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Abstract Background Polymorphisms in and around the CTLA-4 gene have previously been associated to T1D and AITD in several populations. One such single nucleotide polymorphism (SNP), CT60, has been reported to affect the expression level ratio of the soluble (sCTLA-4) to full length CTLA-4 (flCTLA-4) isoforms. The aims of our study were to replicate the association previously published by Ueda et al. of polymorphisms in the CTLA-4 region to T1D and AITD and to determine whether the CT60 polymorphism affects the expression level ratio of sCTLA-4/flCTLA-4 in our population. Methods Three SNPs were genotyped in 253 cases (104 AITD cases and 149 T1D cases) and 865 ethnically matched controls. Blood from 23 healthy individuals was used to quantify mRNA expression of CTLA-4 isoforms in CD4+ cells using real-time PCR. Serum from 102 cases and 59 healthy individuals was used to determine the level of sCTLA-4 protein. Results Here we show association of the MH30, CT60 and JO31 polymorphisms to T1D and AITD in northern Sweden. We also observed a higher frequency of the CT60 disease susceptible allele in our controls compared to the British, Italian and Dutch populations, which might contribute to the high frequency of T1D in Sweden. In contrast to previously published findings, however, we were unable to find differences in the sCTLA-4/flCTLA-4 expression ratio based on the CT60 genotype in 23 healthy volunteers, also from northern Sweden. Analysis of sCTLA-4 protein levels in serum showed no correlation between sCTLA-4 protein levels and disease status or CT60 genotype. Conclusion Association was found between T1D/AITD and all three polymorphisms investigated. However, in contrast to previous investigations, sCTLA-4 RNA and protein expression levels did not differ based on CT60 genotype. Our results do not rule out the CT60 SNP as an important polymorphism in the development of T1D or AITD, but suggest that further investigations are necessary to elucidate the effect of the CTLA-4 region on the development of T1D and AITD.

Background: Phase 1 oncology trials provide access to new therapies and may improve cancer outcomes. Phase 1 trials conducted in the Asian-Pacific region are increasing at a faster rate than the global trend. This study aimed to describe the changing landscape of phase 1 oncology trials in Australia in the last decade., Methods: This cross-sectional study reviewed phase 1 oncology trials registered on ClinicalTrials.gov conducted in Australia. Phase 1 trials were included for analysis if they enrolled adults with solid organ malignancies, used at least one systemic agent, and were first registered between January 1, 2012, and December 31, 2022. The number of trials, site locations, sponsor type, and drug class were analyzed using descriptive statistics., Results: Over the 10-year period, ClinicalTrials.gov included 493 phase 1 clinical trials across 71 Australian sites. Most sites were in metropolitan locations; in Melbourne, trials were concentrated within selected sites, while in Sydney, trials were spread across a larger number of sites. The number of phase 1 trials per annum increased from 18 in 2012 to 75 in 2022. Since 2020, emerging biopharmaceutical companies have become the predominant sponsor type, a trend that is also seen globally. While most trial sponsors were North American (42%), there was increasing representation from Asian sponsors over the 10-year period (6% in 2012 to 39% in 2022). Immunomodulatory (45%) and targeted approaches (44%) accounted for most drug classes used alone or in combination., Conclusions: There are an increasing number of phase 1 trials conducted within Australia. Sponsors of phase 1 trials are increasingly from Asian countries and are more likely to be emerging biopharmaceutical companies., (© 2024 John Wiley & Sons Australia, Ltd.)

Current techniques for the identification of DNA adduct-inducing and DNA interstrand crosslinking agents include electrophoretic crosslinking assays, electrophoretic gel shift assays, DNA and RNA stop assays, mass spectrometry-based methods and 32 P-post-labelling. While these assays provide considerable insight into the site and stability of the interaction, they are relatively expensive, time-consuming and sometimes rely on the use of radioactively-labelled components, and thus are ill-suited to screening large numbers of compounds. A novel medium throughput assay was developed to overcome these limitations and was based on the attachment of a biotin-tagged double stranded (ds) oligonucleotide to Corning DNA-Bind plates. We aimed to detect anthracycline and anthracenedione DNA adducts which form by initial non-covalent intercalation with duplex DNA, and subsequent covalent adduct formation which is mediated by formaldehyde. Following drug treatment, DNA samples were subjected to a denaturation step, washing and then measurement by fluorescence to detect remaining drug-DNA species using streptavidin-europium. This dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) is a time-resolved fluorescence intensity assay where the fluorescence signal arises only from stabilised drug-DNA complexes. We applied this new methodology to the identification of anthracycline-like compounds with the ability to functionally crosslink double-strand oligonucleotides. The entire procedure can be performed by robotics, requiring low volumes of compounds and reagents, thereby reducing costs and enabling multiple compounds to be assessed on a single microtitre plate., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Activating the intrinsic apoptosis pathway with small molecules is now a clinically validated approach to cancer therapy. In contrast, blocking apoptosis to prevent the death of healthy cells in disease settings has not been achieved. Caspases have been favored, but they act too late in apoptosis to provide long-term protection. The critical step in committing a cell to death is activation of BAK or BAX, pro-death BCL-2 proteins mediating mitochondrial damage. Apoptosis cannot proceed in their absence. Here we show that WEHI-9625, a novel tricyclic sulfone small molecule, binds to VDAC2 and promotes its ability to inhibit apoptosis driven by mouse BAK. In contrast to caspase inhibitors, WEHI-9625 blocks apoptosis before mitochondrial damage, preserving cellular function and long-term clonogenic potential. Our findings expand on the key role of VDAC2 in regulating apoptosis and demonstrate that blocking apoptosis at an early stage is both advantageous and pharmacologically tractable.

The cell-cell adhesion protein E-cadherin (CDH1) is a tumor suppressor that is required to maintain cell adhesion, cell polarity and cell survival signalling. Somatic mutations in CDH1 are common in diffuse gastric cancer (DGC) and lobular breast cancer (LBC). In addition, germline mutations in CDH1 predispose to the autosomal dominant cancer syndrome Hereditary Diffuse Gastric Cancer (HDGC). One approach to target cells with mutations in specific tumor suppressor genes is synthetic lethality. To identify novel synthetic lethal compounds for the treatment of cancers associated with E-cadherin loss, we have undertaken a high-throughput screening campaign of ~114,000 lead-like compounds on an isogenic pair of human mammary epithelial cell lines - with and without CDH1 expression. This unbiased approach identified 12 novel compounds that preferentially harmed E-cadherin-deficient cells. Validation of these compounds using both real-time and end-point viability assays identified two novel compounds with significant synthetic lethal activity, thereby demonstrating that E-cadherin loss creates druggable vulnerabilities within tumor cells. In summary, we have identified novel synthetic lethal compounds that may provide a new strategy for the prevention and treatment of both sporadic and hereditary LBC and DGC.

Neutropenia represents one of the major dose-limiting toxicities of many current cancer therapies. To circumvent the off-target effects of cytotoxic chemotherapeutics, kinase inhibitors are increasingly being used as an adjunct therapy to target leukemia. In this study, we conducted a screen of leukemic cell lines in parallel with primary neutrophils to identify kinase inhibitors with the capacity to induce apoptosis of myeloid and lymphoid cell lines whilst sparing primary mouse and human neutrophils. We have utilized a high-throughput live cell imaging platform to demonstrate that cytotoxic drugs have limited effects on neutrophil viability but are toxic to hematopoietic progenitor cells, with the exception of the topoisomerase I inhibitor SN-38. The parallel screening of kinase inhibitors revealed that mouse and human neutrophil viability is dependent on cyclin-dependent kinase (CDK) activity but surprisingly only partially dependent on PI3 kinase and JAK/STAT signaling, revealing dominant pathways contributing to neutrophil viability. Mcl-1 haploinsufficiency sensitized neutrophils to CDK inhibition, demonstrating that Mcl-1 is a direct target for CDK inhibitors. This study reveals a therapeutic window for the kinase inhibitors BEZ235, BMS-3, AZD7762, and (R)-BI-2536 to induce apoptosis of leukemia cell lines whilst maintaining immunocompetence and hemostasis., Competing Interests: Conflicts of Interest The authors declare no financial conflicts of interest.

Purpose: Cyclin E1 ( CCNE1 ) amplification is associated with primary treatment resistance and poor outcome in high-grade serous ovarian cancer (HGSC). Here, we explore approaches to target CCNE1 -amplified cancers and potential strategies to overcome resistance to targeted agents. Experimental Design: To examine dependency on CDK2 in CCNE1 -amplified HGSC, we utilized siRNA and conditional shRNA gene suppression, and chemical inhibition using dinaciclib, a small-molecule CDK2 inhibitor. High-throughput compound screening was used to identify selective synergistic drug combinations, as well as combinations that may overcome drug resistance. An observed relationship between CCNE1 and the AKT pathway was further explored in genomic data from primary tumors, and functional studies in fallopian tube secretory cells. Results: We validate CDK2 as a therapeutic target by demonstrating selective sensitivity to gene suppression. However, we found that dinaciclib did not trigger amplicon-dependent sensitivity in a panel of HGSC cell lines. A high-throughput compound screen identified synergistic combinations in CCNE1 -amplified HGSC, including dinaciclib and AKT inhibitors. Analysis of genomic data from TCGA demonstrated coamplification of CCNE1 and AKT2 Overexpression of Cyclin E1 and AKT isoforms, in addition to mutant TP53 , imparted malignant characteristics in untransformed fallopian tube secretory cells, the dominant site of origin of HGSC. Conclusions: These findings suggest a specific dependency of CCNE1 -amplified tumors for AKT activity, and point to a novel combination of dinaciclib and AKT inhibitors that may selectively target patients with CCNE1 -amplified HGSC. Clin Cancer Res; 23(7); 1862-74. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Resistance to antifungal drugs is an increasingly significant clinical problem. The most common antifungal resistance encountered is efflux pump-mediated resistance of Candida species to azole drugs. One approach to overcome this resistance is to inhibit the pumps and chemosensitize resistant strains to azole drugs. Drug discovery targeting fungal efflux pumps could thus result in the development of azole-enhancing combination therapy. Heterologous expression of fungal efflux pumps in Saccharomyces cerevisiae provides a versatile system for screening for pump inhibitors. Fungal efflux pumps transport a range of xenobiotics including fluorescent compounds. This enables the use of fluorescence-based detection, as well as growth inhibition assays, in screens to discover compounds targeting efflux-mediated antifungal drug resistance. A variety of medium- and high-throughput screens have been used to identify a number of chemical entities that inhibit fungal efflux pumps.

Erythroid enucleation is critical for terminal differentiation of red blood cells, and involves extrusion of the nucleus by orthochromatic erythroblasts to produce reticulocytes. Due to the difficulty of synchronizing erythroblasts, the molecular mechanisms underlying the enucleation process remain poorly understood. To elucidate the cellular program governing enucleation, we utilized a novel chemical screening approach whereby orthochromatic cells primed for enucleation were enriched ex vivo and subjected to a functional drug screen using a 324 compound library consisting of structurally diverse, medicinally active and cell permeable drugs. Using this approach, we have confirmed the role of HDACs, proteasomal regulators and MAPK in erythroid enucleation and introduce a new role for Cyclin-dependent kinases, in particular CDK9, in this process. Importantly, we demonstrate that when coupled with imaging analysis, this approach provides a powerful means to identify and characterize rate limiting steps involved in the erythroid enucleation process.

Frameshifting during translation of viral or in rare cases cellular mRNA results in the synthesis of proteins from two overlapping reading frames within the same mRNA. In HIV-1 the protease, reverse transcriptase, and integrase enzymes are in a second reading frame relative to the structural group-specific antigen (gag), and their synthesis is dependent upon frameshifting. This ensures that a strictly regulated ratio of structural proteins and enzymes, which is critical for HIV-1 replication and viral infectivity, is maintained during protein synthesis. The frameshift element in HIV-1 RNA is an attractive target for the development of a new class of anti HIV-1 drugs. However, a number of examples are now emerging of human genes using -1 frameshifting, such as PEG10 and CCR5. In this study we have compared the HIV-1 and PEG10 frameshift elements and shown they have distinct functional characteristics. Frameshifting occurs at several points within each element. Moreover, frameshift modulators that were isolated by high-throughput screening of a library of 114,000 lead-like compounds behaved differently with the PEG10 frameshift element. The most effective compounds affecting the HIV-1 element enhanced frameshifting by 2.5-fold at 10 μM in two different frameshift reporter assay systems. HIV-1 protease:gag protein ratio was affected by a similar amount in a specific assay of virally-infected cultured cell, but the modulation of frameshifting of the first-iteration compounds was not sufficient to show significant effects on viral infectivity. Importantly, two compounds did not affect frameshifting with the human PEG10 element, while one modestly inhibited rather than enhanced frameshifting at the human element. These studies indicate that frameshift elements have unique characteristics that may allow targeting of HIV-1 and of other viruses specifically for development of antiviral therapeutic molecules without effect on human genes like PEG10 that use the same generic mechanism.

Purpose: Osteosarcoma is the most common cancer of bone occurring mostly in teenagers. Despite rapid advances in our knowledge of the genetics and cell biology of osteosarcoma, significant improvements in patient survival have not been observed. The identification of effective therapeutics has been largely empirically based. The identification of new therapies and therapeutic targets are urgently needed to enable improved outcomes for osteosarcoma patients., Experimental Design: We have used genetically engineered murine models of human osteosarcoma in a systematic, genome-wide screen to identify new candidate therapeutic targets. We performed a genome-wide siRNA screen, with or without doxorubicin. In parallel, a screen of therapeutically relevant small molecules was conducted on primary murine- and primary human osteosarcoma-derived cell cultures. All results were validated across independent cell cultures and across human and mouse osteosarcoma., Results: The results from the genetic and chemical screens significantly overlapped, with a profound enrichment of pathways regulated by PI3K and mTOR pathways. Drugs that concurrently target both PI3K and mTOR were effective at inducing apoptosis in primary osteosarcoma cell cultures in vitro in both human and mouse osteosarcoma, whereas specific PI3K or mTOR inhibitors were not effective. The results were confirmed with siRNA and small molecule approaches. Rationale combinations of specific PI3K and mTOR inhibitors could recapitulate the effect on osteosarcoma cell cultures., Conclusions: The approaches described here have identified dual inhibition of the PI3K-mTOR pathway as a sensitive, druggable target in osteosarcoma, and provide rationale for translational studies with these agents., (©2015 American Association for Cancer Research.)

Following erythrocyte invasion, malaria parasites export a catalogue of remodeling proteins into the infected cell that enable parasite development in the human host. Export is dependent on the activity of the aspartyl protease, plasmepsin V (PMV), which cleaves proteins within the Plasmodium export element (PEXEL; RxL↓xE/Q/D) in the parasite's endoplasmic reticulum. Here, we generated transition state mimetics of the native PEXEL substrate that potently inhibit PMV isolated from Plasmodium falciparum and Plasmodium vivax. Through optimization, we identified that the activity of the mimetics was completely dependent on the presence of P1 Leu and P3 Arg. Treatment of P. falciparum-infected erythrocytes with a set of optimized mimetics impaired PEXEL processing and killed the parasites. The striking effect of the compounds provides a clearer understanding of the accessibility of the PMV active site and reaffirms the enzyme as an attractive target for the design of future antimalarials.

Metastasis accounts for the poor prognosis of the majority of solid tumors. The phenotypic transition of nonmotile epithelial tumor cells to migratory and invasive "mesenchymal" cells (epithelial-to-mesenchymal transition [EMT]) enables the transit of cancer cells from the primary tumor to distant sites. There is no single marker of EMT; rather, multiple measures are required to define cell state. Thus, the multiparametric capability of high-content screening is ideally suited for the comprehensive analysis of EMT regulators. The aim of this study was to generate a platform to systematically identify functional modulators of tumor cell plasticity using the bladder cancer cell line TSU-Pr1-B1 as a model system. A platform enabling the quantification of key EMT characteristics, cell morphology and mesenchymal intermediate filament vimentin, was developed using the fluorescent whole-cell-tracking reagent CMFDA and a fluorescent promoter reporter construct, respectively. The functional effect of genome-wide modulation of protein-coding genes and miRNAs coupled with those of a collection of small-molecule kinase inhibitors on EMT was assessed using the Target Activation Bioapplication integrated in the Cellomics ArrayScan platform. Data from each of the three screens were integrated to identify a cohort of targets that were subsequently examined in a validation assay using siRNA duplexes. Identification of established regulators of EMT supports the utility of this screening approach and indicated capacity to identify novel regulators of this plasticity program. Pathway analysis coupled with interrogation of cancer-related expression profile databases and other EMT-related screens provided key evidence to prioritize further experimental investigation into the molecular regulators of EMT in cancer cells.

We developed a new approach to distinguish distinct protein conformations in live cells. The method, exposable tetracysteine (XTC), involved placing an engineered tetracysteine motif into a target protein that has conditional access to biarsenical dye binding by conformational state. XTC was used to distinguish open and closed regulatory conformations of Src family kinases. Substituting just four residues with cysteines in the conserved SH2 domain of three Src-family kinases (c-Src, Lck, Lyn) enabled open and closed conformations to be monitored on the basis of binding differences to biarsenical dyes FlAsH or ReAsH. Fusion of the kinases with a fluorescent protein tracked the kinase presence, and the XTC approach enabled simultaneous assessment of regulatory state. The c-Src XTC biosensor was applied in a boutique screen of kinase inhibitors, which revealed six compounds to induce conformational closure. The XTC approach demonstrates new potential for assays targeting conformational changes in key proteins in disease and biology.

The Walter and Eliza Hall Institute of Medical Research (WEHI) is Australia's longest serving medical research institute. WEHI's High Throughput Screening (HTS) Facility was established in 2003 with $5 million of infrastructure funds invested by WEHI, and the Victorian State Government's Strategic Technology Initiative through Bio21 Australia Ltd. The Facility was Australia's first truly academic HTS facility and was one of only a handful operating in publicly funded institutions worldwide at that time. The objectives were to provide access to enabling HTS technologies, such as assay design, liquid handling automation, compound libraries and expertise to promote translation of basic research in a national setting that has a relatively young biotech sector and does not have a big Pharma research presence. Ten years on and the WEHI HTS Facility has participated in over 92 collaborative projects, generated over 18 million data points, and most importantly, projects that began in the Facility have been commercialized successfully (due to strong ties with Business Development and emphasis on intellectual property management) and now have molecules progressing in clinical trials.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) plays an essential role in the replication of HCV and is a key target for novel antiviral therapies. Several RdRp inhibitors are in clinical trials and have increased response rates when combined with current interferon-based therapies for genotype 1 (G1) HCV patients. These inhibitors, however, show poor efficacy against non-G1 genotypes, including G3a, which represents ~20% of HCV cases globally. Here, we used a commercially available fluorescent dye to characterize G3a HCV RdRp in vitro. RdRp activity was assessed via synthesis of double-stranded RNA from the single-stranded RNA poly(C) template. The assay was miniaturized to a 384-well microplate format and a pilot high-throughput screen was conducted using 10,208 "lead-like" compounds, randomly selected to identify inhibitors of HCV G3a RdRp. Of 150 compounds demonstrating greatest inhibition, 10 were confirmed using both fluorescent and radioactive assays. The top two inhibitors (HAC001 and HAC002) demonstrated specific activity, with an IC(50) of 12.7 µM and 1.0 µM, respectively. In conclusion, we describe simple, fluorescent-based high-throughput screening (HTS) for the identification of inhibitors of de novo RdRp activity, using HCV G3a RdRp as the target. The HTS system could be used against any positive-sense RNA virus that cannot be cultured.

Background: How antimalarial antibodies are acquired and maintained during pregnancy and boosted after reinfection with Plasmodium falciparum and Plasmodium vivax is unknown., Methods: A nested case-control study of 467 pregnant women (136 Plasmodium-infected cases and 331 uninfected control subjects) in northwestern Thailand was conducted. Antibody levels to P. falciparum and P. vivax merozoite antigens and the pregnancy-specific PfVAR2CSA antigen were determined at enrollment (median 10 weeks gestation) and throughout pregnancy until delivery., Results: Antibodies to P. falciparum and P. vivax were highly variable over time, and maintenance of high levels of antimalarial antibodies involved highly dynamic responses resulting from intermittent exposure to infection. There was evidence of boosting with each successive infection for P. falciparum responses, suggesting the presence of immunological memory. However, the half-lives of Plasmodium antibody responses were relatively short, compared with measles (457 years), and much shorter for merozoite responses (0.8-7.6 years), compared with PfVAR2CSA responses (36-157 years). The longer half-life of antibodies to PfVAR2CSA suggests that antibodies acquired in one pregnancy may be maintained to protect subsequent pregnancies., Conclusions: These findings may have important practical implications for predicting the duration of vaccine-induced responses by candidate antigens and supports the development of malaria vaccines to protect pregnant women.

During immune responses, neutrophils must integrate survival and death signals from multiple sources to regulate their lifespan. Signals that activate either the Bcl-2- or death receptor-regulated apoptosis pathways can provide powerful stimuli for neutrophils to undergo cell death, but whether they act cooperatively in parallel or directly cross-talk in neutrophils is not known. Previous studies suggested that Bcl-2 family proteins are not required for Fas-induced cell death in neutrophils, but did not examine whether they could modulate its rapid onset. By monitoring the rate of change in neutrophil viability associated with activation of the Fas-triggered death receptor pathway using real-time cell imaging, we show that the Bcl-2-related proteins Bid, Bax, and Bak accelerate neutrophil apoptosis but are not essential for cell death. Increased Bcl-2 or Mcl-1 expression prevents efficient induction of apoptosis by Fas stimulation indicating that the Bcl-2-regulated apoptosis pathway can directly interfere with Fas-triggered apoptosis. Fas has been shown to initiate NFκB activation and gene transcription in cell lines, however gene transcription is not altered in Fas-activated Bid(-/-) neutrophils, indicating that apoptosis occurs independently of gene transcription in neutrophils. The specification of kinetics of neutrophil apoptosis by Bid impacts on the magnitude of neutrophil IL-1β production, implicating a functional role for the Bcl-2-regulated pathway in controlling neutrophil responses to FasL. These data demonstrate that the intrinsic apoptosis pathway directly controls the kinetics of Fas-triggered apoptosis in neutrophils.

The current treatment for leishmaniasis is based on chemotherapy, which relies on a handful of drugs with serious limitations, such as high cost, toxicity, and a lack of efficacy in regions of endemicity. Therefore, the development of new, effective, and affordable antileishmanial drugs is a global health priority. Leishmania synthesizes a range of mannose-rich glycoconjugates that are essential for parasite virulence and survival. A prerequisite for glycoconjugate biosynthesis is the conversion of monosaccharides to the activated mannose donor, GDP-mannose, the product of a reaction catalyzed by GDP-mannose pyrophosphorylase (GDP-MP). The deletion of the gene encoding GDP-MP in Leishmania led to a total loss of virulence, indicating that the enzyme is an ideal drug target. We developed a phosphate sensor-based high-throughput screening assay to quantify the activity of GDP-MP and screened a library containing approximately 80,000 lead-like compounds for GDP-MP inhibitors. On the basis of their GDP-MP inhibitory properties and chemical structures, the activities of 20 compounds which were not toxic to mammalian cells were tested against ex vivo amastigotes and in macrophage amastigote assays. The most potent compound identified in the primary screen (compound 3), a quinoline derivative, demonstrated dose-dependent activity in both assays (50% inhibitory concentration = 21.9 microM in the macrophage assay) and was shown to be nontoxic to human fibroblasts. In order to elucidate signs of an early structure-activity relationship (SAR) for this class of compounds, we obtained and tested analogues of compound 3 and undertook limited medicinal chemistry optimization, which included the use of a number of SAR probes of the piperazinyl aryl substituent of compound 3. We have identified novel candidate compounds for the design and synthesis of antileishmanial therapeutics.

Objective: To test the frictional resistance forces (FRS) generated between several archwires and (1) interactive self-ligating (ISL) brackets and (2) conventionally ligated (CL) brackets., Materials and Methods: Frictional forces produced between three different archwire combinations and self-ligating (SL) brackets (ceramic and metal-slot or all-metal) and CL brackets (metal or ceramic) were evaluated in a dry environment. The three ISL brackets tested were In-Ovation-C, In-Ovation-R, and Damon 3. The three CL brackets were Mystique with Neo Clip, Clarity, and Ovation. Each bracket was tested with 0.020'' SS, 0.019'' x 0.025'' SS and 0.018'' x 0.018'' coated SS., Results: The ISL brackets generally exhibited the lowest frictional forces irrespective of the bracket material and the wire size, and CL brackets exhibited consistently higher frictional forces. Mystique with Neo Clip produced the lowest frictional resistance of all brackets. The In-Ovation-C brackets demonstrated significantly lower frictional resistance than the SL brackets In-Ovation-R and Damon 3 as well as the CL brackets Clarity and Ovation., Conclusions: The ISL ceramic brackets produced the lowest frictional resistance of all the self-ligating brackets. The CL ceramic brackets produced the greatest friction.

A recent study found association of one microsatellite and five single nucleotide polymorphisms (SNPs) in intron 3 of the TCF7L2 gene with type 2 diabetes (T2D) in the Icelandic, Danish and American populations. The aim of the present study was to investigate if those SNPs were associated to T2D in two (family- and population-based) cohorts from northern Sweden. We genotyped four of the associated SNPs in a case-control cohort consisting of 872 T2D cases and 857 controls matched with respect to age, sex and geographical origin and in a sample of 59 extended families (148 affected and 83 unaffected individuals). Here, we report replication of association between T2D and three SNPs in the case-control (rs7901695, P=0.003; rs7901346, P=0.00002; and rs12255372, P=0.000004) and two SNPs in the family-based (rs7901695, P=0.01 and rs7901346, P=0.04) samples from northern Sweden. This replication strengthens the evidence for involvement of TCF7L2 in T2D.

Background: Polymorphisms in and around the CTLA-4 gene have previously been associated to T1D and AITD in several populations. One such single nucleotide polymorphism (SNP), CT60, has been reported to affect the expression level ratio of the soluble (sCTLA-4) to full length CTLA-4 (flCTLA-4) isoforms. The aims of our study were to replicate the association previously published by Ueda et al. of polymorphisms in the CTLA-4 region to T1D and AITD and to determine whether the CT60 polymorphism affects the expression level ratio of sCTLA-4/flCTLA-4 in our population., Methods: Three SNPs were genotyped in 253 cases (104 AITD cases and 149 T1D cases) and 865 ethnically matched controls. Blood from 23 healthy individuals was used to quantify mRNA expression of CTLA-4 isoforms in CD4+ cells using real-time PCR. Serum from 102 cases and 59 healthy individuals was used to determine the level of sCTLA-4 protein., Results: Here we show association of the MH30, CT60 and JO31 polymorphisms to T1D and AITD in northern Sweden. We also observed a higher frequency of the CT60 disease susceptible allele in our controls compared to the British, Italian and Dutch populations, which might contribute to the high frequency of T1D in Sweden. In contrast to previously published findings, however, we were unable to find differences in the sCTLA-4/flCTLA-4 expression ratio based on the CT60 genotype in 23 healthy volunteers, also from northern Sweden. Analysis of sCTLA-4 protein levels in serum showed no correlation between sCTLA-4 protein levels and disease status or CT60 genotype., Conclusion: Association was found between T1D/AITD and all three polymorphisms investigated. However, in contrast to previous investigations, sCTLA-4 RNA and protein expression levels did not differ based on CT60 genotype. Our results do not rule out the CT60 SNP as an important polymorphism in the development of T1D or AITD, but suggest that further investigations are necessary to elucidate the effect of the CTLA-4 region on the development of T1D and AITD.

The adsorption of cadmium onto kaolinite and Muloorina illite in the presence of citric acid has been measured as a function of pH and cadmium concentration at 25 degrees C. When citric acid is present in the systems cadmium adsorption is slightly enhanced below pH 5, but significantly suppressed between pH 5 and 8, for both substrates. At higher citric acid concentrations very little cadmium adsorbs onto kaolinite from pH 5 to 8. Above pH 8 adsorption of Cd(II) onto illite is enhanced in the presence of citric acid, especially at lower concentrations, but this does not occur for kaolinite. Adsorption and potentiometric titration data were fitted by simple extended constant-capacitance surface complexation models for the two substrates. Enhancement of adsorption at lower pH values was ascribed to the ternary reaction [X(-)--K(+)](0)+Cd(2+)+L(3-)+2H(+) right arrow over left arrow (0)+K(+) involving outer-sphere complexation with permanently charged X(-) sites on the "silica" faces of both clay minerals. The models suggested that suppression of adsorption in the intermediate pH range was due to the formation of a strong CdL(-) solution complex which adsorbed neither on the permanently charged sites nor on the surface hydroxyl groups at the edges of the clay crystals. At higher pH values the dominant solution complex, CdLOH(2-), apparently adsorbed as an outer-sphere complex at surface hydroxyl groups on illite, SOH+2Cd(2+)+L(3-) right arrow over left arrow [SOCd(+)--CdOHL(2-)](-)+2H(+), but not on kaolinite. This difference in behavior results from the presence of =FeOH groups on the illite surface which can form surface complexes with CdLOH(2-), while the =AlOH groups on the kaolinite surface cannot.

The adsorption of cadmium onto goethite in the presence of citric acid was measured as a function of pH and cadmium concentration at 25 degrees C. Potentiometric titrations were also performed on the system. Cadmium adsorption onto goethite was enhanced above pH 4 in the presence of 50 microM, 100 microM and 1 mM citric acid. While there was little difference between the enhancements caused by 50 and 100 microM citric acid below pH 6, above pH 6 further enhancement is seen in the presence of 100 microM citric acid. When 1 mM citric acid was present, the enhancement of cadmium adsorption was greater below pH 6, with increased Cd(II) adsorption down to pH 3.5. However, above pH 6, 1 mM of citric acid caused slightly less enhancement than the lower citric acid concentrations. ATR-FTIR spectra of soluble and adsorbed citrate-cadmium species were measured as a function of pH. At pH 4.6 there was very little difference between the ternary Cd(II)-citric acid-goethite spectrum and the binary citric acid-goethite spectrum. However, spectra of the ternary system at pH 7.0 and 8.7 indicated the presence of additional surface species. Further analysis of the spectra suggested that these were metal-ligand outer-sphere complexes. Data from the adsorption experiments and potentiometric titrations of the ternary Cd(II)-citric acid-goethite system were fitted by an extended constant-capacitance surface complexation model. The spectroscopic data were used to inform the choice of surface species. Three reactions in addition to those for the binary Cd(II)-goethite and citric acid-goethite systems were required to describe all of the data. They were [formula in text], [formula in text], and [formula in text]. Neither the spectroscopy nor the modeling suggested the formation of a ternary inner-sphere complex or a surface precipitate under the conditions used in this study.

The adsorption of citric acid onto goethite, kaolinite, and illite was measured as a function of pH (adsorption edges) and concentration (adsorption isotherms) at 25 degrees C. The greatest adsorption was onto goethite and the least onto illite. Adsorption onto goethite was at a maximum below pH 5 and decreased as the pH was increased to pH 9. For kaolinite, maximum adsorption occurred between pH 4.5 and pH 7, decreasing below and above this pH region, while for illite maximum adsorption occurred between about pH 5 and pH 7, decreasing at both lower and higher pH. ATR-FTIR spectra of citrate adsorbed to goethite at pH 4.6, pH 7.0, and pH 8.8 were compared with those of citrate solutions between pH 3.5 and pH 9.1. While the spectra of adsorbed citrate resembled those of the fully deprotonated solution species, there were significant differences. In particular the C[bond]O symmetric stretching band of the adsorbed species at pH 4.6 and 7.0 changed shape and was shifted to higher wave number. Further spectral analysis suggested that citrate adsorbed as an inner-sphere complex at pH 4.6 and pH 7.0 with coordination to the surface most probably via one or more carboxyl groups. At pH 8.8 the intensity of the adsorbed bands was much smaller but their shape was similar to those from the deprotonated citrate solution species, suggesting outer-sphere adsorption. Insufficient citric acid adsorbed onto illite or kaolinite to provide spectroscopic information about the mode of adsorption onto these minerals. Data from adsorption experiments, and from potentiometric titrations of suspensions of the minerals in the presence of citric acid, were fitted by extended constant-capacitance surface complexation models. On the goethite surface a monodentate inner-sphere complex dominated adsorption below pH 7.9, with a bidentate outer-sphere complex required at higher pH values. On kaolinite, citric acid adsorption was modeled with a bidentate outer-sphere complex at low pH and a monodentate outer-sphere complex at higher pH. There is evidence of dissolution of kaolinite in the presence of citric acid. For illite two bidentate outer-sphere complexes provided a good fit to all data.

The adsorption of Cd(II) onto goethite, kaolinite, and illite was measured as a function of pH (adsorption edges) and concentration (adsorption isotherms) at 25 degrees C. As the pH was increased, adsorption onto goethite occurred mainly in the pH range 5.5-8, whereas adsorption onto kaolinite occurred in two stages, separated by a plateau in the pH region 5.5 to 7. Adsorption onto illite increased steadily as the pH was increased, with far less Cd(II) adsorbing onto illite than onto goethite or kaolinite per m(2) of mineral surface area. Potentiometric titrations of suspensions of each mineral, with and without Cd(II) present, were also completed. Results from all three types of experiments were modeled using an extended constant- capacitance surface complexation model. The reactions [Formula: see text] [Formula: see text] and [Formula: see text] best described Cd(II) adsorption onto goethite, while [Formula: see text] and [Formula: see text] best described Cd(II) adsorption onto kaolinite. A combination of the first, second, and fourth of these reactions best fitted the data for Cd(II) adsorption onto illite. In each case the model fitted all experimental data well. The results suggest that adsorption onto the variable charge (SOH) sites on illite more closely resembles adsorption onto goethite than onto kaolinite.

A prospective radiological study of 96 patients with mandibular condylar neck fractures was undertaken to assess frequency and nature of mandibular condylar displacement. Data collected included age, gender, aetiology, and anatomical site of fracture and direction of displacement. Men aged 20-29 years sustained the majority of condylar fractures. Assault was the major cause of condylar fracture, followed by motor vehicle accidents and sport accidents. No anterior or posterior displacement of the condyle was noted. Medial displacement of the superior fragment was most frequently observed.

The possibility that personal lead shielding devices can increase absorption of radiation has not been entertained. The purpose of the present investigation specifically was to determine whether pituitary dose might be increased when a leaded apron and thyroid collar are used. Thermoluminescent dosimeters (TLDs) were used to measure absorbed dose. They were calibrated at the kVp used in the clinical situation and a calibration curve relating light output to dose was generated. Lithium fluoride TLD discs were placed in the pituitary gland region of a Rando-Alderson female human phantom. The equivalent of 100 transpharyngeal exposures were delivered. The resultant light output from recovered dosimeters was converted to a uGy value using the calibration curve. The experiment was repeated using a 0.25 mm lead equivalent collar and apron fitted to the phantom in the customary manner. The entire process was repeated in order to have 30 dosimeters for the unshielded and 30 dosimeters for the shielded conditions. A further 30 dosimeters were sham irradiated and served as controls. A statistical comparison between unshielded and shielded conditions was performed. When the leaded apron and thyroid collar were used the absorbed dose to the pituitary gland was increased significantly (P < 0.05). Following this a second group, using a different dosimetry system and a male phantom repeated the experiment. In both cases, the shielded phantom received significantly higher dose to the pituitary region than the unshielded.

The purpose of this study was to assess a possible colour shift in the root surfaces of adult human teeth and if so, whether this colour change is related to chronological age. Teeth extracted from persons of known age and gender were obtained from Ontario dental practitioners and grouped into five-year age ranges. Three experiments were undertaken: (1) to identify a possible difference in yellow colouration between the four surfaces of tooth roots (mesial, distal, lingual, and buccal), (2) to investigate the difference in yellow colouration of tooth roots between non-molar teeth and molar teeth and (3) to assess the correlation between the age of teeth and root colour saturation for yellow, magenta, cyan and black. The teeth in all investigations were scanned by a flat-bed digital colour scanner with a Kodak colour scale control and viewed on a colour computer monitor. In the first two experiments the yellow colour saturation of the root surfaces was measured at six points on each root using Photoshop 5.0 software. A significant difference was observed in the percentage yellow colour saturation between the mesial and the other three anatomical surfaces (p < 0.01), and between the root surfaces of non-molar and molar teeth (p < 0.01) (ANOVA with Bonferroni post-test). The authors then randomly assigned tooth surfaces to select an equivalent number of posterior and anterior teeth in the study, assessing the relationship between age and root colouration. Four points of colour measurement on 40 teeth (sample size permitting, see Table 1) for each known age and gender were assessed for colour saturation (cyan, magenta, yellow and black). The correlation of chronological age to colour saturation was linear for all colours, with correlation coefficients ranging from r = 0.81 to r = 0.94. The high correlation values strongly support the conclusion that chronological age is related to increased root colouration.