Antimicrobial properties of nanostructured surfaces - demonstrating the need for a standard testing methodology

Bioinspired nanostructured materials that exhibit antimicrobial properties are being synthesized and tested at increasing rates for use in healthcare, manufacturing processes, and diagnostics. Although progress has been made in improving and understanding their bactericidal activity, arguably, the biggest problem currently in the field is the lack of a standard testing methodology that allows for optimal characterization and better comparison of emerging nanostructures. Here, we examine two forms of nanostructured silicon that vary in their ability to kill certain bacterial species due to different physical mechanisms and derive guidelines for the comparative testing. We perform a comprehensive evaluation of methodologies used extensively in the field (e.g., colony counting and live dead analysis) and the novel application of high-throughput flow cytometry. The data reveal how the techniques are complementary but not always directly equivalent or correlative. Therefore, comparison of results obtained using different methodologies on different materials can be grossly misleading. We report significant variations in bactericidal efficiencies depending on experimental environments (medium type, etc.) and methodologies employed. In addition, we demonstrate how cytometry is yet another powerful complementary tool that can aid the mechanistic understanding of antimicrobial activities of rough surfaces. Besides standardization for comparison, ultimately, evaluation methods need to consider anticipated applications. Then and only then can the true potential (or limitation) of a novel material be determined for its suitability for advancement in a particular field of use

A study of the effectiveness of thermoradiation sterilization

Effectiveness of thermoradiation sterilization of spacecraft hardwar

Alternative splicing of TGF-betas and their high-affinity receptors TβRI, TβRII and TβRIII (betaglycan) reveal new variants in human prostatic cells

<p>Abstract</p> <p>Background</p> <p>The transforming growth factors (TGF)-β, TGF-β1, TGF-β2 and TGF-β3, and their receptors [TβRI, TβRII, TβRIII (betaglycan)] elicit pleiotropic functions in the prostate. Although expression of the ligands and receptors have been investigated, the splice variants have never been analyzed. We therefore have analyzed all ligands, the receptors and the splice variants TβRIB, TβRIIB and TGF-β2B in human prostatic cells.</p> <p>Results</p> <p>Interestingly, a novel human receptor transcript TβRIIC was identified, encoding additional 36 amino acids in the extracellular domain, that is expressed in the prostatic cancer cells PC-3, stromal hPCPs, and other human tissues. Furthermore, the receptor variant TβRIB with four additional amino acids was identified also in human. Expression of the variant TβRIIB was found in all prostate cell lines studied with a preferential localization in epithelial cells in some human prostatic glands. Similarly, we observed localization of TβRIIC and TGF-β2B mainly in the epithelial cells with a preferential localization of TGF-β2B in the apical cell compartment. Whereas in the androgen-independent hPCPs and PC-3 cells all TGF-β ligands and receptors are expressed, the androgen-dependent LNCaP cells failed to express all ligands. Additionally, stimulation of PC-3 cells with TGF-β2 resulted in a significant and strong increase in secretion of plasminogen activator inhibitor-1 (PAI-1) with a major participation of TβRII.</p> <p>Conclusion</p> <p>In general, expression of the splice variants was more heterogeneous in contrast to the well-known isoforms. The identification of the splice variants TβRIB and the novel isoform TβRIIC in man clearly contributes to the growing complexity of the TGF-β family.</p

Primary Transgenic Bovine Cells and Their Rejuvenated Cloned Equivalents Show Transgene-Specific Epigenetic Differences

Cell-mediated transgenesis, based on somatic cell nuclear transfer (SCNT), provides the opportunity to shape the genetic make-up of cattle. Bovine primary fetal fibroblasts, commonly used cells for SCNT, have a limited lifespan, and complex genetic modifications that require sequential transfections can be challenging time and cost-wise. To overcome these limitations, SCNT is frequently used to rejuvenate the cell lines and restore exhausted growth potential. We have designed a construct to be used in a 2-step cassette exchange experiment. Our transgene contains a puromycin resistance marker gene and an enhanced green fluorescence protein (EGFP) expression cassette, both driven by a strong mammalian promoter, and flanked by loxP sites and sequences from the bovine β-casein locus. Several transgenic cell lines were generated by random insertion into primary bovine cell lines. Two of these original cell lines were rederived by SCNT and new primary cells, with the same genetic makeup as the original donors, were established. While the original cell lines were puromycin-resistant and had a characteristic EGFP expression profile, all rejuvenated cell lines were sensitive to puromycin, and displayed varied EGFP expression, indicative of various degrees of silencing. When the methylation states of individual CpG sites within the transgene were analyzed, a striking increase in transgene-specific methylation was observed in all rederived cell lines. The results indicate that original transgenic donor cells and their rejuvenated derivatives may not be equivalent and differ in the functionality of their transgene sequences

Biological subtyping of early breast cancer : a study comparing RT-qPCR with immunohistochemistry

The biological subtype of breast cancer influences the selection of systemic therapy. Distinction between luminal A and B cancers depends on consistent assessment of Ki-67, but substantial intra-observer and inter-observer variability exists when immunohistochemistry (IHC) is used. We compared RT-qPCR with IHC in the assessment of Ki-67 and other standard factors used in breast cancer subtyping. RNA was extracted from archival breast tumour tissue of 769 women randomly assigned to the FinHer trial. Cancer ESR1, PGR, ERBB2 and MKI67 mRNA content was quantitated with an RT-qPCR assay. Local pathologists assessed ER, PgR and Ki-67 expression using IHC. HER2 amplification was identified with chromogenic in situ hybridization (CISH) centrally. The results were correlated with distant disease-free survival (DDFS) and overall survival (OS). qPCR-based and IHC-based assessments of ER and PgR showed good concordance. Both low tumour MKI67 mRNA (RT-qPCR) and Ki-67 protein (IHC) levels were prognostic for favourable DDFS [hazard ratio (HR) 0.42, 95 % CI 0.25-0.71, P = 0.001; and HR 0.56, 0.37-0.84, P = 0.005, respectively] and OS. In multivariable analyses, cancer MKI67 mRNA content had independent influence on DDFS (adjusted HR 0.51, 95 % CI 0.29-0.89, P = 0.019) while Ki-67 protein expression had not any influence (P = 0.266) whereas both assessments influenced independently OS. Luminal B patients treated with docetaxel-FEC had more favourable DDFS and OS than those treated with vinorelbine-FEC when the subtype was defined by RT-qPCR (for DDFS, HR 0.52, 95 % CI 0.29-0.94, P = 0.031), but not when defined using IHC. Breast cancer subtypes approximated with RT-qPCR and IHC show good concordance, but cancer MKI67 mRNA content correlated slightly better with DDFS than Ki-67 expression. The findings based on MKI67 mRNA content suggest that patients with luminal B cancer benefit more from docetaxel-FEC than from vinorelbine-FEC.Peer reviewe

Transgenic goats producing an improved version of cetuximab in milk [preprint]

Therapeutic monoclonal antibodies (mAbs) represent one of the most important classes of pharmaceutical proteins to treat human diseases. Most are produced in cultured mammalian cells which is expensive, limiting their availability. Goats, striking a good balance between a relatively short generation time and copious milk yield, present an alternative platform for the cost-effective, flexible, large-scale production of therapeutic mAbs. Here, we focused on cetuximab, a mAb against epidermal growth factor receptor, that is commercially produced under the brand name Erbitux and approved for anti-cancer treatments. We generated several transgenic goat lines that produce cetuximab in their milk. Two lines were selected for detailed characterization. Both showed stable genotypes and cetuximab production levels of up to 10g/L. The mAb could be readily purified and showed improved characteristics compared to Erbitux. The goat-produced cetuximab (gCetuximab) lacked a highly immunogenic epitope that is part of Erbitux. Moreover, it showed enhanced binding to CD16 and increased antibody-dependent cell-dependent cytotoxicity compared to Erbitux. This indicates that these goats produce an improved cetuximab version with the potential for enhanced effectiveness and better safety profile compared to treatments with Erbitux. In addition, our study validates transgenic goats as an excellent platform for large-scale production of therapeutic mAbs

An international reproducibility study validating quantitative determination of ERBB2, ESR1, PGR, and MKI67 mRNA in breast cancer using MammaTyper (R)

Background: Accurate determination of the predictive markers human epidermal growth factor receptor 2 (HER2/ERBB2), estrogen receptor (ER/ESR1), progesterone receptor (PgR/PGR), and marker of proliferation Ki67 (MKI67) is indispensable for therapeutic decision making in early breast cancer. In this multicenter prospective study, we addressed the issue of inter- and intrasite reproducibility using the recently developed reverse transcription-quantitative real-time polymerase chain reaction-based MammaTyper (R) test. Methods: Ten international pathology institutions participated in this study and determined messenger RNA expression levels of ERBB2, ESR1, PGR, and MKI67 in both centrally and locally extracted RNA from formalin-fixed, paraffin-embedded breast cancer specimens with the MammaTyper (R) test. Samples were measured repeatedly on different days within the local laboratories, and reproducibility was assessed by means of variance component analysis, Fleiss' kappa statistics, and interclass correlation coefficients (ICCs). Results: Total variations in measurements of centrally and locally prepared RNA extracts were comparable; therefore, statistical analyses were performed on the complete dataset. Intersite reproducibility showed total SDs between 0.21 and 0.44 for the quantitative single-marker assessments, resulting in ICC values of 0.980-0.998, demonstrating excellent agreement of quantitative measurements. Also, the reproducibility of binary single-marker results (positive/negative), as well as the molecular subtype agreement, was almost perfect with kappa values ranging from 0.90 to 1.00. Conclusions: On the basis of these data, the MammaTyper (R) has the potential to substantially improve the current standards of breast cancer diagnostics by providing a highly precise and reproducible quantitative assessment of the established breast cancer biomarkers and molecular subtypes in a decentralized workup.Peer reviewe

Genome editing to the rescue: sustainably feeding 10 billion global human population

Modern animal breeding strategies based on population genetics, molecular tools, artificial insemination, embryo transfer and related technologies have contributed to significant increases in the performance of domestic animals, and are the basis for a regular supply of high quality animal derived food at acceptable prices. However, the current strategy of marker- assisted selection and breeding of animals to introduce novel traits over multiple generations is too pedestrian in responding to unprecedented challenges such as climate change, global pandemics, and feeding an anticipated 33% increase in global population in the next three decades. Here, we propose site-specific genome editing technologies as a basis for “directed” or “rational selection” of agricultural traits. The animal science community envisions genome editing as an essential tool in addressing critical priorities for global food security and environmental sustainability, and seeks additional funding support for development and implementation of these technologies for maximum societal benefit