Nanopore Is Preferable over Illumina for 16S Amplicon Sequencing of the Gut Microbiota When Species-Level Taxonomic Classification, Accurate Estimation of Richness, or Focus on Rare Taxa Is Required.

Nanopore sequencing is a promising technology used for 16S rRNA gene amplicon sequencing as it can provide full-length 16S reads and has a low up-front cost that allows research groups to set up their own sequencing workflows. To assess whether Nanopore with the improved error rate of the Kit 12 chemistry should be adopted as the preferred sequencing technology instead of Illumina for 16S amplicon sequencing of the gut microbiota, we used a mock community and human faecal samples to compare diversity, richness, and species-level community structure, as well as the replicability of the results. Nanopore had less noise, better accuracy with the mock community, a higher proportion of reads from the faecal samples classified to species, and better replicability. The difference between the Nanopore and Illumina results of the faecal bacterial community structure was significant but small compared to the variation between samples. The results show that Nanopore is a better choice for 16S rRNA gene amplicon sequencing when the focus is on species-level taxonomic resolution, the investigation of rare taxa, or an accurate estimation of richness. Illumina 16S sequencing should be reserved for communities with many unknown species, and for studies that require the resolution of amplicon sequence variants.

Neuroendocrine Transdifferentiation in Cutaneous Melanoma: A Case Report and Review of the Literature.

ABSTRACT: Neuroendocrine transdifferentiation refers to the progressive transformation of a nonneuroendocrine tumor to the one showing evidence of neuroendocrine differentiation on morphological and immunohistochemical grounds. Although this phenomenon has been well-documented in certain malignancies, particularly prostatic adenocarcinoma after androgen deprivation, cases of neuroendocrine transdifferentiation in melanomas are exceptionally rare. Herein, we report a case of a conventional superficial spreading melanoma occurring on the skin of the leg in a young male which showed progressive neuroendocrine transdifferentiation as it progressed through 2 in-transit metastases and a nodal metastatic deposit over a 4-year period. The tumor retained the BRAF V600E mutation throughout the disease process, and disease control was achieved through dual BRAF inhibition therapy. The possibility of melanoma masquerading as a high-grade neuroendocrine malignancy when investigating tumors of unknown primary should be kept in the mind of clinicians and histopathologist alike as a potential diagnostic pitfall, thus helping avoid misdiagnosis and guide appropriate treatment strategies.

PKC-mediated phosphorylation and activation of the MEK/ERK pathway as a mechanism of acquired trastuzumab resistance in HER2-positive breast cancer.

Protein expression, activation and stability are regulated through inter-connected signal transduction pathways resulting in specific cellular states. This study sought to differentiate between the complex mechanisms of intrinsic and acquired trastuzumab resistance, by quantifying changes in expression and activity of proteins (phospho-protein profile) in key signal transduction pathways, in breast cancer cellular models of trastuzumab resistance. To this effect, we utilized a multiplex, bead-based protein assay, DigiWest®, to measure around 100 proteins and protein modifications using specific antibodies. The main advantage of this methodology is the quantification of multiple analytes in one sample, utilising input volumes of a normal western blot. The intrinsically trastuzumab-resistant cell line JIMT-1 showed the largest number of concurrent resistance mechanisms, including PI3K/Akt and RAS/RAF/MEK/ERK activation, ß catenin stabilization by inhibitory phosphorylation of GSK3ß, cell cycle progression by Rb suppression, and CREB-mediated cell survival. MAPK (ERK) pathway activation was common to both intrinsic and acquired resistance cellular models. The overexpression of upstream RAS/RAF, however, was confined to JIMT 1; meanwhile, in a cellular model of acquired trastuzumab resistance generated in this study (T15), entry into the ERK pathway seemed to be mostly mediated by PKCα activation. This is a novel observation and merits further investigation that can lead to new therapeutic combinations in HER2-positive breast cancer with acquired therapeutic resistance.

Network and Systems Medicine: Position Paper of the European Collaboration on Science and Technology Action on Open Multiscale Systems Medicine.

Introduction: Network and systems medicine has rapidly evolved over the past decade, thanks to computational and integrative tools, which stem in part from systems biology. However, major challenges and hurdles are still present regarding validation and translation into clinical application and decision making for precision medicine. Methods: In this context, the Collaboration on Science and Technology Action on Open Multiscale Systems Medicine (OpenMultiMed) reviewed the available advanced technologies for multidimensional data generation and integration in an open-science approach as well as key clinical applications of network and systems medicine and the main issues and opportunities for the future. Results: The development of multi-omic approaches as well as new digital tools provides a unique opportunity to explore complex biological systems and networks at different scales. Moreover, the application of findable, applicable, interoperable, and reusable principles and the adoption of standards increases data availability and sharing for multiscale integration and interpretation. These innovations have led to the first clinical applications of network and systems medicine, particularly in the field of personalized therapy and drug dosing. Enlarging network and systems medicine application would now imply to increase patient engagement and health care providers as well as to educate the novel generations of medical doctors and biomedical researchers to shift the current organ- and symptom-based medical concepts toward network- and systems-based ones for more precise diagnoses, interventions, and ideally prevention. Conclusion: In this dynamic setting, the health care system will also have to evolve, if not revolutionize, in terms of organization and management.

Bead-based RNA multiplex panels for biomarker detection in oncology samples.

Patient stratification, prognosis and disease monitoring are three important aspects of personalized cancer medicine. With traditional serum tumour protein biomarkers showing lack of specificity and sensitivity, and tumour heterogeneity affecting the response to targeted therapy based on tissue biomarkers, the focus has shifted to the use of molecular tumour signatures as specific biomarkers. Multiplex microsphere-based panels are robust and cost-effective, high throughput molecular assays, which can accurately characterize tumours even from small amounts of poor quality nucleic acids. Only few studies have reported the use of microspheres (beads) to quantify RNA expression of targets of interest simultaneously (multiplexing). This review is an overview of the various applications of bead-based RNA panels in molecular oncology, with focus on the Invitrogen™ QuantiGene™ Plex Assay (Thermo Fisher Scientific), and provides a comparison with PCR-based and other methodologies. The advantages of multiplex bead assays are exemplified by the quantification of RNA expression in formalin-fixed, paraffin embedded (FFPE) archival tissue and the simultaneous detection of biomarkers in low input samples, including quantification of markers in microdissected tissue material, to characterise heterogeneous tumour sites within a sample, and by the detection of markers in low numbers of circulating tumour cells.

Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material.

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. Gene amplification and expression diagnostic testing using archival material or material that requires transportation to servicing laboratories, needs a more robust and accurate test adapted to current clinical workflows. Our research team optimized the use of Invitrogen™ QuantiGene™ Plex Assay (Thermo Fisher Scientific) to quantify RNA in archival material using branched-DNA (bDNA) technology on Luminex xMAP® magnetic beads. The gene expression assay described in this manuscript is a novel, quick, and multiplex method that can accurately classify breast cancer into the different molecular subtypes, omitting the subjectivity of interpretation inherent in imaging techniques. In addition, due to the low input of material required, heterogeneous tumors can be laser microdissected using Hematoxylin and Eosin (H&E) stained sections. This method has a wide range of possible applications including tumor classification with diagnostic potential and measurement of biomarkers in liquid biopsies, which would allow better patient management and disease monitoring. In addition, the quantitative measurement of biomarkers in archival material is useful in oncology research with access to libraries of clinically-annotated material, in which retrospective studies can validate potential biomarkers and their clinical outcome correlation.

Prevalence and characteristics of community carriage of methicillin-resistant Staphylococcus aureus in Malta.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen worldwide. Malta is one of the countries with the highest MRSA prevalence in Europe, as identified from hospital blood cultures [1]. However, community prevalence of MRSA has never previously been investigated. This study aimed at establishing the prevalence of community MRSA nasal colonization in Maltese individuals and identifying the clonal characteristics of the detected isolates. Nasal swabs were collected from 329 healthy individuals who were also asked to complete a brief questionnaire about risk factors commonly associated with MRSA carriage and infection. The swabs were transported and enriched in a nutrient broth supplemented with NaCl. The presence of MRSA was then determined by culturing on MRSA Select chromogenic agar and then confirming by several assays, including catalase, coagulase and PBP2a agglutination tests. The isolates were assayed for antibiotic susceptibilities and typed by microarray analysis to determine the clonal characteristics of each strain. The prevalence of MRSA nasal colonization in the healthy Maltese population was found to be 8.81% (95% confidence interval [CI], 5.75-11.87%), much higher than that found in other studies carried out in several countries. No statistical association was found between MRSA carriage and demographics or risk factors; however, this was hindered by the small sample size. Almost all the isolates were fusidic-acid resistant. The majority were found to belong to a local endemic clone (CC5) which seems to be replacing the previously prevalent European clone UK-EMRSA-15 in the country. A new clone (CC50-MRSA-V) was also characterized. The presence of such a significant community reservoir of MRSA increases the burdens already faced by the local healthcare system to control the MRSA epidemic. Colonization of MRSA in otherwise healthy individuals may represent a risk for endogenous infection and transmission to hospitalized patients after admission to a healthcare facility, leading to longer hospital stays and, consequently, increased healthcare costs.