Impact of intratumoral heterogeneity of breast cancer tissue on quantitative metabolomics using high‐resolution magic angle spinning 1H NMR spectroscopy

High‐resolution magic angle spinning (HR MAS) nuclear magnetic resonance (NMR) spectroscopy is increasingly being used to study metabolite levels in human breast cancer tissue, assessing, for instance, correlations with prognostic factors, survival outcome or therapeutic response. However, the impact of intratumoral heterogeneity on metabolite levels in breast tumor tissue has not been studied comprehensively. More specifically, when biopsy material is analyzed, it remains questionable whether one biopsy is representative of the entire tumor. Therefore, multi‐core sampling (n = 6) of tumor tissue from three patients with breast cancer, followed by lipid (0.9‐ and 1.3‐ppm signals) and metabolite quantification using HR MAS ¹H NMR, was performed, resulting in the quantification of 32 metabolites. The mean relative standard deviation across all metabolites for the six tumor cores sampled from each of the three tumors ranged from 0.48 to 0.74. This was considerably higher when compared with a morphologically more homogeneous tissue type, here represented by murine liver (0.16–0.20). Despite the seemingly high variability observed within the tumor tissue, a random forest classifier trained on the original sample set (training set) was, with one exception, able to correctly predict the tumor identity of an independent series of cores (test set) that were additionally sampled from the same three tumors and analyzed blindly. Moreover, significant differences between the tumors were identified using one‐way analysis of variance (ANOVA), indicating that the intertumoral differences for many metabolites were larger than the intratumoral differences for these three tumors. That intertumoral differences, on average, were larger than intratumoral differences was further supported by the analysis of duplicate tissue cores from 15 additional breast tumors. In summary, despite the observed intratumoral variability, the results of the present study suggest that the analysis of one, or a few, replicates per tumor may be acceptable, and supports the feasibility of performing reliable analyses of patient tissue.     

Comparative study of ecophysiological and biochemical variation between the Baltic and North Sea populations of the invasive soft shell clam Mya arenaria (L. 1758)

Seasonal variations of environmental factors, such as temperature and salinity, require metabolic acclimatization in sedentary benthic fauna distributed over a wide geographical range. The soft-shell clam Mya arenaria inhabits the coastal waters of the North Atlantic including North America and Europe. In Europe, M. arenaria populations are distributed from Iceland to the Mediterranean Sea, including the North Sea, the Baltic Sea and the Black Sea. Seasonal changes in physiological parameters (gonad index, condition index, biochemical composition and respiration rate) of M. arenaria from the Baltic Sea (the Gulf of Gdańsk, Poland), and the North Sea (Versee Meer, the Netherlands) were studied. The sex ratio of both populations did not differ from 1:1 and the seasonal gonad index was higher in the Baltic population. The average condition index changed seasonally at both studied sites, and was also higher in the Baltic population (except the autumn) compared to the North Sea. In both studied populations, the content of proteins, lipids and carbohydrates in the soft tissue followed the seasonal variations, and it was higher in the Baltic population. The respiration rate was lower in the Baltic population, and seasonal changes in the respiration rate seem to be correlated with changes in the water temperature. Based on the results obtained in the present study, we suggest that Mya arenaria is characterized by a large phenotypic plasticity and differences in the observed physiological traits are due to acclimatization to ambient environmental conditions.     

Large Molecule Specific Assay Operation: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Team

The L2 Global Harmonization Team on large molecule specific assay operation for protein bioanalysis in support of pharmacokinetics focused on the following topics: setting up a balanced validation design, specificity testing, selectivity testing, dilutional linearity, hook effect, parallelism, and testing of robustness and ruggedness. The team additionally considered the impact of lipemia, hemolysis, and the presence of endogenous analyte on selectivity assessments as well as the occurrence of hook effect in study samples when no hook effect had been observed during pre-study validation.     

Assay Formats: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Team

As part of the GBC (Global Bioanalysis Consortium), the L3 assay format team has focused on reviewing common platforms used to support ligand binding assays in the detection of biotherapeutics. The following review is an overview of discussions and presentations from around the globe with a group of experts from different companies to allow an international harmonization of common practices and suggestions for different platforms. Some of the major platforms include Gyrolab, Erenna, RIA, AlphaLISA, Delfia, Immuno-PCR, Luminex, BIAcore, and ELISAs. The review is meant to support bioanalysts in taking decisions between different platforms depending on the needs of the analyte with a number of recommendations to help integration of platforms into a GLP environment.     

Experimental Zika virus infection induces spinal cord injury and encephalitis in newborn Swiss mice

A widespread epidemic of Zika virus (ZIKV) infection was reported in 2015 in South and Central America, with neurological symptons including meningoencephalitis and Guillain-Barré syndrome in adults, besides an apparent increased incidence of microcephaly in infants born to infected mothers. It is becoming a necessity to have a trustworthy animal model to better understand ZIKV infection. In this study we used newborn white Swiss mice as a model to investigate the ZIKV strain recently isolated in Brazil. ZIKV was inoculated via intracerebral and subcutaneous routes and analysed through gross histopathology and immunohistochemistry. Here we demonstrated first that the intracerebral group (ICG) displayed severe cerebral lesions, with neuronal death, presence of apoptotic bodies, white matter degeneration and neutrophil perivascular cuffing. In the subcutaneous group (SCG), we observed moderate cerebral lesions, morphologically similar to that found in ICG and additional myelopathy, with architectural loss, marked by neuronal death and apoptotic bodies. Interestingly, we found an intense astrogliosis in brain of both groups, with increased immunoexpression of GFAP (glial fibrillary acidic protein) and presence of hypertrophic astrocytes. The spinal cord of subcutaneous group (SCG) exhibited reduction of astrocytes, but those positive for GFAP were hypertrophic and presented prolonged cellular processes. Finally significant lesions in the central nervous system (CNS) were present in newborn mice inoculated by both routes, but SCG method led to an important neurological manifestations (including myelopathy), during a longer period of time and appears for us to be a better model for ZIKV infection.     

Consistent mutation status within histologically heterogeneous lung cancer lesions

Mattsson J S M, Imgenberg‐Kreuz J, Edlund K, Botling J & Micke P
(2012) Histopathology  61, 744–748 Consistent mutation status within histologically heterogeneous lung cancer lesions Aims: Activating epidermal growth factor receptor (EGFR) and KRAS mutations characterize molecular subgroups of non‐small‐cell lung cancer (NSCLC) with a strong predictive value for response to EGFR inhibitor therapy. However, the temporal occurrence and clonal stability of these mutations during the course of cancer progression are debated. The aim of this study was to characterize the presence of EGFR and KRAS mutations in histologically different areas of primary NSCLC lesions. Methods and results: Formalin‐fixed paraffin‐embedded cancer specimens from six cases with EGFR mutations and five cases with KRAS mutations were selected from a pool of primary resected NSCLC patients. From each tumour, three morphologically distinct areas were manually microdissected and analysed for the presence of mutations. The results demonstrated consistent EGFR and KRAS mutation status in the different histological areas of all primary tumours. Conclusions: The results support the concept that activating EGFR and KRAS mutations are oncogenic events that are consistently present throughout the primary tumour independently of histological heterogeneity. Thus, for molecular diagnostics, any part of the tumour is likely to be representative for EGFR and KRAS mutation testing.     

Native valve right sided infective endocarditis

Right-sided infective endocarditis (RSIE) accounts for 5–10% of all cases of infective endocarditis (IE), and is predominantly encountered in the injecting drug user (IDU) population, where HIV and HCV coinfections often coexist. Staphylococcus aureus is the most common pathogen. The pathogenesis of RSIE is still not well understood. RSIE usually presents as a persistent fever with respiratory symptoms whilst signs of systemic embolisation as seen in left-sided IE are notably absent. The prompt diagnosis of RSIE thus requires a high index of suspicion. Transthoracic echocardiography (TTE) can detect the majority of RSIE, whilst transoesophageal echocardiography (TOE) can increase sensitivity. Virulence of the causative organism and vegetation size are the major determinants of prognosis. Most cases of RSIE resolve with appropriate antibiotic administration.