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Alloiococcus otitidis is usually detected in children with otitis media (OM) by PCR as it is not often detected by routine culture. Our improved method for its isolation obtained A. otitidis from nearly 50% of 78 children with OM with effusion. The role of A. otitidis in pathogenesis of OM is unclear. This study tested two hypothesis: (1) that fresh isolates of A. otitidis would elicit pro-inflammatory cytokines from THP-1 monocytic cells equivalent to those induced by Streptococcus pneumoniae; (2) priming THP-1 cells with interferon-gamma (IFN-γ) a surrogate for virus infection, would enhance pro-inflammatory responses. Recent clinical isolates of A. otitidis, S. pneumoniae (ATCC 49619) and a blood culture isolate of S. pneumoniae (SP2) were used in the assays. Cytokines were quantified by BioRad bead assay and Luminex 200. IFN-γ priming enhanced cytokine responses. S. pneumoniae ATCC 49619 induced lower responses than SP2 for IL-1β, IL-6, TNF-α. A. otitidis LW 27 elicited higher IL-1β and TNF-α responses than either pneumococcal isolate. Small green colony types of A. otitidis induced higher responses than large white colony types for IL-8 and IL-1β. The hypothesis that A. otitidis elicits cytokines observed in middle ear effusions was supported; the need to use recent clinical isolates in studies of pathogenesis was highlighted.

In our model system using the THP-1 monocytic cell line, whole heat-killed cells of Alloiococcus otitidis elicited several pro-inflammatory cytokines identified in ear effusions of children with otitis media (OM). Levels of these cytokines were equivalent to or greater than those elicited by a standard Gram-positive otopathogen, Streptococcus pneumoniae. The current study examined the hypothesis that extracellular material produced by A. otitidis might also contribute to the inflammatory responses in OM. Cell-free culture filtrates of recent A. otitidis isolates (n = 39) were tested for induction of pro-inflammatory cytokines from THP-1 cells primed with IFN-γ. The highest responses were from IL-8 followed by IL-1β, and the lowest from IL-6. Filtrates from nine isolates were treated with lysozyme or proteinase K to assess the nature of the extracellular stimulants. Peptidoglycan was not a major component eliciting the responses. There was no correlation between colony type or β-haemolysin production. Proteinase K treatment indicated extracellular proteins might induce the inflammatory responses, particularly the 70-75 ku band. Further studies on the role of the extracellular proteins of A. otitidis and cytokine responses in pathogenesis of ear infections are needed.

Background: Slips, trips and falls (STF) are a major cause of workplace injury., Aims: To examine risk factors for STF at a large US chemical manufacturing company., Methods: We conducted a case-control study of occupational STF. Cases were identified from company injury records between 1 April 2009 and 1 May 2011. Four controls per case were randomly selected from all active company workers employed during the same time. Data were collected through a questionnaire and from company medical examinations. Logistic regression was used to calculate odds ratio (OR) and 95% confidence intervals (95% CI) for personal, environmental and health-related risk factors for STF., Results: There were 74 cases and 309 controls. The response rate was 65% for the cases and 68% for the controls. Most STF were unrelated to production activities. When examining all factors in a logistic regression model, increased OR were observed for increased body mass index (OR = 1.44, 95% CI: 1.03-2.02), having arthritis (OR = 2.11, 95% CI: 1.01-4.37), lack of exercise (OR = 2.25, 95% CI: 1.01-5.05), carrying materials (OR = 3.01, 95% CI: 1.41-6.43) and being female (OR = 2.46, 95% CI: 1.17-5.19). Reduced risk of STF was observed for never having smoked (OR = 0.48, 95% CI: 0.24-0.95), long service (OR = 0.53, 95% CI: 0.34-0.81) and persons working over 8h a day (OR = 0.42, 95% CI: 0.20-0.88)., Conclusions: Risk factors for STF in a large US chemical company are similar to those reported from other workplaces, but we found that staying fit and healthy is important for reducing risk.

Introduction: The bromodomain (BRD) and extra-C terminal domain (BET) protein family consists of four members (BRD2, BRD3, BRD4 and BRDT). These "epigenetic readers" bind to acetyllysine (KAc) residues on the tails of histones H3 and H4, and regulate chromatin structure and gene expression. There is increasing evidence of their role in human disease, and recently a number of small-molecule inhibitors have been reported. There is increasing interest in the inhibition of BET proteins for a variety of therapeutic applications that have resulted in considerable patent activity from academia and biotechnology and pharmaceutical companies., Areas Covered: Data supporting the use of BET inhibitors in treating disease are outlined, and the current patent literature is discussed. The survey is focused on patents claiming compounds as BET inhibitors and additional patents covering compounds now reported as BET inhibitors have been included., Expert Opinion: There is now compelling preclinical data demonstrating BET inhibition as a strategy to target processes known to be involved in disease development and progression with clinical trials of two bona fide BET inhibitors now underway. Patent activity in this area is increasing with initial activity focused on variations to reported BET inhibitors and more recent patents disclosing novel chemotypes as BET inhibitors.

The peak age at which sudden infant death syndrome (SIDS) occurs corresponds to the developmental period in which infants are dependent on their innate responses to infection. There is a growing body of evidence indicating that dysregulation of inflammatory responses might contribute to the physiological changes leading to these sudden deaths. This study examined the effects of three important risk factors for SIDS on inflammatory responses: cigarette smoke, virus infection and male sex. Cytokine responses of peripheral monocytic blood cells of healthy, non-smoking males and females to endotoxin were measured. Surrogates for virus infection or cigarette smoke were assessed using IFN-γ or water-soluble cigarette smoke extract (CSE). For most conditions, cells from males had lower pro-inflammatory cytokine responses than those of females. An opposite trend was observed for IL-10. Significantly lower levels of some cytokines were noted for cells from male donors exposed to CSE. In females, there were significant correlations between testosterone levels and levels of pro-inflammatory cytokines, but none for males. Testosterone levels in females correspond to those among male infants in the age range at greatest risk of SIDS. The effects of the testosterone surge in male infants need to be examined in relation to changes in cortisol levels that occur during the same period of infant development.

The prosurvival protein BCL-2 is frequently overexpressed in estrogen receptor (ER)-positive breast cancer. We have generated ER-positive primary breast tumor xenografts that recapitulate the primary tumors and demonstrate that the BH3 mimetic ABT-737 markedly improves tumor response to the antiestrogen tamoxifen. Despite abundant BCL-XL expression, similar efficacy was observed with the BCL-2 selective inhibitor ABT-199, revealing that BCL-2 is a crucial target. Unexpectedly, BH3 mimetics were found to counteract the side effect of tamoxifen-induced endometrial hyperplasia. Moreover, BH3 mimetics synergized with phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitors in eliciting apoptosis. Importantly, these two classes of inhibitor further enhanced tumor response in combination therapy with tamoxifen. Collectively, our findings provide a rationale for the clinical evaluation of BH3 mimetics in therapy for breast cancer., (Copyright © 2013 Elsevier Inc. All rights reserved.)

When formulating a biopharmaceutical protein, its stability in the liquid state is critical. In addition, when preparing biological reference materials the stability, both when lyophilised and after reconstitution, needs to be determined. In order to optimise the stability in aqueous conditions (as indicated by Tmelt or denaturation point) the impact of different excipient choices should be evaluated. Micro differential scanning calorimetry is a well established method for these applications but can be time consuming even when an autosampler is used. Differential scanning fluorimetry (DSF) is a novel technique which measures the fluorescence of a dye when bound to the hydrophobic regions of a denatured protein. We have investigated these techniques for their suitability using alpha-1-protease inhibitor (A1PI) as a model system and found similar trends in terms of the impact of different excipients by both methods. DSF is a promising method and has advantages in terms of speed and quantities of biological material required and can be performed using a PCR instrument., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Serum response factor (SRF) is a transcription factor that regulates the expression of growth-related immediate-early, cytoskeletal, and muscle-specific genes to control growth, differentiation, and cytoskeletal integrity in different cell types. To investigate the role for SRF in epidermal development and homeostasis, we conditionally knocked out SRF in epidermal keratinocytes. We report that SRF deletion disrupted epidermal barrier function leading to early postnatal lethality. Mice lacking SRF in epidermis displayed morphogenetic defects, including an eye-open-at-birth phenotype and lack of whiskers. SRF-null skin exhibited abnormal morphology, hyperplasia, aberrant expression of differentiation markers and transcriptional regulators, anomalous actin organization, enhanced inflammation, and retarded hair follicle (HF) development. Transcriptional profiling experiments uncovered profound molecular changes in SRF-null E17.5 epidermis and revealed that many previously identified SRF target CArG box-containing genes were markedly upregulated in SRF-null epidermis, indicating that SRF may function to repress transcription of a subset of its target genes in epidermis. Remarkably, when transplanted onto nude mice, engrafted SRF-null skin lacked hair but displayed normal epidermal architecture with proper expression of differentiation markers, suggesting that although keratinocyte SRF is essential for HF development, a cross-talk between SRF-null keratinocytes and the surrounding microenvironment is likely responsible for the barrier-deficient mutant epidermal phenotype.

The JAK family of protein tyrosine kinases are now recognized as important participants in a wide range of pathologies, from cancer to inflammatory diseases. In the last decade, the drive to develop drugs targeting members of this family has begun to deliver a panel of small molecule inhibitors of JAK family members, with a range of potencies and specificities. A number of these compounds have already found widespread use as biochemical tools in the elucidation of JAK activity in specific signaling and disease processes; however, many of the first generation compounds are poorly characterized with suboptimal potencies and selectivities.Herein, we present the data for those small molecule JAK inhibitors that have been described in the peer-reviewed literature and the benefits and potential issues that may be associated with the use of these tool compounds.

Assessment of whether pesticide exposure is associated with neurodevelopmental outcomes in children can best be addressed with a systematic review of both the human and animal peer-reviewed literature. This review analyzed epidemiologic studies testing the hypothesis that exposure to pesticides during pregnancy and/or early childhood is associated with neurodevelopmental outcomes in children. Studies that directly queried pesticide exposure (e.g., via questionnaire or interview) or measured pesticide or metabolite levels in biological specimens from study participants (e.g., blood, urine, etc.) or their immediate environment (e.g., personal air monitoring, home dust samples, etc.) were eligible for inclusion. Consistency, strength of association, and dose response were key elements of the framework utilized for evaluating epidemiologic studies. As a whole, the epidemiologic studies did not strongly implicate any particular pesticide as being causally related to adverse neurodevelopmental outcomes in infants and children. A few associations were unique for a health outcome and specific pesticide, and alternative hypotheses could not be ruled out. Our survey of the in vivo peer-reviewed published mammalian literature focused on effects of the specific active ingredient of pesticides on functional neurodevelopmental endpoints (i.e., behavior, neuropharmacology and neuropathology). In most cases, effects were noted at dose levels within the same order of magnitude or higher compared to the point of departure used for chronic risk assessments in the United States. Thus, although the published animal studies may have characterized potential neurodevelopmental outcomes using endpoints not required by guideline studies, the effects were generally observed at or above effect levels measured in repeated-dose toxicology studies submitted to the U.S. Environmental Protection Agency (EPA). Suggestions for improved exposure assessment in epidemiology studies and more effective and tiered approaches in animal testing are discussed.

The first synthesis of a lactam analogue of salicylihalamide A (1) is reported. A key step in the approach was a photochemical acylation coupling between amine 10 and dioxinone 9 to form the amide 19. Acetylation followed by RCM with Grubbs 1st generation catalyst gave the desired E-lactam 23 (E : Z ratio 87 : 13) as the major compound. Conversion of macrolactam 23 into the vinyl iodide 26 followed by Cu catalysed cross coupling with the diene amide 7 gave aza-salicylihalamide analogue 3 in good yield. This compound demonstrated potent activity against several human leukaemia cell lines.

There is significant interest in treating cancers by blocking protein synthesis, to which hematological malignancies seem particularly sensitive. The translation elongation inhibitor homoharringtonine (Omacetaxine mepesuccinate) is undergoing clinical trials for chronic myeloid leukemia, whereas the translation initiation inhibitor silvestrol has shown promise in mouse models of cancer. Precisely how these compounds induce cell death is unclear, but reduction in Mcl-1, a labile pro-survival Bcl-2 family member, has been proposed to constitute the critical event. Moreover, the contribution of translation inhibitors to neutropenia and lymphopenia has not been precisely defined. Herein, we demonstrate that primary B cells and neutrophils are highly sensitive to translation inhibitors, which trigger the Bax/Bak-mediated apoptotic pathway. However, contrary to expectations, reduction of Mcl-1 did not significantly enhance cytotoxicity of these compounds, suggesting that it does not have a principal role and cautions that strong correlations do not always signify causality. On the other hand, the killing of T lymphocytes was less dependent on Bax and Bak, indicating that translation inhibitors can also induce cell death via alternative mechanisms. Indeed, loss of clonogenic survival proved to be independent of the Bax/Bak-mediated apoptosis altogether. Our findings warn of potential toxicity as these translation inhibitors are cytotoxic to many differentiated non-cycling cells.

A qualitative review of the epidemiological literature on the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and health after 2001 is presented. In order to compare the exposure of the general population, bystanders and occupational groups, their urinary levels were also reviewed. In the general population, 2,4-D exposure is at or near the level of detection (LOD). Among individuals with indirect exposure, i.e. bystanders, the urinary 2,4-D levels were also very low except in individuals with opportunity for direct contact with the herbicide. Occupational exposure, where exposure was highest, was positively correlated with behaviors related to the mixing, loading and applying process and use of personal protection. Information from biomonitoring studies increases our understanding of the validity of the exposure estimates used in epidemiology studies. The 2,4-D epidemiology literature after 2001 is broad and includes studies of cancer, reproductive toxicity, genotoxicity, and neurotoxicity. In general, a few publications have reported statistically significant associations. However, most lack precision and the results are not replicated in other independent studies. In the context of biomonitoring, the epidemiology data give no convincing or consistent evidence for any chronic adverse effect of 2,4-D in humans.

Most human health risk assessments are based on animal studies that can be conducted under conditions where exposure to multiple doses of a single chemical can be controlled. Data from epidemiology studies also provide valuable information about human exposure and response to pesticides. Human studies have the potential of evaluating neurobehavioral and other outcomes that may be more difficult to evaluate in animals. The human data together with animal data can contribute to a weight-of-evidence analysis in the characterization of human health risks. Epidemiology data do, however, pose challenges with respect to characterizing human health risks. Similarly, animal data at high doses or routes of exposure not typical for humans also pose challenges to dose-response evaluations needed for risk assessments. This paper summarizes some of the presentations given at a symposium held at the Xi'an, China, International Neurotoxicology Conference held in June 2011. This symposium brought together scientists from government, industry and academia to discuss approaches to evaluating and conducting animal and human neurotoxicity studies for risk assessment purposes, using the pesticides paraquat and chlorpyrifos as case studies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2-yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639-4642, 2009; Mol Cancer Ther 8:3036-3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC(50) 3.7 ± 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.

We describe the structure-activity relationship of the C7-position of pyrano[3,4-b]indole-based inhibitors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the significantly more potent compounds 13 and 14., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Introduction: Macrophages are key drivers of both the innate and adaptive immune systems. The cellular receptor for CSF-1 and IL-34, c-FMS, is a key component of the mechanism(s) by which macrophages are regulated. Several drug discovery programs aimed at uncovering inhibitors of the tyrosine kinase activity of this receptor are now entering clinical phase, and the prospect of readjusting the behavior of macrophages in a number of pathological situations, such as inflammation and cancer, is now on us., Areas Covered: In this review, we evaluate the available patent literature on the topic of small molecule inhibitors of c-FMS. By way of background, we review the biology of c-FMS and make an analysis of the therapeutic opportunities that a small molecule c-FMS inhibitor might present. In order to place the pharmacology in perspective, we examine the literature concerning the role of the CSF-1-IL-34-c-FMS axis in macrophage function as well as cell types related to macrophages, such as the osteoclast, the dendritic cell and microglia, and provide a background to the understanding of the therapeutic opportunities for c-FMS inhibitors as well as potential obstacles that could limit their use., Expert Opinion: The c-FMS receptor is a hot target for the development of novel regulators of macrophage behavior. Some nice candidates have been developed by a number of groups, and their recent entry into clinical phase testing means that we are now on the cusp of a fuller understanding of the role of these important regulators of the innate and adaptive immune systems in the development of cancer and inflammatory diseases.

Background: Occupational studies typically observe a 20% deficit in overall mortality, broadly characterized as the healthy worker effect (HWE). Components of the HWE may be addressed by various analytical approaches., Aims: To explore the HWE in a modern industrial cohort., Methods: Standardized mortality ratios (SMRs) were calculated for 114,683 US chemical industry employees, who worked at least 3 days between 1960 and 2005., Results: SMRs were 79 (95% confidence interval 78-80) for all causes, 81 (95% confidence interval 79-82) for heart disease, 70 (95% confidence interval 67-73) for non-malignant respiratory disease, 83 (95% confidence interval 81-85) for smoking-related cancers (buccal, cervix, oesophagus, stomach, pancreas, lung, larynx, bladder and kidney) combined and 97 (95% confidence interval 95-100) for other cancers., Conclusions: The low SMRs observed in this study are likely due to differential smoking between the cohort and the background population. Future considerations to control for the HWE should take this into account.

Accurate X-ray absorption spectra (XAS) of first row atoms, e.g., O, are notoriously difficult to obtain due to the extreme sensitivity of the measurement to surface contamination, self-absorption, and saturation affects. Herein, we describe a comprehensive approach for determining reliable O K-edge XAS data for ReO(4)(1-) and provide methodology for obtaining trustworthy and quantitative data on nonconducting molecular systems, even in the presence of surface contamination. This involves comparing spectra measured by nonresonant inelastic X-ray scattering (NRIXS), a bulk-sensitive technique that is not prone to X-ray self-absorption and provides exact peak intensities, with XAS spectra obtained by three different detection modes, namely total electron yield (TEY), fluorescence yield (FY), and scanning transmission X-ray microscopy (STXM). For ReO(4)(1-), TEY measurements were heavily influenced by surface contamination, while the FY and STXM data agree well with the bulk NRIXS analysis. These spectra all showed two intense pre-edge features indicative of the covalent interaction between the Re 5d and O 2p orbitals. Density functional theory calculations were used to assign these two peaks as O 1s excitations to the e and t(2) molecular orbitals that result from Re 5d and O 2p covalent mixing in T(d) symmetry. Electronic structure calculations were used to determine the amount of O 2p character (%) in these molecular orbitals. Time dependent-density functional theory (TD-DFT) was also used to calculate the energies and intensities of the pre-edge transitions. Overall, under these experimental conditions, this analysis suggests that NRIXS, STXM, and FY operate cooperatively, providing a sound basis for validation of bulk-like excitation spectra and, in combination with electronic structure calculations, suggest that NaReO(4) may serve as a well-defined O K-edge energy and intensity standard for future O K-edge XAS studies.

Objective: To evaluate the mortality for an occupational cohort and discuss its suitability as an internal comparison population., Methods: The study included 114,683 employees who worked at 1 of 25 US locations between January 1, 1960, and December 31, 2005. Standardized mortality ratios (SMRs) and 95% confidence intervals (CIs) were calculated based on US rates., Results: The SMRs were significantly favorable for all causes, malignant neoplasms, heart disease, and external causes. The most common cancer sites were lung in men (SMR = 83, 95% CI = 80 to 86), and breast in women (SMR = 94, 95% CI = 80 to 111)., Conclusions: Overall, the mortality rates were favorable and robust. Discussed within are topics of historical concern at specific locations that may influence the cohort's appropriateness for future studies.

Employment in the manufacture of the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) is associated with potential exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and elevated serum lipid TCDD concentrations can be measured in workers for decades after terminated occupational exposure. As part of an epidemiological study of 1599 workers employed at a facility in New Plymouth, New Zealand that manufactured 2,4,5-T, serum TCDD concentrations measured in blood samples from 346 workers were used with work history records and a simple pharmacokinetic model in a linear regression to estimate dose rates associated with specific job exposure groups at the facility. The model was used to estimate serum TCDD concentration profiles over time for each individual in the full study group and accounted for 30% of the observed variance in TCDD concentrations in the serum donor subgroup. The model underestimated measured concentrations substantially for eleven individuals in the study group; examination of questionnaire data revealed a variety of activities apart from routine employment at the facility that may have contributed to the measured serum TCDD concentrations. Estimated serum TCDD concentrations were below 300 p.p.t. for all individuals in the cohort over the entire study time period, lower than estimates for other 2,4,5-T worker populations. This finding is consistent with occupational medicine records, which indicated that no cases of chloracne were ever diagnosed among workers employed on the site. The modeled exposures will be used in an evaluation of mortality patterns of workers at this facility.

Activating alleles of Janus kinase 2 (JAK2) such as JAK2(V617F) are central to the pathogenesis of myeloproliferative neoplasms (MPN), suggesting that small molecule inhibitors targeting JAK2 may be therapeutically useful. We have identified an aminopyrimidine derivative (CYT387), which inhibits JAK1, JAK2, and tyrosine kinase 2 (TYK2) at low nanomolar concentrations, with few additional targets. Between 0.5 and 1.5muM CYT387 caused growth suppression and apoptosis in JAK2-dependent hematopoietic cell lines, while nonhematopoietic cell lines were unaffected. In a murine MPN model, CYT387 normalized white cell counts, hematocrit, spleen size, and restored physiologic levels of inflammatory cytokines. Despite the hematologic responses and reduction of the JAK2(V617F) allele burden, JAK2(V617F) cells persisted and MPN recurred upon cessation of treatment, suggesting that JAK2 inhibitors may be unable to eliminate JAK2(V617F) cells, consistent with preliminary results from clinical trials of JAK2 inhibitors in myelofibrosis. While the clinical benefit of JAK2 inhibitors may be substantial, not the least due to reduction of inflammatory cytokines and symptomatic improvement, our data add to increasing evidence that kinase inhibitor monotherapy of malignant disease is not curative, suggesting a need for drug combinations to optimally target the malignant cells.

We describe the structure-activity relationship of the C1-group of pyrano[3,4-b]indole based inhibitors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the significantly more potent compound 12., (2010 Elsevier Ltd. All rights reserved.)

At high and prolonged exposure levels (e.g. >30 ppm), benzene can cause hematological effects. However, there is conflicting evidence on potential hematological effects at lower concentrations. We conducted a study to examine hematological effects at low benzene exposure levels in an occupational setting. Extensive exposure data and data from routine hematology examinations were available for Dow employees at the Terneuzen site in the Netherlands. We compared 8532 blood samples of Dow employees with low benzene exposure to 12,173 samples of employees with no benzene exposure that were available for the period between 1981 and 2007. Based on 21,584 benzene air measurements, a Job Exposure Matrix (JEM) was constructed for all employees with exposure. The JEM was used to estimate benzene exposure in the year in which each blood sample was collected. The average lymphocyte counts for the exposed and non-exposed group were similar. By means of mixed model regression adjustments were made for smoking, age and month of blood sample. These adjustments did not change the results and there was no indication for an adverse effect on any of the hematological parameters under investigation. A further stratification of the exposed population into three subgroups (<0.5 ppm, 0.5-1 ppm and >1 ppm) showed no significant differences for any of the hematological parameters between the three exposure categories or compared with the non-exposed group. The analysis modeling the continuous exposure effect relationship showed similar findings. This study does not indicate that workers exposed to low benzene concentrations are at an increased risk for hematological effects., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Background: Several extensive studies of exposure to 2,4-dichlorophenoxyacetic acid (2,4-D) using urinary concentrations in samples from the general population, farm applicators, and farm family members are now available. Reference doses (RfDs) exist for 2,4-D, and Biomonitoring Equivalents (BEs; concentrations in urine or plasma that are consistent with those RfDs) for 2,4-D have recently been derived and published., Objective: We reviewed the available biomonitoring data for 2,4-D from the United States and Canada and compared them with BE values to draw conclusions regarding the margin of safety for 2,4-D exposures within each population group., Data Sources: Data on urinary 2,4-D excretion in general and target populations from recent published studies are tabulated and the derivation of BE values for 2,4-D summarized., Data Synthesis: The biomonitoring data indicate margins of safety (ratio of BE value to biomarker concentration) of approximately 200 at the central tendency and 50 at the extremes in the general population. Median exposures for applicators and their family members during periods of use appear to be well within acute exposure guidance values., Conclusions: Biomonitoring data from these studies indicate that current exposures to 2,4-D are below applicable exposure guidance values. This review demonstrates the value of biomonitoring data in assessing population exposures in the context of existing risk assessments using the BE approach. Risk managers can use this approach to integrate the available biomonitoring data into an overall assessment of current risk management practices for 2,4-D.

The aim of the current analysis was to examine the determinates of lipid-adjusted body levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from occupational histories, age, body mass index, and self-reported information from a questionnaire. We collected serum from 346 workers at a New Zealand chemical plant that manufactured and formulated the herbicide, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Age, body mass index, and employment history were significant determinates of TCDD. The self-reported data on occupation, residence, and general diet were not predictive of serum levels and we observed no evidence of increased TCDD levels from living close to the site. For participants with putative occupational exposure, employment history and personal factors were important to understand the range of TCDD serum levels. For employees without direct occupational exposure, and resulting lower dioxin levels, we recommend further efforts to develop and validate questionnaires to better evaluate environmental sources of dioxins., ((c) 2009 Elsevier Inc. All rights reserved.)

CYT997 is a wholly synthetic compound that possesses highly potent cytotoxic activity in vitro through inhibition of microtubule polymerization. CYT997 blocks the cell cycle at the G(2)-M boundary, and Western blot analysis indicates an increase in phosphorylated Bcl-2, along with increased expression of cyclin B1. Caspase-3 activation is also observed in cells treated with CYT997 along with the generation of poly(ADP-ribose) polymerase. The compound possesses favorable pharmacokinetic properties, is orally bioavailable, and is efficacious per os in a range of in vivo cancer models, including some refractory to paclitaxel treatment. CYT997 exhibits vascular disrupting activity as measured in vitro by effects on the permeability of human umbilical vein endothelial cell monolayers, and in vivo by effects on tumor blood flow. CYT997 possesses a useful combination of pharmacologic and pharmacokinetic properties and has considerable potential as a novel anticancer agent.

The objective of this study was to evaluate the quantitative relation between measured red blood cell acetylcholinesterase (RBC AChE) and plasma butyrylcholinesterase (BuChE) activities with exposure to chlorpyrifos (CPF) as assessed by measurement of urinary 3,5,6-trichloro-2-pyridinol (TCPy) in a study group of workers occupationally exposed in the manufacture of CPF and a referent group of chemical manufacturing workers. Measures of plasma BuChE and RBC AChE activity and urinary TCPy concentration collected over a year-long study (1999-2000) in CPF-exposed workers (n=53) and referents (n=60) were analyzed using linear mixed models to characterize exposure-response relationships. Intraindividual variability in cholinesterase measures was compared between CPF-exposed workers and referents. Urinary TCPy concentrations in CPF workers were substantially elevated compared to referents, with median and 95th percentile concentrations during typical employment conditions 10-fold and more than 30-fold higher, respectively, than corresponding measures in the referents. Intraindividual variability in cholinesterase activities was substantial, with 17% of unexposed referents experiencing one or more plasma BuChE measures more than 20% below baseline over a year of repeated, periodic measurements. RBC AChE activity, an early biomarker of effect, was unrelated to urinary TCPy concentration over the entire range of exposure, up to 1000 microg TCPy/g creatinine (Cr). Plasma BuChE activity, a non-adverse biomarker of exposure, was negatively related to urinary TCPy concentrations above approximately 110 microg TCPy/g Cr. No-effect levels for inhibition of plasma BuChE and RBC AChE corresponding to absorbed doses of CPF of approximately 5 and greater than 50 microg/kg/day, respectively, were identified. These findings are consistent with previous no-effect level determinations for ChE inhibition in humans and suggest that general population CPF exposure levels are substantially below the identified no-effect levels. The dose-response relationships observed in this study are consistent with predictions from the previously published physiologically based pharmacokinetic/pharmacodynamic model for CPF. Intraindividual variability in measured cholinesterase activities in referents was substantial, suggesting that ongoing monitoring programs may have a substantial rate of false positives.

A series of phenylaminopyrimidines has been identified as inhibitors of Janus kinases (JAKs). Development of this initial series led to the potent JAK2/JAK1 inhibitor CYT387 (N-(cyanomethyl)-4-[2-[[4-(4-morpholinyl)phenyl]amino]-4-pyrimidinyl]-benzamide). Details of synthesis and SAR studies of these compounds are reported.

We describe the use of Cl K-edge X-ray absorption spectroscopy (XAS) and both ground-state and time-dependent hybrid density functional theory (DFT) to probe the electronic structure and determine the degree of orbital mixing in M-Cl bonds for (C(5)Me(5))(2)MCl(2) (M = Ti, 1; Zr, 2; Hf, 3; Th, 4; U, 5), where we can directly compare a class of structurally similar compounds for d- and f-elements. Pre-edge features in the Cl K-edge XAS data for the group IV transition-metals 1-3 provide direct evidence of covalent M-Cl orbital mixing. The amount of Cl 3p character was experimentally determined to be 25%, 23%, and 22% per M-Cl bond for 1-3, respectively. For actinides, we find a pre-edge shoulder for 4 (Th) and distinct and weak pre-edge features for U, 5. The amount of Cl 3p character was determined to be 9% for 5, and we were unable to make an experimental determination for 4. Using hybrid DFT calculations with relativistic effective core potentials, the electronic structures of 1-5 were calculated and used as a guide to interpret the experimental Cl K-edge XAS data. For transition-metal compounds 1-3, the pre-edge features arise due to transitions from Cl 1s electrons into the 3d-, 4d-, and 5d-orbitals, with assignments provided in the text. For Th, 4, we find that 5f- and 6d-orbitals are nearly degenerate and give rise to a single pre-edge shoulder in the XAS. For U, 5, we find the 5f- and 6d-orbitals fall into two distinct energy groupings, and Cl K-edge XAS data are interpreted in terms of Cl 1s transitions into both 5f- and 6d-orbitals. Time-dependent DFT was used to calculate the energies and intensities of Cl 1s transitions into empty metal-based orbitals containing Cl 3p character and provide simulated Cl K-edge XAS spectra for 1-4. For 5, which has two unpaired 5f electrons, simulated spectra were obtained from transition dipole calculations using ground-state Kohn-Sham orbitals. To the best of our knowledge, this represents the first application of Cl K-edge XAS to actinide systems. Overall, this study allows trends in orbital mixing within a well-characterized structural motif to be identified and compared between transition-metals and actinide elements. These results show that the orbital mixing for the d-block compounds slightly decreases in covalency with increasing principal quantum number, in the order Ti > Zr approximately = Hf, and that uranium displays approximately half the covalent orbital mixing of transition elements.

This study examined serum levels of 2,3,7,8-substituted chlorinated dioxins and furans, and 15 PCBs for 346 New Zealand employees who worked at a site that manufactured 2,4,5-trichlorophenol (TCP) and 2,4,5-trichlorophenoxy acetic acid (2,4,5-T). Participants with potential TCP or 2,4,5-T exposures had mean lipid-adjusted 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) levels of 9.9 ng kg(-1) lipid compared to 4.9 ng kg(-1) for workers with no exposure at the site. Among exposed workers, we found evidence of differences in 2,3,7,8-TCDD levels by department and duties. Workers involved in an accidental release had the highest mean 2,3,7,8-TCDD levels, 37.9 ng kg(-1), followed by workers in the trichlorophenol plant, 23.4 ng kg(-1). Workers with potential intermittent exposures to 2,3,7,8-TCDD in construction, maintenance, mechanics, and transport had 2,3,7,8-TCDD levels above New Zealand background levels of 3.9 ng kg(-1), indicating workplace exposures. Among participants with work history indicating no 2,3,7,8-TCDD exposures, we observed some individuals with 2,3,7,8-TCDD levels above background levels. However, in most cases, these workers reported workplace exposures not recorded on their work histories or held other jobs with the potential for 2,3,7,8-TCDD exposures outside the plant. All other dioxin, furan, and PCB levels were similar among the exposed and unexposed workers.

Objective: We examined the overall mortality rates of 1599 workers employed between 1969 and 1988 at a New Zealand site, which manufactured trichlorophenol., Methods: We developed exposure estimates for 2,3,7,8-tetrachlorodibenzo-p-dioxin from a serum dioxin evaluation and used standardized mortality ratios (SMR) and proportional hazards models to evaluate risk from exposure., Results: Among exposed workers, deaths from total cancers (SMR = 1.1, 95% confidence intervals [CI]: 0.9-1.4), non-Hodgkin lymphoma (SMR = 1.6, 95% CI: 03-4.7), and ischemic heart disease (SMR = 1.1, 95% CI: 0.9-1.5) were slightly greater than expected, whereas deaths from lung cancer (SMR = 0.8, 95% CI: 0.4-1.5) were less than expected. We observed no significant trends with exposure levels., Conclusions: Although this study is small, we found no increasing trend of cancer or disease risk with increasing 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure with the possible exception of all cancers combined.

CYT997 was discovered as a potent tubulin polymerization inhibitor possessing potent cytotoxic activity against a range of cancer cells. Details of SAR studies, pharmacokinetic investigations and synthesis of compounds leading to the discovery of CYT997 are reported.

Somatic mutations in Janus kinase 2 (JAK2), including JAK2V617F, result in dysregulated JAK-signal transducer and activator transcription (STAT) signaling, which is implicated in myeloproliferative neoplasm (MPN) pathogenesis. CYT387 is an ATP-competitive small molecule that potently inhibits JAK1/JAK2 kinases (IC(50)=11 and 18 nM, respectively), with significantly less activity against other kinases, including JAK3 (IC(50)=155 nM). CYT387 inhibits growth of Ba/F3-JAK2V617F and human erythroleukemia (HEL) cells (IC(50) approximately 1500 nM) or Ba/F3-MPLW515L cells (IC(50)=200 nM), but has considerably less activity against BCR-ABL harboring K562 cells (IC=58 000 nM). Cell lines harboring mutated JAK2 alleles (CHRF-288-11 or Ba/F3-TEL-JAK2) were inhibited more potently than the corresponding pair harboring mutated JAK3 alleles (CMK or Ba/F3-TEL-JAK3), and STAT-5 phosphorylation was inhibited in HEL cells with an IC(50)=400 nM. Furthermore, CYT387 selectively suppressed the in vitro growth of erythroid colonies harboring JAK2V617F from polycythemia vera (PV) patients, an effect that was attenuated by exogenous erythropoietin. Overall, our data indicate that the JAK1/JAK2 selective inhibitor CYT387 has potential for efficacious treatment of MPN harboring mutated JAK2 and MPL alleles.

Activating Ras mutations occur in a large portion of human tumors. Yet, the signaling pathways involved in Ras-induced tumor formation remain incompletely understood. The mitogen-activated protein kinase pathways are among the best studied Ras effector pathways. The p38 mitogen-activated protein kinase isoforms are important regulators of key biological processes including cell proliferation, differentiation, survival, inflammation, senescence, and tumorigenesis. However, the specific in vivo contribution of individual p38 isoforms to skin tumor development has not been elucidated. Recent studies have shown that p38delta, a p38 family member, functions as an important regulator of epidermal keratinocyte differentiation and survival. In the present study, we have assessed the effect of p38delta deficiency on skin tumor development in vivo by subjecting p38delta knockout mice to a two-stage 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate chemical skin carcinogenesis protocol. We report that mice lacking p38delta gene exhibited a marked resistance to development of 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-induced skin papillomas, with increased latency and greatly reduced incidence, multiplicity, and size of tumors compared with wild-type mice. Our data suggest that the underlying mechanism for reduced susceptibility to skin carcinogenesis in p38delta-null mice involves a defect in proliferative response associated with aberrant signaling through the two major transformation-promoting pathways: extracellular signal-regulated kinase 1/2-activator protein 1 and signal transducer and activator of transcription 3. These findings strongly suggest an in vivo role for p38delta in promoting cell proliferation and tumor development in epidermis and may have therapeutic implication for skin cancer.

Background: Previous studies at the Dow AgroSciences (Formerly Ivon Watkins-Dow) plant in New Plymouth, New Zealand, had raised concerns about the cancer risk in a subset of workers at the site with potential exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. As the plant had been involved in the synthesis and formulation of a wide range of agrochemicals and their feedstocks, we examined the mortality risk for all workers at the site., Aims: To quantify the mortality hazards arising from employment at the Dow AgroSciences agrochemical production site in New Plymouth, New Zealand., Methods: Workers employed between 1 January 1969 and 1 October 2003 were followed up to the end of 2004. Standardized mortality ratios (SMRs) were calculated using national mortality rates by employment duration, sex, period of hire and latency., Results: A total of 1754 employees were followed during the study period and 247 deaths were observed. The all causes and all cancers SMRs were 0.97 (95% CI 0.85-1.10) and 1.01 (95% CI 0.80-1.27), respectively. Mortality due to all causes was higher for short-term workers (SMR 1.23, 95% CI 0.91-1.62) than long-term workers (SMR 0.92, 95% CI 0.80-1.06) and women had lower death rates than men. Analyses by latency and period of hire did not show any patterns consistent with an adverse impact of occupational exposures., Conclusions: The mortality experience of workers at the site was similar to the rest of New Zealand.

A series of 2-(alpha-methylbenzylamino) pyrazines have shown to be potent inhibitors of the FMS tyrosine receptor kinase. Details of SAR studies, modeling and synthesis of compounds within this series are reported.

Venous thromboembolic disease, which includes deep vein thromboses as well as pulmonary emboli, can be a significant complication in the postoperative patient. In particular, colorectal patients often carry a higher risk for venous thromboembolism when compared with patients undergoing other operative procedures. Features unique to colorectal patients are the high incidence of inflammatory bowel disease or malignancy. Typically, these patients will undergo lengthy pelvic procedures, which also contribute to a cumulative risk of venous thrombosis. It is critical that all patients and the proposed operative procedure are appropriately risk stratified. Risk stratification allows for easier implementation of an appropriate prophylactic strategy. There are a wide range of safe and effective mechanical and pharmacologic measures available. The authors provide very specific recommendations, but note that clinical judgment plays a significant role.

M-CSF/CSF-1 supports the proliferation and differentiation of monocytes and macrophages. In mice, CSF-1 also promotes proinflammatory responses in vivo by regulating mature macrophage functions, but little is known about the acute effects of this growth factor on mature human macrophages. Here, we show that in contrast to its effects on mouse bone marrow-derived macrophages, CSF-1 did not induce expression of urokinase plasminogen activator mRNA, repress expression of apolipoprotein E mRNA, or prime LPS-induced TNF and IL-6 secretion in human monocyte-derived macrophages (HMDM) from several independent donors. Instead, we show by expression profiling that CSF-1 modulates the HMDM transcriptome to favor a proatherogenic environment. CSF-1 induced expression of the proatherogenic chemokines CXCL10/IFN-inducible protein 10, CCL2, and CCL7 but repressed expression of the antiatherogenic chemokine receptor CXCR4. CSF-1 also up-regulated genes encoding enzymes of the cholesterol biosynthetic pathway (HMGCR, MVD, IDI1, FDPS, SQLE, CYP51A1, EBP, NSDHL, DHCR7, and DHCR24), and expression of ABCG1, encoding a cholesterol efflux transporter, was repressed. Consistent with these effects, CSF-1 increased levels of free cholesterol in HMDM, and the selective CSF-1R kinase inhibitor GW2580 ablated this response. These data demonstrate that CSF-1 represents a further link between inflammation and cardiovascular disease and suggest two distinct mechanisms by which CSF-1, which is known to be present in atherosclerotic lesions, may contribute to plaque progression.

Migratory insertion of diphenyldiazomethane into both metal-carbon bonds of the bis(alkyl) and bis(aryl) complexes (C(5)Me(5))(2)AnR(2) yields the first f-element bis(hydrazonato) complexes (C(5)Me(5))(2)An[eta(2)-(N,N')-R-N-N=CPh(2)](2) [An = Th, R = CH(3) (18), PhCH(2) (15), Ph (16); An = U, R = CH(3) (17), PhCH(2) (14)], which have been characterized by a combination of spectroscopy, electrochemistry, and X-ray crystallography. The two hydrazonato ligands adopt an eta(2)-coordination mode leading to 20-electron (for Th) and 22-electron (for U) complexes that have no transition-metal analogues. In fact, reaction of (C(5)H(5))(2)Zr(CH(3))(2) or (C(5)Me(5))(2)Hf(CH(3))(2) with diphenyldiazomethane is limited to the formation of the corresponding mono(hydrazonato) complex (C(5)R(5))(2)M[eta(2)-(N,N')-CH(3)-N-N=CPh(2)](CH(3)) (M = Zr, R = H or M = Hf, R = CH(3)). The difference in the reactivities of the group 4 metal complexes and the actinides was used as a unique platform for investigating in depth the role of 5f orbitals on the reactivity and bonding in actinide organometallic complexes. The electronic structure of the (C(5)H(5))(2)M[eta(2)-(N,N')-CH(3)-N-N=CH(2)](2) (M = Zr, Th, U) model complexes was studied using density functional theory (DFT) calculations and compared to experimental structural, electrochemical, and spectroscopic results. Whereas transition-metal bis(cyclopentadienyl) complexes are known to stabilize three ligands in the metallocene girdle to form saturated (C(5)H(5))(2)ML(3) species, in a bis(hydrazonato) system, a fourth ligand is coordinated to the metal center to give (C(5)H(5))(2)ML(4). DFT calculations have shown that 5f orbitals in the actinide complexes play a crucial role in stabilizing this fourth ligand by stabilizing both the sigma and pi electrons of the two eta(2)-coordinated hydrazonato ligands. In contrast, the stabilization of the hydrazonato ligands was found to be significantly less effective for the putative bis(hydrazonato) zirconium(IV) complex, yielding a higher energy structure. However, the difference in the reactivities of the group 4 metal and actinide complexes does not arise on thermodynamic grounds but is primarily of kinetic origin. Unfavorable steric factors have been ruled out as the sole influence to explain these different behaviors, and electronic factors were shown to govern the reactivity. For the actinides, both the C(5)H(5) and more realistic C(5)Me(5) ligands have been taken into account in computing the energy surface. The reaction profile for the C(5)Me(5) system differs from that with the C(5)H(5) ligand by a uniform shift of approximately 5 kcal/mol in the relative energies of the transition state and products. The insertion of a second diazoalkane molecule into the sole metal-carbon bond in the mono(hydrazonato) complexes involves a high energy barrier (approximately 20 kcal/mol) for the zirconium(IV) system, whereas the actinides can facilitate the approach of the diazoalkane by coordination (formation of an adduct) and its insertion into the An-C bond with a very low barrier on the potential energy surface.

A selection of physicochemical and biological assays were investigated for their utility in detecting changes in preparations of Interferon alpha-2a and Interferon alpha-2b (IFN-alpha 2a, IFN-alpha 2b), which had been subjected to stressed conditions, in order to create models of biopharmaceutical products containing product-related impurities. The stress treatments, which included oxidation of methionine residues and storage at elevated temperatures for different periods of time, were designed to induce various degrees of degradation, aggregation or oxidation of the interferon. Biological activity of the stressed preparations was assessed in three different in vitro cell-based bioassay systems: a late-stage anti-proliferative assay and early-stage assays measuring reporter gene activation or endogenous gene expression by quantitative real time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Relevant physicochemical methods such as SDS-PAGE, reverse phase (RP) chromatography, size-exclusion chromatography (SEC) and dynamic light scattering (DLS), proved their complementarity in detecting structural changes in the stressed preparations which were reflected by reductions in biological activity.

Cardiovascular disease is a major cause of mortality with the underlying process being atherosclerosis. Modified proteoglycans bind low-density lipoproteins (LDL), a critical initial step in the atherosclerotic cascade, representing a potential therapeutic target. Platelet-derived growth factor (PDGF) stimulates proteoglycan synthesis and is strongly implicated in atherogenesis. In human vascular smooth muscle cells (VSMCs), CYC10424 (Cytopia Research Ltd), a pyrido-pyrimidine derivative, dose-dependently decreased PDGF-mediated radiolabel incorporation into proteoglycans associated with an increase in electrophoretic mobility by SDS-PAGE. PDGF stimulated increases in both chemically-cleaved and xyloside-associated glycosaminoglycan (GAG) chain size, which were inhibited in the presence of CYC10424 by size exclusion chromatography (Sepharose CL-6B). CYC10424 treatment inhibited the PDGF effect to increase the 6:4 position sulfation ratio of monosulfated disaccharides by fluorophore-assisted carbohydrate electrophoresis. Proteoglycans derived from cells treated with CYC10424 had a decreased binding affinity and capacity to human LDL by gel mobility shift assay. CYC10424 and related compounds are possible candidates as therapeutic agents for the prevention of lipid deposition as characteristic of diseases such as atherosclerosis.

In Type 1 diabetes mellitus the insulin-secreting beta-cells in pancreatic islets of Langerhans are selectively destroyed by autoimmune assault. Because diabetes is caused by the loss of a single cell type it is amenable to treatment by cell replacement therapy. Advances in islet transplantation procedures have demonstrated that people with Type 1 diabetes can be cured by human islet transplantation, but the severely limited availability of donor islets has restricted the widespread application of this approach, and driven the search for substitute transplant tissues. Recent experimental studies suggest that three separate sources of tissue show therapeutic potential--xenografts from other species, tissue stem cells and embryonic stem cells. Of these, xenografts are closest to clinical application but there are still major obstacles to be overcome. Insulin-expressing cells have been derived from a number of different stem cell populations but embryonic stem cells offer the major advantage of being able, in principle, to provide the vast numbers of cells required for transplantation therapy.

We examined the serum lipid adjusted levels of 2,3,7,8-substituted chlorinated dioxins and furans, and four coplanar PCBs for 98 workers. We found workers who worked only in the trichlorophenol units had mean lipid adjusted 2,3,7,8-TCDD levels of 36.8 ppt significantly higher (p<0.05) than 6.0 ppt in the reference group. Workers who worked only in the pentachlorophenol units had mean lipid adjusted levels for 123478-HxCDD of 14.8 ppt, 123678-HxCDD of 156.4 ppt,123789-HxCDD of 23.7 ppt, 1234678-HpCDD of 234.6 ppt, and OCDD of 2,778.2 ppt significantly higher (p<0.05) than the reference group levels for the same congeners of 7.5, 71.8, 8.0, 67.5, and 483.2 ppt, respectively. While we did find 12378-PeCDD levels higher than the reference group in trichlorophenol and pentachlorophenol workers, the differences are small, and could be attributed to normal variation. All furan levels among the trichlorophenol or pentachlorophenol only workers were not significantly different than the reference group. Workers with both trichlorophenol and pentachlorophenol exposures had mean dioxin levels consistent with complex chlorophenol exposures. Tradesmen who worked throughout the plant had congener profiles consistent with both trichlorophenol and pentachlorophenol exposures. PCB 169, 23478-PeCDF, 123478-HxCDF, and 123678-HxCDF levels were also significantly greater (p<0.05) in these tradesmen than in the reference group. We found distinct patterns of dioxin congeners many years after exposure among workers with different chlorophenol exposures. We were effectively able to distinguish past trichlorophenol exposures from pentachlorophenol exposures based on differing serum dioxin profiles among workers.

We have investigated the use of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) as an alternative to a selection of late-stage functional bioassays for determination of the potency of preparations of vascular endothelial growth factor (VEGF). Responses were measured in cultures of human umbilical vein endothelial cells (HUVECs). Late-stage responses measured were cell survival and proliferation, and production of interleukin-8 (IL-8), interleukin-6 (IL-6), and tissue factor. The dose-response range was similar across the assays, increasing from 2 ng/mL VEGF and reaching a maximum between 30 ng/mL and 125 ng/mL VEGF. A number of VEGF-induced mRNA species demonstrated dose-response curves suitable for VEGF potency determination. IL-8 mRNA induction after 45 min incubation with VEGF, which showed maximal responses between 15.6 ng/mL and 62.5 ng/mL VEGF, was selected for further characterization. This gene-expression bioassay was robust across a range of cell seeding densities and could be used for samples processed immediately following incubation with VEGF and for cell lysates stored at -80 degrees C for 3 months. We also compared this gene-expression bioassay and the assays of late-stage responses in the potency measurement of the inhibitors of VEGF activity, anti-VEGF monoclonal antibody MAB293, and a VEGF soluble receptor VEGFsR1 preparation. We present a critical evaluation of the use of qRT-PCR in assaying the potency of VEGF and its inhibitors, and of the potential of this platform for measuring the potency of other biological therapeutics.

For 3-5d transition-metal ions, the (C5R5)2MCl2 (R = H, Me for M = Ti, Zr, Hf) bent metallocenes represent a series of compounds that have been central in the development of organometallic chemistry and homogeneous catalysis. Here, we evaluate how changes in the principal quantum number for the group IV (C5H5)2MCl2 (M = Ti, Zr, Hf; 1- 3, respectively) complexes affects the covalency of M-Cl bonds through application of Cl K-edge X-ray Absorption Spectroscopy (XAS). Spectra were recorded on solid samples dispersed as a thin film and encapsulated in polystyrene matrices to reliably minimize problems associated with X-ray self-absorption. The data show that XAS pre-edge intensities can be quantitatively reproduced when analytes are encapsulated in polystyrene. Cl K-edge XAS data show that covalency in M-Cl bonding changes in the order Ti > Zr > Hf and demonstrates that covalency slightly decreases with increasing principal quantum number in 1-3. The percent Cl 3p character was experimentally determined to be 26, 23, and 18% per M-Cl bond in the thin-film samples for 1-3 respectively and was indistinguishable from the polystyrene samples, which analyzed as 25, 25, and 19% for 1-3, respectively. To aid in interpretation of Cl K-edge XAS, 1-3 were also analyzed by ground-state and time-dependent density functional theory (TD-DFT) calculations. The calculated spectra and percent chlorine character are in close agreement with the experimental observations, and show 20, 18, and 17% Cl 3p character per M-Cl bond for 1-3, respectively. Polystyrene matrix encapsulation affords a convenient method to safely contain radioactive samples to extend our studies to include actinide elements, where both 5f and 6d orbitals are expected to play a role in M-Cl bonding and where transition assignments must rely on accurate theoretical calculations.