Managing Operating Procedures in Distributed Collaborative Projects

In recent years, large distributed collaborative projects have become very prominent in scientific research, allowing exchanges between laboratories located in different institutions and countries and between various domains of competence. Particularly the work on nanotoxicity – a field which has only been under investigation for a few years and is still lacking regulatory framework – highlighted the need for well-controlled methods, as well as rules for the handling and disposal of used materials. To obtain comparable and reproducible results of experiments conducted in a distributed context, the standardisation and proper documentation of the applied methods is crucial. The European project NanoDiaRA, whose aim is to develop nanoparticles and biomarkers for the early diagnosis of inflammatory disease, faces this situation as it involves 15 European partners and brings together different scientific cultures and professional backgrounds. Protocols especially developed for Superparamagnetic Iron Oxide Nanoparticles and a management system were designed and implemented within the NanoDiaRA project to fulfil those needs. The main goals were the establishment of standardised Standard Operating Procedures assuring transparency and reproducibility and the provision of access to these protocols to every project partner, as well as their clear allocation to carry out precise measurements and production steps

A common genetic variant of a mitochondrial RNA processing enzyme predisposes to insulin resistance

Mitochondrial energy metabolism plays an important role in the pathophysiology of insulin resistance. Recently, a missense N437S variant was identified in the MRPP3 gene, which encodes a mitochondrial RNA processing enzyme within the RNase P complex, with predicted impact on metabolism. We used CRISPR-Cas9 genome editing to introduce this variant into the mouse Mrpp3 gene and show that the variant causes insulin resistance on a high-fat diet. The variant did not influence mitochondrial gene expression markedly, but instead, it reduced mitochondrial calcium that lowered insulin release from the pancreatic islet β cells of the Mrpp3 variant mice. Reduced insulin secretion resulted in lower insulin levels that contributed to imbalanced metabolism and liver steatosis in the Mrpp3 variant mice on a high-fat diet. Our findings reveal that the MRPP3 variant may be a predisposing factor to insulin resistance and metabolic disease in the human population

Chemically-Induced RAT Mesenchymal Stem Cells Adopt Molecular Properties of Neuronal-Like Cells but Do Not Have Basic Neuronal Functional Properties

Induction of adult rat bone marrow mesenchymal stem cells (MSC) by means of chemical compounds (β-mercaptoethanol, dimethyl sulfoxide and butylated hydroxyanizole) has been proposed to lead to neuronal transdifferentiation, and this protocol has been broadly used by several laboratories worldwide. Only a few hours of MSC chemical induction using this protocol is sufficient for the acquisition of neuronal-like morphology and neuronal protein expression. However, given that cell death is abundant, we hypothesize that, rather than true neuronal differentiation, this particular protocol leads to cellular toxic effects. We confirm that the induced cells with neuronal-like morphology positively stained for NF-200, S100, β-tubulin III, NSE and MAP-2 proteins. However, the morphological and molecular changes after chemical induction are also associated with an increase in the apoptosis of over 50% of the plated cells after 24 h. Moreover, increased intracellular cysteine after treatment indicates an impairment of redox circuitry during chemical induction, and in vitro electrophysiological recordings (patch-clamp) of the chemically induced MSC did not indicate neuronal properties as these cells do not exhibit Na+ or K+ currents and do not fire action potentials. Our findings suggest that a disruption of redox circuitry plays an important role in this specific chemical induction protocol, which might result in cytoskeletal alterations and loss of functional ion-gated channels followed by cell death. Despite the neuronal-like morphology and neural protein expression, induced rat bone marrow MSC do not have basic functional neuronal properties, although it is still plausible that other methods of induction and/or sources of MSC can achieve a successful neuronal differentiation in vitro

Global wealth disparities drive adherence to COVID-safe pathways in head and neck cancer surgery

Peer reviewe

Changes in oxygen tension affect cardiac mitochondrial respiration rate via changes in the rate of mitochondrial hydrogen peroxide production

The capacity of mitochondria to respond to changes in oxygen delivery has the potential to affect the ability of the heart to tolerate decreased oxygen delivery. Respiration by mitochondria is typically regarded as independent of oxygen tension (pO2) until critically low oxygen concentrations limit the activity of cytochrome oxidase. Paradoxically, there is evidence that cellular and mitochondrial oxygen consumption (respiration) can decline at oxygen tensions well above this critical pO2. We tested the hypothesis that oxygen sensitive decreases in mitochondrial hydrogen peroxide production can decrease cardiac mitochondrial respiration rate. Consistent with previous work, an acute decline in pO2 from 146 mm Hg to 10-13 mm Hg in less than 10 min did not affect mitochondrial respiration rate. In contrast, sustained incubation of mitochondria at a pO2 of 10-13 mm Hg for 30 min caused a 50% decrease in mitochondrial respiration rate. This decrease in mitochondrial respiration rate was mimicked by incubation with the hydrogen peroxide scavenger catalase and the decrease in mitochondrial respiration rate was fully reversible by reintroducing oxygen or by adding hydrogen peroxide. Incubation at low pO2 was also associated with a decreased rate of mitochondrial reactive oxygen species production. These findings indicate that oxygen-dependent decreases in the rate of mitochondrial hydrogen peroxide production can decrease cardiac mitochondrial respiration

Mitochondrial mistranslation modulated by metabolic stress causes cardiovascular disease and reduced lifespan

Changes in the rate and fidelity of mitochondrial protein synthesis impact the metabolic and physiological roles of mitochondria. Here we explored how environmental stress in the form of a high-fat diet modulates mitochondrial translation and affects lifespan in mutant mice with error-prone (Mrps12ep/ep) or hyper-accurate (Mrps12ha/ha) mitochondrial ribosomes. Intriguingly, although both mutations are metabolically beneficial in reducing body weight, decreasing circulating insulin and increasing glucose tolerance during a high-fat diet, they manifest divergent (either deleterious or beneficial) outcomes in a tissue-specific manner. In two distinct organs that are commonly affected by the metabolic disease, the heart and the liver, Mrps12ep/ep mice were protected against heart defects but sensitive towards lipid accumulation in the liver, activating genes involved in steroid and amino acid metabolism. In contrast, enhanced translational accuracy in Mrps12ha/ha mice protected the liver from a high-fat diet through activation of liver proliferation programs, but enhanced the development of severe hypertrophic cardiomyopathy and led to reduced lifespan. These findings reflect the complex transcriptional and cell signalling responses that differ between post-mitotic (heart) and highly proliferative (liver) tissues. We show trade-offs between the rate and fidelity of mitochondrial protein synthesis dictate tissue-specific outcomes due to commonly encountered stressful environmental conditions or aging

Evaluation of Emergent Mutations in Circulating Cell-Free DNA and Clinical Outcomes in Patients with Metastatic Colorectal Cancer Treated with Panitumumab in the ASPECCT Study

Background: Mutations in EGFR pathway genes are poor prognostic indicators in patients with metastatic colorectal cancer. Plasma analysis of cell-free DNA is a minimally invasive and highly sensitive method to detect somatic mutations in tumors.Methods: Plasma samples collected from panitumumab-treated patients in the ASPECCT study at baseline and safety follow-up (SFU) were analyzed by a next-generation sequencing-based approach for extended RAS mutant allele frequency as a continuous variable and their association with clinical outcomes and the mutational prevalence of 63 cancer-related genes. The correlation between patient outcome and baseline mutational status of EGFR pathway genes was also examined.Results: Overall, 261 patients in the panitumumab arm had evaluable plasma samples. Patients with a higher RAS mutant allele frequency at baseline had worse clinical outcomes than those with a lower frequency (P < 0.001, Cox PH model); however, RAS mutations did not necessarily preclude patients from deriving benefits. The objective response rate (complete or partial response) was 10.8% for patients with baseline RAS mutations and 21.7% for those with BRAF mutations. The 63-gene panel analysis revealed an increase in tumor mutational burden from baseline to SFU (P < 0.001, Wilcoxon signed rank test). Baseline mutations in EGFR pathway genes, when analyzed both categorically and continuously, were associated with shorter survival.Conclusions: When mutations in EGFR pathway genes were analyzed continuously, higher mutant allele frequency correlated with poorer outcomes. However, extended RAS mutation, by itself, did not preclude clinical responses to panitumumab in a monotherapy setting