Diagnostic value of in situ polymerase chain reaction in childhood leprosy

Objective: Our aim was to assess the diagnostic value of in situ polymerase chain reaction (PCR) in leprosy, particularly for enhancing histopathological diagnosis. Methods: We prospectively studied 20 children (aged <16 years) with leprosy. Clinical examination of each case was performed, and skin smear for acid-fast bacillus was prepared. A biopsy of the lesion site was performed for histopathological examination and in situ PCR testing. Results: Histopathological examination confirmed the clinical diagnosis in only 45% of the cases; nonspecific histopathology was reported for the remaining 55% of the cases. In situ PCR showed a positivity of 57.1% in the early/localized form of leprosy (indeterminate/borderline tuberculoid) and 61.5% in the borderline borderline/borderline lepromatous group. When compared with the histopathological examination, a significant enhancement of 15% in diagnosis was seen. With in situ PCR, the diagnosis could be confirmed in 4 (36.3%) of 11 cases with nonspecific histopathological features (which is common in early disease) in addition to confirmation of 8 (88.8%) of 9 histopathologically confirmed tissue sections. Histopathology and in situ PCR combined together confirmed the diagnosis in 13 (65%) of the 20 cases. Conclusions: In situ PCR is an important diagnostic tool, especially in early and doubtful cases of leprosy

Integrative analyses identify modulators of response to neoadjuvant aromatase inhibitors in patients with early breast cancer

Introduction Aromatase inhibitors (AIs) are a vital component of estrogen receptor positive (ER+) breast cancer treatment. De novo and acquired resistance, however, is common. The aims of this study were to relate patterns of copy number aberrations to molecular and proliferative response to AIs, to study differences in the patterns of copy number aberrations between breast cancer samples pre- and post-AI neoadjuvant therapy, and to identify putative biomarkers for resistance to neoadjuvant AI therapy using an integrative analysis approach. Methods Samples from 84 patients derived from two neoadjuvant AI therapy trials were subjected to copy number profiling by microarray-based comparative genomic hybridisation (aCGH, n = 84), gene expression profiling (n = 47), matched pre- and post-AI aCGH (n = 19 pairs) and Ki67-based AI-response analysis (n = 39). Results Integrative analysis of these datasets identified a set of nine genes that, when amplified, were associated with a poor response to AIs, and were significantly overexpressed when amplified, including CHKA, LRP5 and SAPS3. Functional validation in vitro, using cell lines with and without amplification of these genes (SUM44, MDA-MB134-VI, T47D and MCF7) and a model of acquired AI-resistance (MCF7-LTED) identified CHKA as a gene that when amplified modulates estrogen receptor (ER)-driven proliferation, ER/estrogen response element (ERE) transactivation, expression of ER-regulated genes and phosphorylation of V-AKT murine thymoma viral oncogene homolog 1 (AKT1). Conclusions These data provide a rationale for investigation of the role of CHKA in further models of de novo and acquired resistance to AIs, and provide proof of concept that integrative genomic analyses can identify biologically relevant modulators of AI response

Hexanuclear Ln6L6 Complex Formation by using an Unsymmetric Ligand

Multinuclear, self‐assembled lanthanide complexes present clear opportunities as sensors and imaging agents. Despite the widely acknowledged potential of this class of supramolecule, synthetic and characterization challenges continue to limit systematic studies into their self‐assembly restricting the number and variety of lanthanide architectures reported relative to their transition metal counterparts. Here we present the first study evaluating the effect of ligand backbone symmetry on multinuclear lanthanide complex self‐assembly. Replacement of a symmetric ethylene linker with an unsymmetric amide at the centre of a homoditopic ligand governs formation of an unusual Ln6L6 complex with coordinatively unsaturated metal centres. The choice of triflate as a counterion, and the effect of ionic radii are shown to be critical for formation of the Ln6L6 complex. The atypical Ln6L6 architecture is characterized using a combination of mass spectrometry, luminescence, DOSY NMR and EPR spectroscopy measurements. Luminescence experiments support clear differences between comparable Eu6L6 and Eu2L3 complexes, with relatively short luminescent lifetimes and low quantum yields observed for the Eu6L6 structure indicative of non‐radiative decay processes. Synthesis of the Gd6L6analogue allows three distinct Gd···Gd distance measurements to be extracted using homo‐RIDME EPR experiments

The status of epidermal growth factor receptor in borderline ovarian tumours

The majority of borderline ovarian tumours (BOTs) behave in a benign fashion, but some may show aggressive behavior. The reason behind this has not been elucidated. The epidermal growth factor receptor (EGFR) is known to contribute to cell survival signals as well as metastatic potential of some tumours. EGFR expression and gene status have not been thoroughly investigated in BOTs as it has in ovarian carcinomas. In this study we explore protein expression as well as gene mutations and amplifications of EGFR in BOTs in comparison to a subset of other epithelial ovarian tumours. We studied 85 tumours, including 61 BOTs, 10 low grade serous carcinomas (LGSCs), 9 high grade serous carcinomas (HGSCs) and 5 benign epithelial tumours. EGFR protein expression was studied using immunohistochemistry. Mutations were investigated by Sanger sequencing exons 18-21 of the tyrosine kinase domain of EGFR. Cases with comparatively higher protein expression were examined for gene amplification by chromogenic in situ hybridization. We also studied the tumours for KRAS and BRAF mutations. Immunohistochemistry results revealed both cytoplasmic and nuclear EGFR expression with variable degrees between tumours. The level of nuclear localization was relatively higher in BOTs and LGSCs as compared to HGSCs or benign tumours. The degree of nuclear expression of BOTs showed no significant difference from that in LGSCs (mean ranks 36.48, 33.05, respectively, p=0.625), but was significantly higher than in HGSCs (mean ranks: 38.88, 12.61 respectively, p<0.001) and benign tumours (mean ranks: 35.18, 13.00 respectively, p=0.010). Cytoplasmic expression level was higher in LGSCs. No EGFR gene mutations or amplification were identified, yet different polymorphisms were detected. Five different types of point mutations in the KRAS gene and the V600E BRAF mutation were detected exclusively in BOTs and LGSCs. Our study reports for the first time nuclear localization of EGFR in BOTs. The nuclear localization similarities between BOTs and LGSCs and not HGSCs support the hypothesis suggesting evolution of LGSCs from BOTs. We also confirm that EGFR mutations and amplifications are not molecular events in the pathogenesis of BOTs

Lanthanide speciation in potential SANEX and GANEX actinide/ 2 lanthanide separations using Tetra-N-Donor extractants

Lanthanide(III) complexes with N-donor ex-tractants, which exhibit the potential for the separation of minor actinides from lanthanides in the management of spent nuclear fuel, have been directly synthesized and characterized in both solution and solid states. Crystal structures of the Pr3+, Eu3+, Tb3+, and Yb3+ complexes of 6,6′-bis(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2,4-benzotriazin3-yl)-1,10-phenanthroline(CyMe4-BTPhen) and the Pr3+, Eu3+, and Tb3+ complexes of 2,9-bis(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2,4-benzotria-zin-3-yl)-2,2′-bypyridine (CyMe4-BTBP) were obtained. The majority of these structures displayed coordination of two ofthe tetra-N-donor ligands to each Ln3+ ion, even when in some cases the complexations were performed with equimolar amounts of lanthanide and N-donor ligand. The structures showed that generally the lighter lanthanides had their coordination spheres completed by a bidentate nitrate ion, giving a 2+ charged complex cation, whereas the structures of the heavier lanthanides displayed tricationic complex species with a single water molecule completing their coordination environments. Electronic absorption spectroscopic titrations showed formation of the 1:2 Ln3+/LN4‑donor species (Ln = Pr3+, Eu3+, Tb3+) in methanol when the N-donor ligand was in excess. When the Ln3+ ion was in excess, evidence for formation of a 1:1 Ln3+/LN4‑donor complex species was observed. Luminescent lifetime studies of mixtures of Eu3+ with excess CyMe4-BTBP and CyMe4-BTPhen in methanol indicated that the nitrate-coordinated species is dominant in solution. X-ray absorption spectra of Eu3+ and Tb3+ species, formed by extraction from an acidic aqueous phase into an organic solution consisting of excess N-donor extractant in pure cyclohexanone or 30% tri-n-butyl phosphate (TBP) in cyclohexanone, were obtained. The presence of TBP in the organic phase did not alter lanthanide speciation. Extended X-ray absorption fine structure data from these spectra were fitted using chemical models established by crystallography and solution spectroscopy and showed the dominant lanthanide species in the bulk organic phase was a 1:2 Ln3+/LN‑donor species

Retinoid X receptor activation reverses age-related deficiencies in myelin debris phagocytosis and remyelination.

The efficiency of central nervous system remyelination declines with age. This is in part due to an age-associated decline in the phagocytic removal of myelin debris, which contains inhibitors of oligodendrocyte progenitor cell differentiation. In this study, we show that expression of genes involved in the retinoid X receptor pathway are decreased with ageing in both myelin-phagocytosing human monocytes and mouse macrophages using a combination of in vivo and in vitro approaches. Disruption of retinoid X receptor function in young macrophages, using the antagonist HX531, mimics ageing by reducing myelin debris uptake. Macrophage-specific RXRα (Rxra) knockout mice revealed that loss of function in young mice caused delayed myelin debris uptake and slowed remyelination after experimentally-induced demyelination. Alternatively, retinoid X receptor agonists partially restored myelin debris phagocytosis in aged macrophages. The agonist bexarotene, when used in concentrations achievable in human subjects, caused a reversion of the gene expression profile in multiple sclerosis patient monocytes to a more youthful profile and enhanced myelin debris phagocytosis by patient cells. These results reveal the retinoid X receptor pathway as a positive regulator of myelin debris clearance and a key player in the age-related decline in remyelination that may be targeted by available or newly-developed therapeutics.This work was supported by grants from the UK Multiple Sclerosis Society, Wellcome-Trust, NINDS/NIH Intramural Research Program, Health Research Board Scholars Program, Gates-Cambridge Scholarship, and Spanish Ministry of Economy and Competitiveness (SAF2012- 31483).S

Modelling Conformational Flexibility in a Spectrally Addressable Molecular Multi-Qubit Model System

Dipolar coupled multi-spin systems have the potential to be used as molecular qubits. Herein we report the synthesis of a molecular multi-qubit model system with three individually addressable, weakly interacting, spin (Formula presented.) centres of differing g-values. We use pulsed Electron Paramagnetic Resonance (EPR) techniques to characterise and separately address the individual electron spin qubits; CuII, Cr7Ni ring and a nitroxide, to determine the strength of the inter-qubit dipolar interaction. Orientation selective Relaxation-Induced Dipolar Modulation Enhancement (os-RIDME) detecting across the CuII spectrum revealed a strongly correlated CuII-Cr7Ni ring relationship; detecting on the nitroxide resonance measured both the nitroxide and CuII or nitroxide and Cr7Ni ring correlations, with switchability of the interaction based on differing relaxation dynamics, indicating a handle for implementing EPR-based quantum information processing (QIP) algorithms

Integrative molecular and functional profiling of ERBB2-amplified breast cancers identifies new genetic dependencies.

Overexpression of the receptor tyrosine kinase ERBB2 (also known as HER2) occurs in around 15% of breast cancers and is driven by amplification of the ERBB2 gene. ERBB2 amplification is a marker of poor prognosis, and although anti-ERBB2-targeted therapies have shown significant clinical benefit, de novo and acquired resistance remains an important problem. Genomic profiling has demonstrated that ERBB2+ve breast cancers are distinguished from ER+ve and 'triple-negative' breast cancers by harbouring not only the ERBB2 amplification on 17q12, but also a number of co-amplified genes on 17q12 and amplification events on other chromosomes. Some of these genes may have important roles in influencing clinical outcome, and could represent genetic dependencies in ERBB2+ve cancers and therefore potential therapeutic targets. Here, we describe an integrated genomic, gene expression and functional analysis to determine whether the genes present within amplicons are critical for the survival of ERBB2+ve breast tumour cells. We show that only a fraction of the ERBB2-amplified breast tumour lines are truly addicted to the ERBB2 oncogene at the mRNA level and display a heterogeneous set of additional genetic dependencies. These include an addiction to the transcription factor gene TFAP2C when it is amplified and overexpressed, suggesting that TFAP2C represents a genetic dependency in some ERBB2+ve breast cancer cell