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Additional file 3: Fig. E3. SDS-PAGE and immunoblot is shown for both bovine collagen after duodenal digestion preceded by gastric digestion at pH 1.2/PPR 0.1. Under these conditions, bovine collagen is fully resistant to duodenal digestion.

Additional file 1: Fig. E1. Selected SDS-PAGE and immunoblot samples are shown for both albumins, Ara h 2 and pea albumin PA2. Panel a illustrates that pea albumin PA2 is truncated at low PPR but truncated molecules are still detected on immunoblot. Panel b shows gastric digestion of Ara h 2 at low pH and high PPR. Under these conditions the upper band is quite stable but the lower band is truncated. Truncated molecules are not recognized on immunoblot. Panel c and d illustrates that Ara h 2 (panel c) and pea albumin PA2 (panel d) behave differently when gastric digestion at pH 1.2 and pH 2.5 are compared. Ara h 2 is quite stable under both conditions, but if anything slightly more susceptible to digestion at pH 2.5. For pea albumin PA2 this is the other way around. It is clearly more resistant to digestion at pH 2.5 than pH 1.2.

Discussion of organelle inheritance

Discussion of establishment of sex in multicellular organisms

Discussion of ROS producing processes during photosynthesis

Vanishing white matter (VWM) is a leukodystrophy with predominantly early-childhood onset. Affected children display various neurological signs, including ataxia and spasticity, and die early. VWM patients have bi-allelic mutations in any of the five genes encoding the subunits of the eukaryotic translation factor 2B (eIF2B). eIF2B regulates protein synthesis rates under basal and cellular stress conditions. The underlying molecular mechanism of how mutations in eIF2B result in VWM is unknown. Previous studies suggest that brain white matter astrocytes are primarily affected in VWM. We hypothesized that the translation rate of certain astrocytic mRNAs is affected by the mutations, resulting in astrocytic dysfunction. Here we subjected primary astrocyte cultures of wild type (wt) and VWM (2b5ho) mice to pulsed labeling proteomics based on stable isotope labeling with amino acids in cell culture (SILAC) with an L-azidohomoalanine (AHA) pulse to select newly synthesized proteins. AHA was incorporated into newly synthesized proteins in wt and 2b5ho astrocytes with similar efficiency, without affecting cell viability. We quantified proteins synthesized in astrocytes of wt and 2b5ho mice. This proteomic profiling identified a total of 80 proteins that were regulated by the eIF2B mutation.We confirmed increased expression of PROS1 in 2b5ho astrocytes and brain. A DAVID enrichment analysis showed that approximately 50% of the eIF2B-regulated proteins used the secretory pathway. A small-scale metabolic screen further highlighted a significant change in the metabolite 6-phospho-gluconate, indicative of an altered flux through the pentose phosphate pathway (PPP). Some of the proteins migrating through the secretory pathway undergo oxidative folding reactions in the endoplasmic reticulum (ER), which produces reactive oxygen species (ROS). The PPP produces NADPH to remove ROS. The proteomic and metabolomics data together suggest a deregulation of ER function in 2b5ho mouse astrocytes.