Degradation Parameters from Pulse-Chase Experiments

Pulse-chase experiments are often used to study the degradation of macromolecules such as proteins or mRNA. Considerations for the choice of pulse length include the toxicity of the pulse to the cell and maximization of labeling. In the general case of non-exponential decay, varying the length of the pulse results in decay patterns that look different. Analysis of these patterns without consideration to pulse length would yield incorrect degradation parameters. Here we propose a method that constructively includes pulse length in the analysis of decay patterns and extracts the parameters of the underlying degradation process. We also show how to extract decay parameters reliably from measurements taken during the pulse phase

A mouse model of gestational diabetes shows dysregulated lipid metabolism post-weaning, after return to euglycaemia

Background: Gestational diabetes is associated with increased risk of type 2 diabetes mellitus and cardiovascular disease for the mother in the decade after delivery. However, the molecular mechanisms that drive these effects are unknown. Recent studies in humans have shown that lipid metabolism is dysregulated before diagnosis of and during gestational diabetes and we have shown previously that lipid metabolism is also altered in obese female mice before, during and after pregnancy. These observations led us to the hypothesis that this persistent dysregulation reflects an altered control of lipid distribution throughout the organism.Methods: We tested this in post-weaning (PW) dams using our established mouse model of obese GDM (high fat, high sugar, obesogenic diet) and an updated purpose-built computational tool for plotting the distribution of lipid variables throughout the maternal system (Lipid Traffic Analysis v2.3).Results: This network analysis showed that unlike hyperglycaemia, lipid distribution and traffic do not return to normal after pregnancy in obese mouse dams. A greater range of phosphatidylcholines was found throughout the lean compared to obese post-weaning dams. A range of triglycerides that were found in the hearts of lean post-weaning dams were only found in the livers of obese post-weaning dams and the abundance of odd-chain FA-containing lipids differed locally in the two groups. We have therefore shown that the control of lipid distribution changed for several metabolic pathways, with evidence for changes to the regulation of phospholipid biosynthesis and FA distribution, in a number of tissues.Conclusions: We conclude that the control of lipid metabolism is altered following an obese pregnancy. These results support the hypothesis that obese dams that developed GDM maintain dysregulated lipid metabolism after pregnancy even when glycaemia returned to normal, and that these alterations could contribute to the increased risk of later type 2 diabetes and cardiovascular disease

Quantitative assessment of ribosome drop-off in E. coli

Premature ribosome drop-off is one of the major errors in translation of mRNA by ribosomes. However, repeated analyses of Ribo-seq data failed to quantify its strength in E. coli. Relying on a novel highly sensitive data analysis method we show that a significant rate of ribosome drop-off is measurable and can be quantified also when cells are cultured under non-stressing conditions. Moreover, we find that the drop-off rate is highly variable, depending on multiple factors. In particular, under environmental stress such as amino acid starvation or ethanol intoxication, the drop-off rate markedly increases

Molecular genetic features and risk assessment in a series of 30 patients who underwent an operation for gastrointestinal stromal tumours

Background: The objective of the study was to investigate the relationship between molecular genetic features and the standard criteria of risk assessment in patients affected by gastrointestinal stromal tumours (GISTs). Methods: A review was conducted of a series of 30 patients, with a mean age of 67 years, who underwent surgery for primary GISTs. R0 resection was accomplished in 27 patients. CD117, CD34 desmin, vimentin, S-100 and smooth muscle actin were immunohistochemically tested to achieve a diagnosis of GIST. The loss of wild-type KIT or platelet-derived growth factor receptor alpha (PDGFRα) genes was investigated by sequencing the tumour DNA. Results: Tumour genes mutations were reported in 23 patients (77%), and wild-type in seven. Mutations on the KIT gene occurred in 18 patients, and mutations on the PDGFRα gene in five. The average sizes of the GIST were 8.7 cm, 5.4 cm and 5.9 cm for KIT gene-mutated, PDGFRα gene-mutated and wild-type tumours, respectively. KIT gene mutations were detected in 50% of gastric and in 70% of extragastric GISTs. Moreover, 70% of tumours with a mitotic rate ≥ 5 x 50 highpower fields (HPFs) underwent KIT gene mutations. Conversely, PDGFRα mutations were observed only in gastric GISTs with a mitotic rate ≤ 5 x 50 HPFs. By stratifying GISTs according to classes of risk, KIT mutation was shown in most of the high-risk tumours. PDGFRα mutations occurred exclusively in lower classes of risk. Conclusion: Molecular analysis data might have a role as a prognostic variable in models of risk assessment for patients with GISTs