Mechanistic Insights into Bioengineered Antibiofilm Enamel Pellicles.

Dental caries remains the most widespread chronic disease worldwide. Basically, caries originates within biofilms accumulated on dental enamel. Despite the nonrenewable nature of the enamel tissue, targeted preventive strategies are still very limited. We previously introduced customized multifunctional proteinaceous pellicles (coatings) for controlling bacterial attachment and subsequent biofilm succession. Stemmed from our whole proteome/peptidome analysis of the in vivo acquired enamel pellicle, we designed these pellicles using hybrid mixtures of the most abundant and complementary-acting antimicrobial and antifouling proteins/peptides for synergetic suppression of early biofilms. In conjugating these domains synthetically, their bioinhibitory efficacy was remarkably boosted. Herein, we sought to explore the key structure-function relationship of these potent de novo hybridized conjugates in comparison with their individual domains, solely or in physical mixtures. Specifically, we interrelated the following facets: physicochemical and 3-dimensional folding characteristics via molecular dynamics simulations, adopted secondary structure by circular dichroism, immobilization capacity on enamel through high-spatial resolution multiphoton microscopy, and biofilm suppression potency. Our data showed consistent associations among the increased preference for protein folding structures, α-helix content, and enamel-immobilization capacity; all were inversely correlated with the attached bioburden. The expressed phenotypes could be explained by the adopted strongly amphipathic helical conformation upon conjugation, mediated by the highly anionic and acidic N-terminal pentapeptide shared region/motif for enhanced immobilization on enamel. In conclusion, conjugating bioactive proteins/peptides is a novel translational approach to engineer robust antibiofilm pellicles for caries prevention. The adopted α-helical conformation is key to enhance the antibiofilm efficacy and immobilization capacity on enamel that are promoted by certain physicochemical properties of the constituent domains. These data are valuable for bioengineering versatile therapeutics to prevent/arrest dental caries, a condition that otherwise requires invasive treatments with substantial health care expenditures.

Sensitivity of the Kß″ X-ray Emission Line to Coordination Changes in GeO2 and TiO2.

The hard X-ray Kß″ emission line shows sensitivity with respect to a wide range of cation-ligand coordination, which we investigate in the cases of GeO2 and TiO2 on the basis of ab initio spectral calculations on amorphous and crystalline structures. In compressed amorphous GeO2, the sampling of a large number of instantaneous coordination polyhedra from an ab initio molecular dynamics trajectory reveals that the functional relation between the Kß″ shift and coordination is close to linear between 4-fold and 7-fold coordination. A similar sensitivity of the Kß″ emission line exists in the coordination range between six and nine of crystalline high-pressure TiO2 polymorphs. Our results demonstrate the potential of the Kß″ emission line in research on the structure of amorphous oxide material.

Extracellular vimentin mimics VEGF and is a target for anti-angiogenic immunotherapy.

Anti-angiogenic cancer therapies possess immune-stimulatory properties by counteracting pro-angiogenic molecular mechanisms. We report that tumor endothelial cells ubiquitously overexpress and secrete the intermediate filament protein vimentin through type III unconventional secretion mechanisms. Extracellular vimentin is pro-angiogenic and functionally mimics VEGF action, while concomitantly acting as inhibitor of leukocyte-endothelial interactions. Antibody targeting of extracellular vimentin shows inhibition of angiogenesis in vitro and in vivo. Effective and safe inhibition of angiogenesis and tumor growth in several preclinical and clinical studies is demonstrated using a vaccination strategy against extracellular vimentin. Targeting vimentin induces a pro-inflammatory condition in the tumor, exemplified by induction of the endothelial adhesion molecule ICAM1, suppression of PD-L1, and altered immune cell profiles. Our findings show that extracellular vimentin contributes to immune suppression and functions as a vascular immune checkpoint molecule. Targeting of extracellular vimentin presents therefore an anti-angiogenic immunotherapy strategy against cancer.

Heterogenous bone response to biologic DMARD therapies in rheumatoid arthritis patients and their relationship to functional indices.

Objectives: Previous studies of high-resolution peripheral quantitative computed tomography (HR-pQCT) imaging of hand joints in patients with rheumatoid arthritis (RA) have suggested that erosion healing may occur. Our objective was to examine changes in erosion volume, joint space width (JSW), bone mineral density (BMD), and bone remodelling, and their association with clinical outcomes and measures of patient hand function.Method: We examined 48 patients who achieved a good response to a newly initiated biologic therapy. HR-pQCT images of the dominant hands' second and third metacarpophalangeal joints were obtained 3 and 12 months after therapy initiation. Bone erosion volume, JSW, BMD, and bone remodelling were quantified from HR-pQCT images, with improvement, no change (unchanged), or progression in these measures determined by least significant change. Disease activity and hand function measures were collected.Results: There were no significant group changes in HR-pQCT outcomes over the 9 month period. Twenty-two patients had total erosion volumes that remained unchanged, nine showed improvement, and two progressed. The majority of JSW and BMD measures remained unchanged. There was a significant association between the baseline Health Assessment Questionnaire score and the change in minimum JSW, but no other significant associations between HR-pQCT outcomes and function were observed.Conclusions: The vast majority of patients maintained unchanged JSW and BMD over the course of follow-up. Significant improvements in total erosion volume occurred in 27% of patients, suggesting that biologic therapies may lead to erosion healing in some patients, although this did not have an impact on self-reported and demonstrated hand function.

Integrating Phenotypic Search and Phosphoproteomic Profiling of Active Kinases for Optimization of Drug Mixtures for RCC Treatment.

Combined application of multiple therapeutic agents presents the possibility of enhanced efficacy and reduced development of resistance. Definition of the most appropriate combination for any given disease phenotype is challenged by the vast number of theoretically possible combinations of drugs and doses, making extensive empirical testing a virtually impossible task. We have used the streamlined-feedback system control (s-FSC) technique, a phenotypic approach, which converges to optimized drug combinations (ODC) within a few experimental steps. Phosphoproteomics analysis coupled to kinase activity analysis using the novel INKA (integrative inferred kinase activity) pipeline was performed to evaluate ODC mechanisms in a panel of renal cell carcinoma (RCC) cell lines. We identified different ODC with up to 95% effectivity for each RCC cell line, with low doses (ED5-25) of individual drugs. Global phosphoproteomics analysis demonstrated inhibition of relevant kinases, and targeting remaining active kinases with additional compounds improved efficacy. In addition, we identified a common RCC ODC, based on kinase activity data, to be effective in all RCC cell lines under study. Combining s-FSC with a phosphoproteomic profiling approach provides valuable insight in targetable kinase activity and allows for the identification of superior drug combinations for the treatment of RCC.

Shielding commissioning factors in nuclear medicine facilities.

This article reviews the essential considerations in planning and designing nuclear medicine departments. There are four proposed categories to consider as 'shielding commissioning factors' (SCF). The first SCF: 'Patient flow optimisation and workload' emphasises the importance of carefully considering patient flow in the departmental design, which would impact the cost of the shielding and the management of radioactive patients. The second SCF: 'Equipment and space allocation' discusses the principles of space allocations in the department for cost-effective designs. The third SCF: 'Shielding calculation methods' reviews the methodologies of shielding calculations in nuclear medicine to offer a standardised approach. The fourth SCF: 'Shielding integrity' reviews the plan to inspect, eyewitness and verify shielding integrity. All discussions were supplemented by practical examples. Overall, this article aims to be a practical manual which health or medical physicists can use when providing counsels to the design committee.

Hybrid ligands with calixarene and thiodigalactoside groups: galectin binding and cytotoxicity.

Galectins have diverse functions and are involved in many biological processes because of their complex intra- and extracellular activities. Selective and potent inhibitors for galectins will be valuable tools to investigate the biological functions of these proteins. Therefore, we describe here the synthesis of galectin inhibitors with a potential "chelate effect". These compounds are designed to bind to two different binding sites on galectins simultaneously. In this paper a series of asymmetric "hybrid" compounds are prepared, which combine two galectin ligands (1) a substituted thiodigalactoside derivative and (2) an antagonist calixarene-based therapeutic agent. NMR spectroscopy was used to evaluate the interactions of these compounds with Galectin-1 and -3. In addition, cellular experiments were conducted to compare the cytotoxic effects of the hybrids with those of a calixarene derivative. While only the thiodigalactoside part of the hybrids showed strong binding, the calixarene part was responsible for observed cytoxoxicity effects, suggesting that the calixarene moiety may also be addressing a non-galectin target.

Position paper: recommendations for a digital mammography quality assurance program V4.0.

In 2001 the ACPSEM published a position paper on quality assurance in screen film mammography which was subsequently adopted as a basis for the quality assurance programs of both the Royal Australian and New Zealand College of Radiologists (RANZCR) and of BreastScreen Australia. Since then the clinical implementation of digital mammography has been realised and it has become evident that existing screen-film protocols were not appropriate to assure the required image quality needed for reliable diagnosis or to address the new dose implications resulting from digital technology. In addition, the advantages and responsibilities inherent in teleradiology are most critical in mammography and also need to be addressed. The current document is the result of a review of current overseas practice and local experience in these areas. At this time the technology of digital imaging is undergoing significant development and there is still a lack of full international consensus about some of the detailed quality control (QC) tests that should be included in quality assurance (QA) programs. This document describes the current status in digital mammography QA and recommends test procedures that may be suitable in the Australasian environment. For completeness, this document also includes a review of the QA programs required for the various types of digital biopsy units used in mammography. In the future, international harmonisation of digital quality assurance in mammography and changes in the technology may require a review of this document. Version 2.0 represented the first of these updates and key changes related to image quality evaluation, ghost image evaluation and interpretation of signal to noise ratio measurements. In Version 3.0 some significant changes, made in light of further experience gained in testing digital mammography equipment were introduced. In Version 4.0, further changes have been made, most notably digital breast tomosynthesis (DBT) testing and QC have been addressed. Some additional testing for conventional projection imaging has been added in order that sites may have the capability to undertake dose surveys to confirm compliance with diagnostic reference levels (DRLs) that may be established at the National or State level. A key recommendation is that dosimetry calculations are now to be undertaken using the methodology of Dance et al. Some minor changes to existing facility QC tests have been made to ensure the suggested procedures align with those most recently adopted by the Royal Australian and New Zealand College of Radiologists and BreastScreen Australia. Future updates of this document may be provided as deemed necessary in electronic format on the ACPSEM's website ( https://www.acpsem.org.au/whatacpsemdoes/standards-position-papers and see also http://www.ranzcr.edu.au/quality-a-safety/radiology/practice-quality-activities/mqap ).

A genomic screen for angiosuppressor genes in the tumor endothelium identifies a multifaceted angiostatic role for bromodomain containing 7 (BRD7).

Tumor angiogenesis is characterized by deregulated gene expression in endothelial cells (EC). While studies until now have mainly focused on overexpressed genes in tumor endothelium, we here describe the identification of transcripts that are repressed in tumor endothelium and thus have potential suppressive effects on angiogenesis. We identified nineteen putative angiosuppressor genes, one of them being bromodomain containing 7 (BRD7), a gene that has been assigned tumor suppressor properties. BRD7 was studied in more detail, and we demonstrate that BRD7 expression is inversely related to EC activation. Ectopic expression of BRD7 resulted in a dramatic reduction of EC proliferation and viability. Furthermore, overexpression of BRD7 resulted in a bromodomain-dependent induction of NFκB-activity and NFκB-dependent gene expression, including ICAM1, enabling leukocyte-endothelial interactions. In silico functional annotation analysis of genome-wide expression data on BRD7 knockdown and overexpression revealed that the transcriptional signature of low BRD7 expressing cells is associated with increased angiogenesis (a.o. upregulation of angiopoietin-2, VEGF receptor-1 and neuropilin-1), cytokine activity (a.o. upregulation of CXCL1 and CXCL6), and a reduction of immune surveillance (TNF-α, NFκB, ICAM1). Thus, combining in silico and in vitro data reveals multiple pathways of angiosuppressor and anti-tumor activities of BRD7.

Apelin: A putative novel predictive biomarker for bevacizumab response in colorectal cancer.

Bevacizumab (bvz) is currently employed as an anti-angiogenic therapy across several cancer indications. Bvz response heterogeneity has been well documented, with only 10-15% of colorectal cancer (CRC) patients benefitting in general. For other patients, clinical efficacy is limited and side effects are significant. This reinforces the need for a robust predictive biomarker of response. To identify such a biomarker, we performed a DNA microarray-based transcriptional profiling screen with primary endothelial cells (ECs) isolated from normal and tumour colon tissues. Thirteen separate populations of tumour-associated ECs and 10 of normal ECs were isolated using fluorescence-activated cell sorting. We hypothesised that VEGF-induced genes were overexpressed in tumour ECs; these genes could relate to bvz response and serve as potential predictive biomarkers. Transcriptional profiling revealed a total of 2,610 differentially expressed genes when tumour and normal ECs were compared. To explore their relation to bvz response, the mRNA expression levels of top-ranked genes were examined using quantitative PCR in 30 independent tumour tissues from CRC patients that received bvz in the adjuvant setting. These analyses revealed that the expression of MMP12 and APLN mRNA was significantly higher in bvz non-responders compared to responders. At the protein level, high APLN expression was correlated with poor progression-free survival in bvz-treated patients. Thus, high APLN expression may represent a novel predictive biomarker for bvz unresponsiveness.

Combination of ruthenium(II)-arene complex [Ru(η6-p-cymene)Cl2(pta)] (RAPTA-C) and the epidermal growth factor receptor inhibitor erlotinib results in efficient angiostatic and antitumor activity.

Ruthenium-based compounds show strong potential as anti-cancer drugs and are being investigated as alternatives to other well-established metal-based chemotherapeutics. The organometallic compound [Ru(η6-p-cymene)Cl2(pta)], where pta = 1,3,5-triaza-7-phosphaadamantane (RAPTA-C) exhibits broad acting anti-tumor efficacy with intrinsic angiostatic activity. In the search for an optimal anti-angiogenesis drug combination, we identified synergistic potential between RAPTA-C and the epidermal growth factor receptor (EGFR) inhibitor, erlotinib. This drug combination results in strong synergistic inhibition of cell viability in human endothelial (ECRF24 and HUVEC) and human ovarian carcinoma (A2780 and A2780cisR) cells. Additionally, erlotinib significantly enhances the cellular uptake of RAPTA-C relative to treatment with RAPTA-C alone in human ovarian carcinoma cells, but not endothelial cells. Drug combinations induce the formation of chromosome bridges that persist after mitotic exit and delay abscission in A2780 and A2780cisR, therefore suggesting initiation of cellular senescence. The therapeutic potential of these compounds and their combination is further validated in vivo on A2780 tumors grown on the chicken chorioallantoic membrane (CAM) model, and in a preclinical model in nude mice. Immunohistochemical analysis confirms effective anti-angiogenic and anti-proliferative activity in vivo, based on a significant reduction of microvascular density and a decrease in proliferating cells.

A key role for galectin-1 in sprouting angiogenesis revealed by novel rationally designed antibodies.

Galectins are carbohydrate binding proteins that function in many key cellular processes. We have previously demonstrated that galectins are essential for tumor angiogenesis and their expression is associated with disease progression. Targeting galectins is therefore a potential anti-angiogenic and anti-cancer strategy. Here, we used a rational approach to generate antibodies against a specific member of this conserved protein family, i.e. galectin-1. We characterized two novel mouse monoclonal antibodies that specifically react with galectin-1 in human, mouse and chicken. We demonstrate that these antibodies are excellent tools to study galectin-1 expression and function in a broad array of biological systems. In a potential diagnostic application, radiolabeled antibodies showed specific targeting of galectin-1 positive tumors. In a therapeutic setting, the antibodies inhibited sprouting angiogenesis in vitro and in vivo, underscoring the key function of galectin-1 in this process.

Mechanisms for pressure-induced crystal-crystal transition, amorphization, and devitrification of SnI4.

The pressure-induced amorphization and subsequent recrystallization of SnI4 have been investigated using first principles molecular dynamics calculations together with high-pressure (119)Sn nuclear resonant inelastic x-ray scattering measurements. Above ∼8 GPa, we observe a transformation from an ambient crystalline phase to an intermediate crystal structure and a subsequent recrystallization into a cubic phase at ∼64 GPa. The crystalline-to-amorphous transition was identified on the basis of elastic compatibility criteria. The measured tin vibrational density of states shows large amplitude librations of SnI4 under ambient conditions. Although high pressure structures of SnI4 were thought to be determined by random packing of equal-sized spheres, we detected electron charge transfer in each phase. This charge transfer results in a crystal structure packing determined by larger than expected iodine atoms.

Rapid optimization of drug combinations for the optimal angiostatic treatment of cancer.

Drug combinations can improve angiostatic cancer treatment efficacy and enable the reduction of side effects and drug resistance. Combining drugs is non-trivial due to the high number of possibilities. We applied a feedback system control (FSC) technique with a population-based stochastic search algorithm to navigate through the large parametric space of nine angiostatic drugs at four concentrations to identify optimal low-dose drug combinations. This implied an iterative approach of in vitro testing of endothelial cell viability and algorithm-based analysis. The optimal synergistic drug combination, containing erlotinib, BEZ-235 and RAPTA-C, was reached in a small number of iterations. Final drug combinations showed enhanced endothelial cell specificity and synergistically inhibited proliferation (p < 0.001), but not migration of endothelial cells, and forced enhanced numbers of endothelial cells to undergo apoptosis (p < 0.01). Successful translation of this drug combination was achieved in two preclinical in vivo tumor models. Tumor growth was inhibited synergistically and significantly (p < 0.05 and p < 0.01, respectively) using reduced drug doses as compared to optimal single-drug concentrations. At the applied conditions, single-drug monotherapies had no or negligible activity in these models. We suggest that FSC can be used for rapid identification of effective, reduced dose, multi-drug combinations for the treatment of cancer and other diseases.

Angiostatic treatment prior to chemo- or photodynamic therapy improves anti-tumor efficacy.

Tumor vasculature is known to be poorly organized leading to increased leakage of molecules to the extravascular space. This process can potentially increase interstitial fluid pressure impairing intra-tumoral blood flow and oxygen supply, and can affect drug uptake. Anti-angiogenic therapies are believed to reduce vascular permeability, potentially reducing interstitial fluid pressure and improving the extravasation of small molecule-based chemotherapeutics. Here we show that pretreatment of human ovarian carcinoma tumors with sub-optimal doses of the VEGFR targeting tyrosine kinase inhibitor axitinib, but not the EGFR targeting kinase inhibitor erlotinib, induces a transient period of increased tumor oxygenation. Doxorubicin administered within this window was found to enter the extravascular tumor space more rapidly compared to doxorubicin when applied alone or outside this time window. Treatment with the chemotherapeutics, doxorubicin and RAPTA-C, as well as applying photodynamic therapy during this period of elevated oxygenation led to enhanced tumor growth inhibition. Improvement of therapy was not observed when applied outside the window of increased oxygenation. Taken together, these findings further confirm the hypothesis of angiostasis-induced vascular normalization and also help to understand the interactions between anti-angiogenesis and other anti-cancer strategies.

Rituximab: an emerging treatment for recurrent diffuse alveolar hemorrhage in systemic lupus erythematosus.

Diffuse alveolar hemorrhage (DAH) is a rare manifestation of systemic lupus erythematosus (SLE) and is associated with high mortality rates. Treatment typically consists of aggressive immunosuppression with pulse-dose steroids, cyclophosphamide, and plasma exchange therapy. Mortality rates remain high despite use of multiple medical therapies. We present a case of recurrent DAH in a 52-year-old female with SLE after a deceased donor renal transplant who was successfully treated with rituximab. Our report highlights the pathophysiologic importance of B-cell-mediated immunosuppression in SLE-associated DAH and suggests that rituximab may represent a viable alternative to cyclophosphamide in the treatment of this disease. We also review eight other reported cases of rituximab use in SLE-associated DAH.

Novel characterization of bEnd.3 cells that express lymphatic vessel endothelial hyaluronan receptor-1.

Murine bEnd.3 endothelioma cell line has been widely used in vascular research and here we report the novel finding that bEnd.3 cells express lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and vascular endothelial growth factor receptor-3 (VEGFR-3). Moreover, these cells express progenitor cell markers of Sca-1 and CD133. Upon stimulation with tumor necrosis factor-alpha (TNF-alpha), the bEnd.3 cells demonstrate enhanced formation of capillary-type tubes, which express LYVE-1. As the bEnd.3 cell line is derived from murine endothelioma, we further examined human tissues of endothelioma and identified lymphatic vessels in the tumor samples which express both LYVE-1 and podoplanin. Moreover, a significantly higher number of lymphatic vessels were detected in the endothelioma samples compared with normal control. Taken together, this study not only redefines bEnd.3 cells for vascular research, but also indicates a broader category of human diseases that are associated with lymphatics, such as endothelioma.