer was found to be almost entirely restricted to corneal endothelial cells, with scattered expression in epithelial cells. Following these ex vivo studies, genetically modified corneas were transplanted as orthotopic allografts in rabbits. Similar kinetics of gene expression were seen after transplantation as in the ex vivo experiment, with maximal levels of gene expression in endothelial cells on days 1-4 after grafting. Corneal function following transplantation was not affected by the gene transfer, with the corneas attaining clarity within 1 day of grafting, and thereafter showing the expected thinning on ultrasonic pachymetry. In the absence of any immunosuppression, no inflammation was evident in graft recipient eyes, with the exception of allograft rejection in 1 animal 23 days after grafting. In this study we show that gene transfer to nonreplicating corneal endothelial cells is feasible using recombinant adenovirus vectors, and so may have potential application in the setting of corneal transplantation.

Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement.

BACKGROUND: The antiviral effect of anti-influenza drugs such as zanamivir may be demonstrated in patients as an increased rate of decline in viral load over a time course of treatment as compared with placebo. Historically this was measured using plaque assays, or Culture Enhanced Enzyme Linked Immunosorbent Assay (CE-ELISA). OBJECTIVES: to develop and characterise real time quantitative PCR (qPCR) assays to measure influenza A and B viral load in clinical samples, that offer improvements over existing methods, in particular virus infectivity assays. STUDY DESIGN: The dynamic range and robustness were established for the real time qPCR assays along with stability of the assay components. Cross validation of the real time PCR assays with CE-ELISA was performed by parallel testing of both serial dilutions of three different subtypes of cultured virus and a panel of influenza positive throat swab specimens. RESULTS: the assays were specific for influenza A and B and the dynamic ranges were at least seven logs. The assay variability was within acceptable limits but increased towards the lower limit of quantification, which was 3.33 log(10) viral cDNA copies/ml of virus transport medium (ten viral RNA copies/PCR). The components of the assay were robust enough to withstand extended storage and several freeze-thaw cycles. For the real time PCR assays the limit of quantification was equivalent to the virus infectivity cut off, which equates to a 93-fold increase in sensitivity. CONCLUSION: Well characterised real time PCR assays offer significant improvements over the existing methods for measuring the viral load of strains of influenza A and B in clinical specimens.

The effect of visual deprivation and proprioceptive change on postural sway in healthy adults.

In 39 healthy active people aged 17 to 79 who had not fallen, anteroposterior sway during quiet standing increased slightly with age; there was no increase with age in lateral sway. Sway increased on deprivation of visual information and altered proprioception due to foot pressure sensory change in all age groups, and especially when both forms of sensation were altered concurrently; but the increase of sway on pressure sensory change was no greater in the older than in the younger subjects.

The effects of an oral multivitamin combination with calcium, magnesium, and zinc on psychological well-being in healthy young male volunteers: a double-blind placebo-controlled trial.

RATIONALE: Vitamin and mineral supplements may be associated with improved psychological status. OBJECTIVE: The present study tested the effects of a multivitamin and mineral supplement (Berocca) on psychological well-being. METHODS: In a double-blind randomised-control trial, 80 healthy male volunteers were assigned to either Berocca or placebo. Questionnaires measuring psychological state were completed and a blood sample taken to determine plasma zinc concentration on day 1 (pre-treatment) and again on day 28 (post-treatment), following 28 days of treatments, which were administered at a dosage of one tablet daily. At the end of the study, the acceptability of the treatment and participants' awareness of treatment condition were assessed, as was habitual dietary behaviour. RESULTS: Relative to placebo, treatment with Berocca was associated with consistent and statistically significant reductions in anxiety and perceived stress. Participants in the Berocca group also tended to rate themselves as less tired and better able to concentrate following treatment. In addition, participants registered more somatic symptoms following placebo than following Berocca. These effects cannot be attributed to differences in the acceptability of the two treatments or to participants guessing what treatment they received. CONCLUSION: These findings demonstrate that Berocca significantly reduces anxiety and perceived stress.

Photo amidoglycosylation of an allal azidoformate. Synthesis of beta-2-amido allopyranosides.

[figure: see text] Photolysis of an allal C-3 azidoformate provoked intramolecular nitrene insertion into the glycal C=C unit and allowed direct incorporation of alcohol nucleophiles as beta-disposed substituents at C-1. The 2-amido allopyranoside products were elaborated via N-acylation and selective oxazolidinone hydrolysis, providing N-Boc-protected 2-amino sugars and simplifying stereochemical assignments. Synthesis of the potentially labile allal azidoformate was achieved via reaction of the corresponding carbonyl imidazolide with trimethylsilyl azide, facilitated by dibutyltin oxide.