Skin Dendritic Cell Targeting via Microneedle Arrays Laden with Antigen-Encapsulated Poly-D, L-lactide-co-Glycolide Nanoparticles Induces Efficient Antitumor and Antiviral Immune Responses

The efficacious delivery of antigens to antigen-presenting cells (APCs), in particular, to dendritic cells (DCs), and their subsequent activation remains a significant challenge in the development of effective vaccines. This study highlights the potential of dissolving microneedle (MN) arrays laden with nanoencapsulated antigen to increase vaccine immunogenicity by targeting antigen specifically to contiguous DC networks within the skin. Following in situ uptake, skin-resident DCs were able to deliver antigen-encapsulated poly-d,l-lactide-co-glycolide (PGLA) nanoparticles to cutaneous draining lymph nodes where they subsequently induced significant expansion of antigen-specific T cells. Moreover, we show that antigen-encapsulated nanoparticle vaccination via microneedles generated robust antigen-specific cellular immune responses in mice. This approach provided complete protection in vivo against both the development of antigen-expressing B16 melanoma tumors and a murine model of para-influenza, through the activation of antigen-specific cytotoxic CD8(+) T cells that resulted in efficient clearance of tumors and virus, respectively. In addition, we show promising findings that nanoencapsulation facilitates antigen retention into skin layers and provides antigen stability in microneedles. Therefore, the use of biodegradable polymeric nanoparticles for selective targeting of antigen to skin DC subsets through dissolvable MNs provides a promising technology for improved vaccination efficacy, compliance, and coverage.

Domain configuration of permalloy ellipses in a rotating magnetic field

The domain configuration of micron-sized permalloy ellipses was studied under the influence of an in-plane rotating magnetic field using magnetic force microscopy. The field amplitude was chosen such that when the field is applied parallel to the long axis of the ellipses they are saturated, but when the field is perpendicular to the long axis they exhibit multi-domain states. The rotation angle for nucleation and annihilation of domains was determined for different magnitudes of the applied magnetic field and for two different lateral sizes of ellipses, 6 Am x 2 Am and 3 Am x 1 Am. It was found that both nucleation and annihilation occur over a range of angles for both lateral sizes of ellipses. Saturated states are stable for a wider range of angles for larger values of the applied field.

Exotic Domain States in Ferroelectrics: Searching for Vortices and Skyrmions

Very recent experimental and theoretical work has sought to find out whether or not complex arrangements of dipoles (such as flux-closure, vortex or skyrmion patterns) exist in ferroelectrics. While there is undoubtedly still considerable work to be done, enough insight has now been gained to warrant a brief discussion of progress to date. This review article attempts to undertake such a discussion.

Multiferroic domain dynamics in strained strontium titanate

Multiferroicity can be induced in strontium titanate by applying biaxial strain. Using optical second harmonic generation, we report a transition from 4/mmm to the ferroelectric mm2 phase, followed by a transition to a ferroelastic-ferroelectric mm2 phase in a strontium titanate thin film. Piezoelectric force microscopy is used to study ferroelectric domain switching. Second harmonic generation, combined with phase-field modeling, is used to reveal the mechanism of coupled ferroelectric-ferroelastic domain wall motion. These studies have relevance to multiferroics with coupled polar and axial phenomena.

Substrate Clamping Effects on Irreversible Domain Wall Dynamics in Lead Zirconate Titanate Thin Films

The role of long-range strain interactions on domain wall dynamics is explored through macroscopic and local measurements of nonlinear behavior in mechanically clamped and released polycrystalline lead zirconate-titanate (PZT) films. Released films show a dramatic change in the global dielectric nonlinearity and its frequency dependence as a function of mechanical clamping. Furthermore, we observe a transition from strong clustering of the nonlinear response for the clamped case to almost uniform nonlinearity for the released film. This behavior is ascribed to increased mobility of domain walls. These results suggest the dominant role of collective strain interactions mediated by the local and global mechanical boundary conditions on the domain wall dynamics. The work presented in this Letter demonstrates that measurements on clamped films may considerably underestimate the piezoelectric coefficients and coupling constants of released structures used in microelectromechanical systems, energy harvesting systems, and microrobots.

Stripe domain structure in epitaxial (001) BiFeO3 thin films on orthorhombic TbScO3 substrate

We have analyzed the ferroelastic and ferroelectric domain structure of high crystalline quality (001) BiFeO3 films on orthorhombic (110) TbScO3 substrates. Two domains were present in stripes separated by (010) vertical boundaries, with spontaneous polarizations in adjacent domains rotated by 109 degrees. The striped morphology was caused by nucleation of only two ferroelastic domains on the low symmetry GdFeO3-type substrate. Domain engineering through substrate symmetry is an important finding for rhombohedral ferroelectric epitaxial thin films. The stripe pattern with vertical walls may be useful for extracting domain wall contributions to magnetism and electrical transport properties of BiFeO3 materials.

Targeting distributed systems in FastFlow

FastFlow is a structured parallel programming framework targeting shared memory multi-core architectures. In this paper we introduce a FastFlow extension aimed at supporting also a network of multi-core workstations. The extension supports the execution of FastFlow programs by coordinating-in a structured way-the fine grain parallel activities running on a single workstation. We discuss the design and the implementation of this extension presenting preliminary experimental results validating it on state-of-the-art networked multi-core nodes. © 2013 Springer-Verlag.

The Interlaboratory Robustness Of Next-Generation Sequencing (IRON) Study Phase II: Deep-Sequencing Analyses Of Hematological Malignancies Performed In 8,867 Cases By An International Network Involving 27 Laboratories

Introduction: Amplicon deep-sequencing using second-generation sequencing technology is an innovative molecular diagnostic technique and enables a highly-sensitive detection of mutations. As an international consortium we had investigated previously the robustness, precision, and reproducibility of 454 amplicon next-generation sequencing (NGS) across 10 laboratories from 8 countries (Leukemia, 2011;25:1840-8).

Aims: In Phase II of the study, we established distinct working groups for various hematological malignancies, i.e. acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and multiple myeloma. Currently, 27 laboratories from 13 countries are part of this research consortium. In total, 74 gene targets were selected by the working groups and amplicons were developed for a NGS deep-sequencing assay (454 Life Sciences, Branford, CT). A data analysis pipeline was developed to standardize mutation interpretation both for accessing raw data (Roche Amplicon Variant Analyzer, 454 Life Sciences) and variant interpretation (Sequence Pilot, JSI Medical Systems, Kippenheim, Germany).

Results: We will report on the design, standardization, quality control aspects, landscape of mutations, as well as the prognostic and predictive utility of this assay in a cohort of 8,867 cases. Overall, 1,146 primer sequences were designed and tested. In detail, for example in AML, 924 cases had been screened for CEBPA mutations. RUNX1 mutations were analyzed in 1,888 cases applying the deep-sequencing read counts to study the stability of such mutations at relapse and their utility as a biomarker to detect residual disease. Analyses of DNMT3A (n=1,041) were focused to perform landscape investigations and to address the prognostic relevance. Additionally, this working group is focusing on TET2, ASXL1, and TP53 analyses. A novel prognostic model is being developed allowing stratification of AML into prognostic subgroups based on molecular markers only. In ALL, 1,124 pediatric and adult cases have been screened, including 763 assays for TP53 mutations both at diagnosis and relapse of ALL. Pediatric and adult leukemia expert labs developed additional content to study the mutation incidence of other B and T lineage markers such as IKZF1, JAK2, IL7R, PAX5, EP300, LEF1, CRLF2, PHF6, WT1, JAK1, PTEN, AKT1, IL7R, NOTCH1, CREBBP, or FBXW7. Further, the molecular landscape of CLL is changing rapidly. As such, a separate working group focused on analyses including NOTCH1, SF3B1, MYD88, XPO1, FBXW7 and BIRC3. Currently, 922 cases were screened to investigate the range of mutational burden of NOTCH1 mutations for their prognostic relevance. In MDS, RUNX1 mutation analyses were performed in 977 cases. The prognostic relevance of TP53 mutations in MDS was assessed in additional 327 cases, including isolated deletions of chromosome 5q. Next, content was developed targeting genes of the cellular splicing component, e.g. SF3B1, SRSF2, U2AF1, and ZRSR2. In BCR-ABL1-negative MPN, nine genes of interest (JAK2, MPL, TET2, CBL, KRAS, EZH2, IDH1, IDH2, ASXL1) have been analyzed in a cohort of 155 primary myelofibrosis cases searching for novel somatic mutations and addressing their relevance for disease progression and leukemia transformation. Moreover, an assay was developed and applied to CMML cases allowing the simultaneous analysis of 25 leukemia-associated target genes in a single sequencing run using just 20 ng of starting DNA. Finally, nine laboratories are studying CML, applying ultra-deep sequencing of the BCR-ABL1 tyrosine kinase domain. Analyses were performed on 615 cases investigating the dynamics of expansion of mutated clones under various tyrosine kinase inhibitor therapies.

Conclusion: Molecular characterization of hematological malignancies today requires high diagnostic sensitivity and specificity. As part of the IRON-II study, a network of laboratories analyzed a variety of disease entities applying amplicon-based NGS assays. Importantly, the consortium not only standardized assay design for disease-specific panels, but also achieved consensus on a common data analysis pipeline for mutation interpretation. Distinct working groups have been forged to address scientific tasks and in total 8,867 cases had been analyzed thus far.

Comparison of Wavelet Transform and Time-Domain Analysis of Second Trimester Uterine Artery Doppler Waveforms in Screening for Pre-Eclampsia

The aim of this study was to compare time-domain waveform analysis of second-trimester uterine artery Doppler using the resistance index (RI) with waveform analysis using a mathematical tool known as wavelet transform for the prediction of pre-eclampsia (PE). This was a retrospective, nested case-cohort study of 336 women, 37 of whom subsequently developed PE. Uterine artery Doppler waveforms were analysed using both RI and waveform analysis. The utility of these indices in screening for PE was then evaluated using receiver operating characteristic curves. There were significant differences in uterine artery RI between the PE women and those with normal pregnancy outcome. After wavelet analysis, significant difference in the mean amplitude in wavelet frequency band 4 was noted between the 2 groups. The sensitivity for both Doppler RI and frequency band 4 for the detection of PE at a 10% false-positive rate was 45%. This small study demonstrates the application of wavelet transform analysis of uterine artery Doppler waveforms in screening for PE. Further prospective studies are needed in order to clearly define if this analytical approach to waveform analysis may have the potential to improve the detection of PE by uterine artery Doppler screening.

Targeting EGFR Induced Oxidative Stress by PARP1 Inhibition in Glioblastoma Therapy

Despite the critical role of Epidermal Growth Factor Receptor (EGFR) in glioblastoma pathogenesis [1,2], EGFR targeted therapies have achieved limited clinical efficacy [3]. Here we propose an alternate therapeutic strategy based on the conceptual framework of non-oncogene addiction [4,5]. A directed RNAi screen revealed that glioblastoma cells overexpressing EGFRvIII [6], an oncogenic variant of EGFR, become hyper-dependent on a variety of DNA repair genes. Among these, there was an enrichment of Base Excision Repair (BER) genes required for the repair of Reactive Oxygen Species (ROS)-induced DNA damage, including poly-ADP ribose polymerase 1 (PARP1). Subsequent studies revealed that EGFRvIII overexpression in glioblastoma cells caused increased levels of ROS, DNA strand break accumulation, and genome instability. In a panel of primary glioblastoma lines, sensitivity to PARP1 inhibition correlated with the levels of EGFR activation and oxidative stress. Gene expression analysis indicated that reduced expression of BER genes in glioblastomas with high EGFR expression correlated with improved patient survival. These observations suggest that oxidative stress secondary to EGFR hyperactivation necessitates increased cellular reliance on PARP1 mediated BER, and offer critical insights into clinical trial design.

The Novel Tubulin-Targeting Agent Pyrrolo-1,5-Benzoxazepine-15 Induces Apoptosis in Poor Prognostic Subgroups of Chronic Lymphocytic Leukemia

Pyrrolo-1,5-benzoxazepine-15 (PBOX-15) is a novel microtubule depolymerization agent that induces cell cycle arrest and subsequent apoptosis in a number of cancer cell lines. Chronic lymphocytic leukemia (CLL) is characterized by clonal expansion of predominately nonproliferating mature B cells. Here, we present data suggesting PBOX-15 is a potential therapeutic agent for CLL. We show activity of PBOX-15 in samples taken from a cohort of CLL patients (n = 55) representing both high-risk and low-risk disease. PBOX-15 exhibited cytotoxicity in CLL cells (n = 19) in a dose-dependent manner, with mean IC(50) of 0.55 mu mol/L. PBOX-15 significantly induced apoptosis in CLL cells (n = 46) including cells with poor prognostic markers: unmutated IgV(II) genes, CD38 and zeta-associated protein 70 (ZAP-70) expression, and fludarabine-resistant cells with chromosomal deletions in 17p. In addition, PBOX-15 was more potent than fludarabine in inducing apoptosis in fludarabine-sensitive cells. Pharmacologic inhibition and small interfering RNA knockdown of caspase-8 significantly inhibited PBOX-15-induced apoptosis. Pharmacologic inhibition of c-jun NH(2)-terminal kinase inhibited PBOX-15-induced apoptosis in mutated IgV(II) and ZAP-70(-) CLL cells but not in unmutated IgV(II) and ZAP-70(+) cells. PBOX-15 exhibited selective cytotoxicity in CLL cells compared with normal hematopoietic cells. Our data suggest that PBOX-15 represents a novel class of agents that are toxic toward both high-risk and low-risk CLL cells. The need for novel treatments is acute in CLL, especially for the subgroup of patients with poor clinical outcome and drug-resistant disease. This study identifies a novel agent with significant clinical potential.

PBOX-15, a novel microtubule targeting agent, induces apoptosis, upregulates death receptors, and potentiates TRAIL-mediated apoptosis in multiple myeloma cells

Background: In recent years, much progress has been made in the treatment of multiple myeloma. However, a major limitation of existing chemotherapeutic drugs is the eventual emergence of resistance; hence, the development of novel agents with new mechanisms of action is pertinent. Here, we describe the activity and mechanism of action of pyrrolo-1,5-benzoxazepine-15 (PBOX-15), a novel microtubule-targeting agent, in multiple myeloma cells.

Methods: The anti-myeloma activity of PBOX-15 was assessed using NCI-H929, KMS11, RPMI8226, and U266 cell lines, and primary myeloma cells. Cell cycle distribution, apoptosis, cytochrome c release, and mitochondrial inner membrane depolarisation were analysed by flow cytometry; gene expression analysis was carried out using TaqMan Low Density Arrays; and expression of caspase-8 and Bcl-2 family of proteins was assessed by western blot analysis.

Results: Pyrrolo-1,5-benzoxazepine-15 induced apoptosis in ex vivo myeloma cells and in myeloma cell lines. Death receptor genes were upregulated in both NCI-H929 and U266 cell lines, which displayed the highest and lowest apoptotic responses, respectively, following treatment with PBOX-15. The largest increase was detected for the death receptor 5 (DR5) gene, and cotreatment of both cell lines with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), the DR5 ligand, potentiated the apoptotic response. In NCI-H929 cells, PBOX-15-induced apoptosis was shown to be caspase-8 dependent, with independent activation of extrinsic and intrinsic apoptotic pathways. A caspase-8-dependent decrease in expression of Bim(EL) preceded downregulation of other Bcl-2 proteins (Bid, Bcl-2, Mcl-1) in PBOX-15-treated NCI-H929 cells.

Conclusion: PBOX-15 induces apoptosis and potentiates TRAIL-induced cell death in multiple myeloma cells. Thus, PBOX-15 represents a promising agent, with a distinct mechanism of action, for the treatment of this malignancy. British Journal of Cancer (2011) 104, 281-289. doi: 10.1038/sj.bjc.6606035 www.bjcancer.com Published online 21 December 2010 (C) 2011 Cancer Research UK

Targeting treatment resistant breast cancer stem cells with FKBPL and its peptide derivative, AD-01, via the CD44 pathway

PURPOSE: FKBPL and its peptide derivative, AD-01, have already demonstrated tumour growth inhibition and CD44 dependent anti-angiogenic activity. Here we explore the ability of AD-01 to target CD44 positive breast cancer stem cells (BCSCs). EXPERIMENTAL DESIGN: Mammosphere assays and flow cytometry were utilized to analyse the effect of FKBPL overexpression/knockdown and AD-01 treatment ± other anti-cancer agents on BCSCs using breast cancer cell lines (MCF-7/MDA-231/ZR-75), primary patient samples and xenografts. Delays in tumour initiation were evaluated in vivo. The anti-stem cell mechanisms were determined using clonogenic assays, qPCR and immunofluorescence. RESULTS: AD-01 treatment was highly effective at inhibiting the BCSC population by reducing mammosphere forming efficiency (MFE) and ESA+/CD44+/CD24- or ALDH+ cell subpopulations in vitro and tumour initiation in vivo. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed; mammospheres were completely eradicated by the third generation. The mechanism appears to be due to AD-01-mediated BCSC differentiation demonstrated by a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones; the stem cell markers, Nanog, Oct4 and Sox2, were also significantly reduced. Furthermore, we demonstrated additive inhibitory effects when AD-01 was combined with the Notch inhibitor, DAPT. AD-01 was also able to abrogate a chemo- and radiotherapy induced enrichment in BCSCs. Finally, FKBPL knockdown led to an increase in Nanog/Oct4/Sox2 and an increase in BCSCs, highlighting a role for endogenous FKBPL in stem cell signalling. CONCLUSIONS: AD-01 has dual anti-angiogenic and anti-BCSC activity which will be advantageous as this agent enters clinical trial.