Azidopropyl functionalized mesoporous silica SBA-15 were prepared with variable azide loadings of 0.03 – 0.7 mmol g−1 (ca. 2 – 50% of maximal surface coverage) through a direct synthesis, co-condensation approach. These materials are functionalized selectively with ethynylated organic moieties through the copper-catalyzed azide alkyne cycloaddition (CuAAC) or “click” reaction. Specific loading within a material can be regulated by either the azide loading or limiting the alkyne reagent relative to the azide loading. The immobilization of ferrocene, pyrene, tris(pyridylmethyl)amine (TPA), and iron porphyrin (FeTPP) demonstrates the robust nature and reproducibility of this two step synthetic attachment strategy. Loading-sensitive pyrene fluorescence correlates with a theoretically random. surface distribution, rather than a uniform one; full site-isolation of tethered moieties ca. 15 Å in length. occurs at loadings less than 0.02 mmol g−1. The effect of surface loading on reactivity is observed in oxygenation of SBA-15-[CuI(TPA)]. SBA-15-[MnII(TPA)]-catalyzed epoxidation exhibits a systematic dependence on surface loading. A comparison of homogeneous, site-isolated and site-dense complexes provides insight into catalyst speciation and ligand activity.

Peptidic oligomers that contain both α- and β-amino acid residues, in regular patterns throughout the backbone, are emerging as structural mimics of α-helix-forming conventional peptides (composed exclusively of α-amino acid residues). Here we describe a comprehensive evaluation of diverse α/β-peptide homologues of the Bim BH3 domain in terms of their ability to bind to the BH3-recognition sites on two partner proteins, Bcl-xL and Mcl-1. These proteins are members of the anti-apoptotic Bcl-2 family, and both bind tightly to the Bim BH3 domain itself. All α/β-peptide homologues retain the side chain sequence of the Bim BH3 domain, but each homologue contains periodic α-residue → β3-residue substitutions. Previous work has shown that the ααβαααβ pattern, which aligns the β3-residues in a 'stripe' along one side of the helix, can support functional α-helix mimicry, and the results reported here support this conclusion. The present study provides the first evaluation of functional mimicry by ααβ and αααβ patterns, which cause the β3-residues to spiral around the helix periphery. We find that the αααβ pattern can support effective mimicry of the Bim BH3 domain, as manifested by the crystal structure of an α/β-peptide bound to Bcl-xL, affinity for a variety of Bcl-2 family proteins, and induction of apoptotic signaling in mouse embryonic fibroblast extracts. The best αααβ homologue shows substantial protection from proteolytic degradation relative to the Bim BH3 α-peptide.

μ-Conotoxin KIIIA (μ-KIIIA) blocks mammalian voltage-gated sodium channels (VGSCs) and is a potent analgesic following systemic administration in mice. Previous structure-activity studies of μ-KIIIA identified a helical pharmacophore for VGSC blockade. This suggested a route for designing truncated analogues of μ-KIIIA by incorporating the key residues into an α-helical scaffold. As (i, i+4) lactam bridges constitute a proven approach for stabilizing α-helices, we designed and synthesized six truncated analogues of μ-KIIIA containing single lactam bridges at various locations. The helicity of these lactam analogues was analysed by NMR spectroscopy, and their activities were tested against mammalian VGSC subtypes NaV1.1 through 1.7. Two of the analogues, Ac-cyclo9/13[Asp9,Lys13]KIIIA7–14 and Ac-cyclo9/13[Lys9,Asp13]KIIIA7–14, displayed µM activity against VGSC subtypes NaV1.2 and NaV1.6; importantly, the subtype selectivity profile for these peptides matched that of μ-KIIIA. Our study highlights structure-activity relationships within these helical mimetics and provides a basis for the design of additional truncated peptides as potential analgesics.

CpG oligonucleotide 7909 (CpG 7909, PF-03512676), a synthetic 24mer single stranded agonist of TLR9 expressed by B cells and plasmacytoid dendritic cells, is immunomodulatory and can cause activation-induced death of chronic lymphocytic leukemia (CLL) cells. We report a phase I study of CpG 7909 in 41 patients with early relapsed CLL. A single intravenous dose of CpG 7909 was well tolerated with no clinical effects and no significant toxicity up to 1.05 mg/kg. Single dose subcutaneous CpG 7909 had a maximum tolerated dose (MTD) of 0.45 mg/kg with dose limiting toxicity of myalgia and constitutional effects. Multiple weekly subcutaneous doses at the MTD were well tolerated. CpG 7909 administration induced immunologic changes in CLL and non-malignant cells that were dose and route dependent. We conclude that multidose therapy with subcutaneous CpG 7909 (0.45 mg/kg) could be used in future phase II combination clinical trials for CLL.

The crystal structure of a complex between the pro-survival protein Bcl-xL and an α/β-peptide 21-mer is described. The α/β-peptide contains six β-amino acid residues distributed periodically throughout the sequence and adopts an α-helix-like conformation that mimics the bioactive shape of the Puma BH3 domain. The α/β-peptide forms all of the noncovalent contacts that have previously been identified as necessary for recognition of the pro-survival protein by an authentic BH3 domain. Comparison of our α/β-peptide:Bcl-xL structure with structures of complexes between native BH3 domains and Bcl-2 family proteins reveals how subtle adjustments, including variations in helix radius and helix bowing, allow the α/β-peptide to engage Bcl-xL with high affinity. Geometric comparisons of the BH3-mimetic α/β-peptide with α/β-peptides in helix-bundle assemblies provide insight on the conformational plasticity of backbones that contain combinations of α- and β-amino acid residues. The BH3-mimetic α/β-peptide displays pro-survival protein-binding preferences distinct from those of Puma BH3 itself, even though these two oligomers have identical side chain sequences. Our results suggest origins for this backbone-dependent change in selectivity.

Objectives

To assess the effectiveness of an intervention package comprising intense education, a range of reporting options, changes in report management and enhanced feedback, in order to improve incident‐reporting rates and change the types of incidents reported.

Design, setting and participants

Non‐equivalent group controlled clinical trial involving medical and nursing staff working in 10 intervention and 10 control units in four major cities and two regional hospitals in South Australia.

Main outcome measures

Comparison of reporting rates by type of unit, profession, location of hospital, type of incident reported and reporting mechanism between baseline and study periods in control and intervention units.

Results

The intervention resulted in significant improvement in reporting in inpatient areas (additional 60.3 reports/10 000 occupied bed days (OBDs); 95% CI 23.8 to 96.8, p<0.001) and in emergency departments (EDs) (additional 39.5 reports/10 000 ED attendances; 95% CI 17.0 to 62.0, p<0.001). More reports were generated (a) by doctors in EDs (additional 9.5 reports/10 000 ED attendances; 95% CI 2.2 to 16.8, p = 0.001); (b) by nurses in inpatient areas (additional 59.0 reports/10 000 OBDs; 95% CI 23.9 to 94.1, p<0.001) and (c) anonymously (additional 20.2 reports/10 000 OBDs and ED attendances combined; 95% CI 12.6 to 27.8, p<0.001). Compared with control units, the study resulted in more documentation, clinical management and aggression‐related incidents in intervention units. In intervention units, more reports were submitted on one‐page forms than via the call centre (1005 vs 264 reports, respectively).

Conclusions

A greater variety and number of incidents were reported by the intervention units during the study, with improved reporting by doctors from a low baseline. However, there was considerable heterogeneity between reporting rates in different types of units.

It has been hypothesized that ionizing radiation-induced disruptions in mitochondrial O2 metabolism lead to persistent heritable increases in steady-state levels of intracellular superoxide (O2•−) and hydrogen peroxide (H2O2) that contribute to the biological effects of radiation. Hamster fibroblasts (B9 cells) expressing a mutation in the gene coding for the mitochondrial electron transport chain protein succinate dehydrogenase subunit C (SDHC) demonstrate increases in steady-state levels of O2•− and H2O2. When B9 cells were exposed to low-dose/low-LET radiation (5–50 cGy), they displayed significantly increased clonogenic cell killing compared with parental cells. Clones derived from B9 cells overexpressing a wild-type human SDHC (T4, T8) demonstrated significantly increased surviving fractions after exposure to 5–50 cGy relative to B9 vector controls. In addition, pretreatment with polyethylene glycol-conjugated CuZn superoxide dismutase and catalase as well as adenoviral-mediated overexpression of MnSOD and/or mitochondria-targeted catalase resulted in significantly increased survival of B9 cells exposed to 10 cGy ionizing radiation relative to vector controls. Adenoviral-mediated overexpression of either MnSOD or mitochondria-targeted catalase alone was equally as effective as when both were combined. These results show that mammalian cells over expressing mutations in SDHC demonstrate low-dose/low-LET radiation sensitization that is mediated by increased levels of O2•− and H2O2. These results also support the hypothesis that mitochondrial O2•− and H2O2 originating from SDH are capable of playing a role in low-dose ionizing radiation-induced biological responses.

Background

This research develops methods for determining the effect of geocoding quality on relationships between environmental exposures and health. The likelihood of detecting an existing relationship – statistical power – between measures of environmental exposures and health depends not only on the strength of the relationship but also on the level of positional accuracy and completeness of the geocodes from which the measures of environmental exposure are made. This paper summarizes the results of simulation studies conducted to examine the impact of inaccuracies of geocoded addresses generated by three types of geocoding processes: a) addresses located on orthophoto maps, b) addresses matched to TIGER files (U.S Census or their derivative street files); and, c) addresses from E-911 geocodes (developed by local authorities for emergency dispatch purposes).

Results

The simulated odds of disease using exposures modelled from the highest quality geocodes could be sufficiently recovered using other, more commonly used, geocoding processes such as TIGER and E-911; however, the strength of the odds relationship between disease exposures modelled at geocodes generally declined with decreasing geocoding accuracy.

Conclusion

Although these specific results cannot be generalized to new situations, the methods used to determine the sensitivity of results can be used in new situations. Estimated measures of positional accuracy must be used in the interpretation of results of analyses that investigate relationships between health outcomes and exposures measured at residential locations. Analyses similar to those employed in this paper can be used to validate interpretation of results from empirical analyses that use geocoded locations with estimated measures of positional accuracy.

Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca2+-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of 32[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca2+-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component.

Author Summary

Apicomplexan parasites are a group of obligate intracellular pathogens of wide medical and agricultural significance. Included within this phylum is Plasmodium spp, the causative agents to malaria and the ubiquitous parasite Toxoplasma, which inflicts disease burden on AIDS patients, transplant recipients and the unborn fetus. No matter the host cell that they target, all apicomplexan parasites must activate invasion upon host cell contact. Calcium-mediated signal transduction pathways modulate this process, yet the molecular processes are largely unknown. Using a range of proteomics approaches we reveal proteins in Toxoplasma that are phosphorylated upon calcium signaling, and furthermore, identify phosphorylation sites on a range of proteins that may play crucial roles in regulating parasite motility and microneme secretion. By quantitatively monitoring phosphorylation deposition upon calcium signaling we define putative regulatory domains of GAP45 and MLC1 and further show evidence that the invasion motor potentially more strongly associates upon calcium signaling. We also identified that a new Calmodulin-like protein is part of the invasion motor and this suggests that direct Ca2+ binding may also modulate motor activity.

K-ras mutations occur in as high as 95% of patients with pancreatic cancer. K-ras activates Rac1-dependent NADPH oxidase, a key source of superoxide. Superoxide plays an important role in pancreatic cancer cell proliferation and scavenging or decreasing the levels of superoxide inhibits pancreatic cancer cell growth both in vitro and in vivo. DNA microarray analysis and RT-PCR has demonstrated that Rac1 is also upregulated in pancreatic cancer. The aim of this study was to determine if inhibiting Rac1 would alter pancreatic tumor cell behavior. Human pancreatic cancer cells with mutant K-ras (MIA PaCa-2), wild-type K-ras (BxPC-3), and the immortal H6c7 cell line (pancreatic ductal epithelium) expressing K-ras oncogene (H6c7eR-KrasT) that is tumorigenic, were infected with a dominant/negative Rac1 construct (AdN17Rac1). In cells with mutant K-ras, AdN17Rac1 decreased rac activity, decreased superoxide levels, and inhibited in vitro growth. However in the BxPC-3 cell line, AdN17Rac1 did not change rac activity, superoxide levels, or in vitro cell growth. Additionally, AdN17Rac1 decreased superoxide levels and inhibited in vitro growth in the KrasT tumorigenic cell line, but had no effect in the immortalized H6c7 cell line. In human pancreatic tumor xenografts, intratumoral injections of AdN17Rac1 inhibited tumor growth. These results suggest that activation of Rac1-dependent superoxide generation leads to pancreatic cancer cell proliferation. In pancreatic cancer inhibition of Rac1 may be a potential therapeutic target.

This study evaluates the predictive value of post-therapy FDG-PET, including indeterminate studies, following curative intent therapy in Diffuse Large B-cell Lymphoma (DLBCL).

Consecutive, patients from September 2002-December 2005 were prospectively offered enrollment in an observational registry. Available FDG-PET reports after primary therapy were interpreted by hematologists-oncologists as positive, negative or indeterminate.

125 DLBCL patients had median follow-up of 35.2 months. Ninety-three percent were treated with R-CHOP-like therapy. Twenty percent of PET reports were judged indeterminate. EFS at 3 years for the negative and indeterminate groups was 85% and 71% respectively (p=0.28 by log-rank). OS at 3 years for negative, indeterminate, and positive groups was 89%, 88%, and 48%. Combining pre-therapy International Prognostic Index (IPI) with the post-therapy FDG-PET result added to the predictive value of the study for patients. Three-year EFS for patients with low or low-intermediate IPI risk and an indeterminate FDG-PET report was 93% while high or high-intermediate pre-therapy IPI patients had a 3 year EFS of 45%(p<0.02). Interpreting FDG-PET reports following curative intent chemotherapy in patients is informative but imprecise, and incorporation of pre-therapy prognosis can improve predictive utility.

Purpose

DNA strand breaks appear to be important in mediating radiosensitization during thymidine deprivation. This work examines the role of DNA repair, and altered thymidine analogs in altering the response to radiation during thymidine deprivation.

Methods

Mismatch repair deficient and proficient cell lines HEC59 and HC-2.4 were treated with FUdR, AZT and irradiation either alone or in combination and outcomes of clonogenic survival and cell cycle distributions were determined.

Results

Survival outcomes for all treatments were similar for both cell lines, suggesting hMSH2 does not significantly influence thymidine deprivation toxicity or radiosensitiation. The chain terminating thymidine analog azidothymidine (AZT) increased the toxicity of FUdR and increased DNA fragmentation. The combination of FUDR and AZT afforded greater radiosensitization than either drug alone. Drug enhancement ratios, the degree of excess radiation induced cell death in drug treated cultures compared to radiation alone for HEC59 was 1.2, 1.4 and 1.8 for AZT, FUdR and the combination. Enhancement ratios for HC-2.4 were 1.3, 1.5 and 1.8 for AZT, FUdR and the combination.

Conclusion

AZT, a chain terminating thymidine analog, can enhance the radiosensitizing affects of thymidine deprivation. DNA strand breaks may play an important role in the mechanism of thymidine deprivation induced radiosensitization.

Purpose

Sunburns are an important risk factor for melanoma and those occurring in childhood are often cited as posing the greatest risk. We conducted a meta-analysis to quantify the magnitude of association for melanoma and sunburns during childhood, adolescence, adulthood and over a lifetime.

Methods

After reviewing over 1300 article titles and evaluating 270 articles in detail, we pooled ORs from 51 independent study populations for “ever” sunburned and risk of cutaneous melanoma. Among these, 26 studies reported results from dose-response analyses. Dose-response analyses were examined using both fixed-effects models and Bayesian random-effects models.

Results

An increased risk of melanoma was seen with increasing number of sunburns for all time-periods (childhood, adolescence, adulthood and lifetime). In an attempt to understand how risk between life-periods compares, we also report these same linear models on a scale of 5 sunburns per decade for each life-period. The magnitude of risk for 5 sunburns per decade is highest for adult and lifetime sunburns.

Conclusions

Overall, these results show an increased risk of melanoma with increasing number of sunburns during all life-periods, not just childhood. Prevention efforts should focus on reducing sunburns during all life-periods.

Overexpression of MnSOD when combined with certain chemicals that inhibit peroxide removal increases cancer cell cytotoxicity. Elevating MnSOD levels in cells enhances the conversion of superoxide (O2•−) to hydrogen peroxide (H2O2), combined with inhibiting the removal of H2O2, further increases H2O2 levels, leading to increased cytotoxicity. We hypothesized that increasing endogenous O2•− production in cells that were pretreated with adenoviral MnSOD (AdMnSOD) plus BCNU would lead to an increased level of intracellular H2O2 accumulation and increased cell killing. The cytotoxic effects of adriamycin or radiation, agents known to produce O2•−, were determined in MDA-MB-231 breast cancer cells pretreated with AdMnSOD plus BCNU both in vitro and in vivo. In vitro, AdMnSOD plus BCNU sensitized cells to the cytotoxicity of adriamycin or radiation. In vivo, AdMnSOD, BCNU, and adriamycin or ionizing radiation inhibited tumor growth and prolonged survival. The results suggest that agents that produce O2•− in combination with AdMnSOD plus BCNU may represent a powerful new antitumor regimen against breast cancer.

Justification for article appearing in Angewandte Chemie:

Foldamers are currently being explored by a number of groups as antagonists of protein-protein interactions. Here we report the first high-resolution structure of a foldamer in complex with its target protein, the anti-apoptotic protein Bcl-xL. The structure demonstrates that α/β-peptide foldamers can accurately mimic natural peptide ligands and that the β-amino acid residues can make unique contacts at the binding interface. This structural information provides a basis for the design of foldamers with enhanced Bcl-xL-binding properties and, more generally, encouragement for continued efforts to develop foldameric inhibitors of other protein-protein interactions.

Several hundred malaria parasite proteins are exported beyond an encasing vacuole and into the cytosol of the host erythrocyte, a process that is key to the virulence and viability of the causative Plasmodium species. The trafficking machinery responsible for this export is unknown. Here, we identify a Plasmodium Translocon of EXported proteins (PTEX), which is located in the vacuole membrane. The PTEX complex is ATP-powered and comprises HSP101, which is a ClpA/B-like AAA+ ATPase of a type commonly associated with protein translocons, a novel protein termed PTEX150 and a known parasite protein EXP2. EXP2 is the potential channel as it is the membrane-associated component of the core PTEX complex. Two other proteins, a novel protein PTEX88 and a thioredoxin known as TRX2, were also identified as PTEX components. As a common portal for numerous crucial processes, this novel translocon offers an exciting new avenue for therapeutic intervention.

Increased reactive oxygen species (ROS) such as superoxide have been implicated as causal elements of oncogenesis. A variety of cancers have displayed changes in steady state levels of key antioxidant enzymes with the mitochondrial form of superoxide dismutase (MnSOD) being commonly implicated. Increasing MnSOD expression suppresses the malignant phenotype in various cancer cell lines and suppresses tumor formation in xenograft and transgenic mouse models. We examined the impact of MnSOD expression in the development of T cell lymphoma in mice expressing pro-apoptotic Bax. Lck-Bax38/1 transgenic mice were crossed to mice overexpressing MnSOD (Lck-MnSOD) as well as MnSOD +/− mice. The effect of MnSOD on apoptosis, cell cycle, chromosomal instability (CIN), and lymphoma development was determined. The apoptotic and cell cycle phenotypes observed in thymocytes from control and Bax transgenic mice were unaffected by variations in MnSOD levels. Remarkably, increased gene dosage of MnSOD significantly decreased aneuploidy in pre-malignant thymocytes as well as the onset of tumor formation in Lck-Bax38/1 mice. The observed effects of MnSOD support a role for ROS in CIN and tumor formation in this mouse model of T cell lymphoma.

Purpose

Determine if the response of human head and neck cancer xenografts to cisplatin (CIS) could be enhanced with 2-deoxyglucose (2DG) and determine if 2-[F-18]-fluoro-2-deoxy-D-glucose (FDG) uptake correlated with responses to this drug combination. Determine if 2DG would enhance CIS-induced radiosensitization.

Experimental Design

Clonogenic survival responses to CIS + 2DG were determined in FaDu and Cal-27 cells and GSH/GSSG levels were monitored as parameters indicative of oxidative stress. The efficacy of CIS + 2DG was determined in FaDu and Cal-27 xenografts and FDG uptake was determined with Positron Emission Tomography (PET).

Results

CIS + 2DG enhanced cell killing of FaDu and Cal-27 cells, compared to either drug alone, while increasing %GSSG in vitro. CIS + 2DG inhibited FaDu and Cal-27 tumor growth and increased disease free survival, compared to either drug alone. Cal-27 tumors demonstrated greater pretreatment FDG uptake and increased disease free survival when treated with 2DG + CIS, relative to FaDu tumors. 2DG treatment enhanced CIS-induced radiosensitization in FaDu tumor cells grown in vitro and in vivo and resulted in apparent cures in 50% of tumors.

Conclusions

These results demonstrate the enhanced therapeutic efficacy of CIS + 2DG in human head and neck cancer cells in vitro and in vivo when compared to either drug alone as well as demonstrating the potential for FDG uptake to predict tumor sensitivity to 2DG + CIS. These findings provide a strong rationale for evaluating 2DG + CIS in combined modality head and neck cancer therapy with radiation in a clinical setting.

It is important to assess the effectiveness of patient education programs for people with COPD (chronic obstructive pulmonary disease) to ensure that limited health resources are being spent effectively. We aimed to assess the effectiveness of programs reported to date, and to look for ways of designing more effective programs.

COPD patient education to date has produced little demonstrated success, but studies, education programs and study reports all show limitations. To demonstrate links between outcomes and patient education program components, there is a need for more trials which combine process and outcome evaluation. Programs to date have relied too heavily on the provision of medical information to patients. Programs which also aim to improve disease management self-efficacy hold promise but further determinants of health behavior should be included also, as part of more systematic program design. Program components need to be clearly described and the rationale for their use justified in trial reports. This will produce an evidence base that should show what role education programs can play in improving outcomes, and inform the development of more effective programs.

Background

Studies investigating adverse events have traditionally been principally undertaken from a medical perspective. The impact that experience of an adverse event has on consumer confidence in health care is largely unknown. The objectives of the study were to seek public opinion on 1) the rate and severity of adverse events experienced in hospitals; and 2) the perception of safety in hospitals, so that predictors of lack of safety could be identified.

Methods

A multistage, clustered survey of persons residing in South Australia (2001), using household interviews (weighted n = 2,884).

Results

A total of 67% of respondents aged over forty years reported having at least one member of their household hospitalised in the past five years; with the average being two hospital admissions in five years. Respondents stated that 7.0% (95%CI: 6.2% to 7.9%) of those hospital admissions were associated with an adverse event; 59.7% of respondents (95% CI: 51.4% to 67.5%) rated the adverse event as really serious and 48.5% (95% CI: 40.4% to 56.8%) stated prolonged hospitalisation was required as a consequence of the adverse event. Perception of safety in hospitals was largely affected by the experience of an adverse event; really serious events were the most significant predictor of lack of safety in those aged 40 years and over (RR 2.38; p<0.001).

Conclusion

The experience of adverse events negatively impacted on public confidence in hospitals. The consumer-reported adverse event rate in hospitals (7.0%) is similar to that identified using medical record review. Based on estimates from other studies, self-reported claims of adverse events in hospital by consumers appear credible, and should be considered when developing appropriate treatment regimes.