Variability of the reverse transcription step: practical implications.

BACKGROUND: The reverse transcription (RT) of RNA to cDNA is a necessary first step for numerous research and molecular diagnostic applications. Although RT efficiency is known to be variable, little attention has been paid to the practical implications of that variability. METHODS: We investigated the reproducibility of the RT step with commercial reverse transcriptases and RNA samples of variable quality and concentration. We quantified several mRNA targets with either singleplex SYBR Green I or dualplex probe-based reverse transcription real-time quantitative PCR (RT-qPCR), with the latter used to calculate the correlation between quantification cycles (Cqs) of mRNA targets amplified in the same real-time quantitative PCR (qPCR) assay. RESULTS: RT efficiency is enzyme, sample, RNA concentration, and assay dependent and can lead to variable correlation between mRNAs from the same sample. This translates into relative mRNA expression levels that generally vary between 2- and 3-fold, although higher levels are also observed. CONCLUSIONS: Our study demonstrates that the variability of the RT step is sufficiently large to call into question the validity of many published data that rely on quantification of cDNA. Variability can be minimized by choosing an appropriate RTase and high concentrations of RNA and characterizing the variability of individual assays by use of multiple RT replicates.

Use of an action-selection framework for human-carnivore conflict in the Bangladesh Sundarbans.

Human-carnivore conflict is manifested in the death of humans, livestock, and carnivores. The resulting negative local attitudes and retribution killings imperil the future of many endangered carnivores. We tailored existing management tools to create a framework to facilitate the selection of actions to alleviate human-carnivore conflict and applied the framework to the human-tiger conflict in the Bangladesh Sundarbans. We identified potential actions that consider previous management efforts, local knowledge, cost-effectiveness, fieldwork experience of authors and project staff, previous research on tiger ecology by the authors, and recommendations from human-carnivore conflict studies in other countries. Our framework includes creation of a profile to improve understanding of the nature of the conflict and its underlying causality. Identified actions include deterrents, education, direct tiger management, and response teams. We ranked actions by their potential to reduce conflict and the monetary cost of their implementation. We ranked tiger-response teams and monitoring problem tigers as the two best actions because both had relatively high impact and cost-effectiveness. We believe this framework could be used under a wide range of human-wildlife conflict situations because it provides a structured approach to selection of mitigating actions.

Perturbation of T-cell development by insertional mutation of a PrP transgene.

The normal cellular form of the prion protein PrP(C) is a glycosylphosphatidylinositol-linked cell-surface glycoprotein expressed primarily by cells of the nervous and immune systems. There is evidence to suggest that PrP(C) is involved in cell signalling and cellular homeostasis. We have investigated the immune composition of peripheral lymphoid tissue in PrP-/-, wild-type, tg19 and tga20 strains of mice, which express 0, 1-, 3-5- and 4-7-fold higher levels of PrP(C), respectively, relative to wild-type mice. Our data show that tga20 mice have a reduced number of spleen T-cell receptor (TCR)-alphabeta(+) T cells and an increased number of TCR-gammadelta(+) T cells compared with wild-type mice. This was not seen in tg19 mice, which also express elevated levels of PrP(C). In addition, we have found that the Prnp transgene in the tga20 genome is located centrally on chromosome 17, in or around genes involved in T-cell development. Significantly, mRNA transcripts from pre-TCR-alpha (pTalpha), a T-cell development gene located on mouse chromosome 17, are drastically reduced in tga20 mice, indicative of a perturbation in pTalpha gene regulation. We propose that the immune cell phenotype of tga20 mice may be caused by the insertional mutation of the Prnp transgene into the pTalpha gene or its regulatory elements.

Influence of Study Design on Outcomes Following Reflexology Massage: An Integrative and Critical Review of Interventional Studies.

BACKGROUND: Interpretation of the efficacy of reflexology is hindered by inconsistent research designs and complicated by professional views that criteria of randomized controlled trials (RCTs)are not ideal to research holistic complementary and alternative medicine practice. The influence of research designs on study outcomes is not known. This integrative review sought to evaluate this possibility. MATERIALS AND METHODS: Thirty-seven interventional studies (2000-2014) were identified; they had RCT or non-RCT design and compared reflexology outcomes against a control/comparison group. Viability of integrating RCT and non-RCT studies into a single database was first evaluated by appraisal of 16 reporting fields related to study setting and objectives, sample demographics, methodologic design, and treatment fidelity and assessment against Jadad score quality criteria for RCTs. For appraisal, the database was stratified into RCT/non-RCT or Jadad score of 3 or more or less than 3. Deficits in reporting were identified for blind assignment of participants, dropout/completion rate, and School of Reflexology. For comparison purposes, these fields were excluded from subsequent analysis for evidence of association between design fields and of fields with study outcomes. RESULTS: Thirty-one studies applied psychometric tools and 20 applied biometric tools (14 applied both). A total of 116 measures were used. Type of measure was associated with study objectives (p < 0.001; chi-square), in particular of psychometric measures with a collated "behavioral/cognitive" objective. Significant outcomes were more likely (p < 0.001; chi-square) for psychometric than for biometric measures. Neither type of outcome was associated with choice of RCT or non-RCT method, but psychometric responses were associated (p = 0.007) with a nonmassage control strategy. CONCLUSIONS: The review supports psychometric responses to reflexology when study design uses a nonmassage control strategy. Findings suggest that an evaluation of outcomes against sham reflexology massage and other forms of massage, as well as a narrower focus of study objective, may clarify whether there is a relationship between study design and efficacy of reflexology.

Homogeneous and digital proximity ligation assays for the detection of Clostridium difficile toxins A and B.

BACKGROUND: The proximity ligation assay (PLA) detects proteins via their interaction with pairs of proximity probes, which are antibodies coupled to noncomplementary DNA oligonucleotides. The binding of both proximity probes to their epitopes on the target protein brings the oligonucleotides together, allowing them to be bridged by a third oligonucleotide with complementarity to the other two. This enables their ligation and the detection of the resulting amplicon by real-time quantitative PCR (qPCR), which acts as a surrogate marker for the protein of interest. Hence PLA has potential as a clinically relevant diagnostic tool for the detection of pathogens where nucleic acid based tests are inconclusive proof of infection. METHODS: We prepared monoclonal and polyclonal proximity probes targeting Clostridium difficile toxins A (TcdA) and B (TcdB) and used hydrolysis probe-based qPCR and digital PCR (dPCR) assays to detect antibody/antigen interactions. RESULTS: The performance of the PLA assays was antibody-dependent but both TcdA and TcdB assays were more sensitive than comparable ELISAs in either single- or dualplex formats. Both PLAs could be performed using single monoclonal antibodies coupled to different oligonucleotides. Finally, we used dPCR to demonstrate its potential for accurate and reliable quantification of TcdA. CONCLUSIONS: PLA with either qPCR or dPCR readout have potential as new diagnostic applications for the detection of pathogens where nucleic acid based tests do not indicate viability or expression of toxins. Importantly, since it is not always necessary to use two different antibodies, the pool of potential antibodies useful for PLA diagnostic assays is usefully enhanced.

Targeted Cancer Therapy: Correlative Light-Electron Microscopy Shows RGD-Targeted ZnO Nanoparticles Dissolve in the Intracellular Environment of Triple Negative Breast Cancer Cells and Cause Apoptosis with Intratumor Heterogeneity (Adv. Healthcare Mater. 11/2016).

On page 1310 J. S. Merzaban, A. E. Porter, and co-workers present fluorescently labeled RGD-targeted ZnO nanoparticles (NPs; green) for the targeted delivery of cytotoxic ZnO to integrin αvß3 receptors expressed on triple negative breast cancer cells. Correlative light-electron microscopy shows that NPs dissolve into ionic Zn(2+) (blue) upon uptake and cause apoptosis (red) with intra-tumor heterogeneity, thereby providing a possible strategy for targeted breast cancer therapy. Cover design by Ivan Gromicho.

Correlative Light-Electron Microscopy Shows RGD-Targeted ZnO Nanoparticles Dissolve in the Intracellular Environment of Triple Negative Breast Cancer Cells and Cause Apoptosis with Intratumor Heterogeneity.

ZnO nanoparticles (NPs) are reported to show a high degree of cancer cell selectivity with potential use in cancer imaging and therapy. Questions remain about the mode by which the ZnO NPs cause cell death, whether they exert an intra- or extracellular effect, and the resistance among different cancer cell types to ZnO NP exposure. The present study quantifies the variability between the cellular toxicity, dynamics of cellular uptake, and dissolution of bare and RGD (Arg-Gly-Asp)-targeted ZnO NPs by MDA-MB-231 cells. Compared to bare ZnO NPs, RGD-targeting of the ZnO NPs to integrin αvß3 receptors expressed on MDA-MB-231 cells appears to increase the toxicity of the ZnO NPs to breast cancer cells at lower doses. Confocal microscopy of live MDA-MB-231 cells confirms uptake of both classes of ZnO NPs with a commensurate rise in intracellular Zn(2+) concentration prior to cell death. The response of the cells within the population to intracellular Zn(2+) is highly heterogeneous. In addition, the results emphasize the utility of dynamic and quantitative imaging in understanding cell uptake and processing of targeted therapeutic ZnO NPs at the cellular level by heterogeneous cancer cell populations, which can be crucial for the development of optimized treatment strategies.

Recent progress in developing proximity ligation assays for pathogen detection.

The effective management of infectious diseases depends on the early detection of the microbes responsible, since pathogens are most effectively eliminated in the initial stages of infection. Current immunodiagnostic methods lack the sensitivity for earliest possible diagnosis. Nucleic acid-based tests (NATs) are more sensitive, but the detection of microbial DNA does not definitively prove the presence of a viable microorganism capable of causing a given infection. Proximity assays combine the specificity of antibody-based detection of proteins with the sensitivity and dynamic range of NATs, and their use may allow earlier as well as more clinically relevant detection than is possible with current NATs or immunoassays. However, the full potential of proximity assays for pathogen detection remains to be fulfilled, mainly due to the challenges associated with identifying suitable antibodies and antibody combinations, sensitivity issues arising from non-specific interactions of proximity probes and the longer incubation times required to carry out the assays.

Proximity assays for sensitive quantification of proteins.

Proximity assays are immunohistochemical tools that utilise two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridisation of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein-protein interactions. Assays can be carried out in homogeneous or solid phase formats and in situ assays can visualise single protein molecules or complexes with high spatial accuracy. These properties highlight the potential of proximity assays in research, diagnostic, pharmacological and many other applications that require sensitive, specific and accurate assessments of protein expression.

Biology of stress revisited: intracellular mechanisms and the conceptualization of stress.

Application of allostatic theory to stress during the 1990s refocused attention on internal responses to a perceived hazard, and the last 20 years has seen considerable developments in the biological contexts of stress. Evidence from neuroscience now suggests that secretion of the hormone cortisol is not only stimulated by the outcomes of cognitive transaction but it also feeds back and contributes positively to the cognitive adaptation that is a feature of stress resilience. More recently, the operative intracellular mechanisms are beginning to be understood and provide an insight into the regulation and maintenance of intracellular homeostasis that underpins adaptive change and vulnerability. The maintenance or appropriate modulation of intracellular homeostasis usually provides a buffering of potential adverse interactions. However, the capacity to do so is diminished during chronic stress leading to intracellular and subsequently systemic, homeostatic failure and hence maladaptation. This area of research seems far removed from cognitive theory, but placing intracellular homeostasis at the core of cognitive and biological responses supports the concept of stress as a genuinely psycho-biological phenomenon.

CD74-dependent deregulation of the tumor suppressor scribble in human epithelial and breast cancer cells.

The γ subunit of the major histocompatibility complex (MHC) class II complex, CD74, is overexpressed in a significant proportion of metastatic breast tumors, but the mechanistic foundation and biologic significance of this phenomenon are not fully understood. Here, we show that when CD74 is overexpressed in human cancer and noncancerous epithelial cells, it interacts and interferes with the function of Scribble, a product of a well-known tumor suppressor gene. Furthermore, using epithelial cell lines expressing CD74 under the control of tetracycline-inducible promoter and quantitative high-resolution mass spectrometry, we demonstrate that, as a result of CD74 overexpression, the phosphorylation pattern of the C-terminal part of Scribble undergoes specific changes. This is accompanied with a translocation of the protein from the sites of cell-to-cell contacts at the plasma membrane to the cytoplasm, which is likely to effectively enhance the motility and invasiveness of the cancer cells.

Stat1 and CD74 overexpression is co-dependent and linked to increased invasion and lymph node metastasis in triple-negative breast cancer.

Triple-negative breast cancer is difficult to treat because of the lack of rationale-based therapies. There are no established markers and targets that can be used for stratification of patients and targeted therapy. Here we report the identification of novel molecular features, which appear to augment metastasis of triple negative breast tumors. We found that triple-negative breast tumors can be segregated into 2 phenotypes based on their genome-wide protein abundance profiles. The first is characterized by high expression of Stat1, Mx1, and CD74. Seven out of 9 tumors from this group had invaded at least 2 lymph nodes while only 1 out of 10 tumors in group 2 was lymph node positive. In vitro experiments showed that the interferon-induced increase in Stat1 abundance correlates with increased migration and invasion in cultured cells. When CD74 was overexpressed, it increased cell adhesion on matrigel. This effect was accompanied with a marked increase in the membrane expression of beta-catenin, MUC18, plexins, integrins, and other proteins involved in cell adhesion and cancer metastasis. Taken together, our results show that Stat1/CD74 positive triple-negative tumors are more aggressive and suggest an approach for development of better diagnostics and more targeted therapies for triple negative breast cancer. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Optimized Protocol for Protein Extraction from the Breast Tissue that is Compatible with Two-Dimensional Gel Electrophoresis.

Proteomics is a highly informative approach to analyze cancer-associated transformation in tissues. The main challenge to use a tissue for proteomics studies is the small sample size and difficulties to extract and preserve proteins. The choice of a buffer compatible with proteomics applications is also a challenge. Here we describe a protocol optimized for the most efficient extraction of proteins from the human breast tissue in a buffer compatible with two-dimensional gel electrophoresis (2D-GE). This protocol is based on mechanically assisted disintegration of tissues directly in the 2D-GE buffer. Our method is simple, robust and easy to apply in clinical practice. We demonstrate high quality of separation of proteins prepared according to the reported here protocol.

Meta-data analysis as a strategy to evaluate individual and common features of proteomic changes in breast cancer.

BACKGROUND: Individual differences among breast tumours in patients is a significant challenge for the treatment of breast cancer. This study reports a strategy to assess these individual differences and the common regulatory mechanisms that may underlie breast tumourigenesis. MATERIALS AND METHODS: The two-step strategy was based firstly on a full-scale proteomics analysis of individual cases, and secondly on the analysis of common features of the individual proteome-centred networks (meta-data). RESULTS: Proteomic profiling of human invasive ductal carcinoma tumours was performed and each case was analysed individually. Analysis of primary datasets for common cancer-related proteins identified keratins. Analysis of individual networks built with identified proteins predicted features and regulatory mechanisms involved in each individual case. Validation of these findings by immunohistochemistry confirmed the predicted deregulation of expression of CK2α, PDGFRα, PYK and p53 proteins. CONCLUSION: Meta-data analysis allowed efficient evaluation of both individual and common features of the breast cancer proteome.

A peptide-centric approach to breast cancer biomarker discovery utilizing label-free multiple reaction monitoring mass spectrometry.

We report an approach for multiplex analysis of cancer biomarkers based on the measurement of diagnostic peptides in whole tissue protein digests. Label-free quantitation with MS3 multiple reaction monitoring (MRM) was developed to afford accurate analysis of prospective marker peptides in a panel of breast tumors. This approach provides an economical and robust alternative to stable isotope-based methods. It is equally applicable to the analysis of samples derived from tissue biopsy, aspirate, or plasma and can be easily translated to clinic.

Proteome profiling of breast tumors by gel electrophoresis and nanoscale electrospray ionization mass spectrometry.

We have conducted proteome-wide analysis of fresh surgery specimens derived from breast cancer patients, using an approach that integrates size-based intact protein fractionation, nanoscale liquid separation of peptides, electrospray ion trap mass spectrometry, and bioinformatics. Through this approach, we have acquired a large amount of peptide fragmentation spectra from size-resolved fractions of the proteomes of several breast tumors, tissue peripheral to the tumor, and samples from patients undergoing noncancer surgery. Label-free quantitation was used to generate protein abundance maps for each proteome and perform comparative analyses. The mass spectrometry data revealed distinct qualitative and quantitative patterns distinguishing the tumors from healthy tissue as well as differences between metastatic and non-metastatic human breast cancers including many established and potential novel candidate protein biomarkers. Selected proteins were evaluated by Western blotting using tumors grouped according to histological grade, size, and receptor expression but differing in nodal status. Immunohistochemical analysis of a wide panel of breast tumors was conducted to assess expression in different types of breast cancers and the cellular distribution of the candidate proteins. These experiments provided further insights and an independent validation of the data obtained by mass spectrometry and revealed the potential of this approach for establishing multimodal markers for early metastasis, therapy outcomes, prognosis, and diagnosis in the future.