Inbred Mouse Populations Exhibit Intergenerational Changes in Intestinal Microbiota Composition and Function Following Introduction to a Facility

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Inbred mice are used to investigate many aspects of human physiology, including susceptibility to disease and response to therapies. Despite increasing evidence that the composition and function of the murine intestinal microbiota can substantially influence a broad range of experimental outcomes, relatively little is known about microbiome dynamics within experimental mouse populations. We investigated changes in the intestinal microbiome between C57BL/6J mice spanning six generations (assessed at generations 1, 2, 3, and 6), following their introduction to a stringently controlled facility. Fecal microbiota composition and function were assessed by 16S rRNA gene amplicon sequencing and liquid chromatography mass spectrometry, respectively. Significant divergence of the intestinal microbiota between founder and second generation mice, as well as continuing inter-generational variance, was observed. Bacterial taxa whose relative abundance changed significantly through time included Akkermansia, Turicibacter, and Bifidobacterium (p < 0.05), all of which are recognized as having the potential to substantially influence host physiology. Shifts in microbiota composition were mirrored by corresponding differences in the fecal metabolome (r = 0.57, p = 0.0001), with notable differences in levels of tryptophan pathway metabolites and amino acids, including glutamine, glutamate and aspartate. We related the magnitude of changes in the intestinal microbiota and metabolome characteristics during acclimation to those observed between populations housed in separate facilities, which differed in regards to husbandry, barrier conditions and dietary intake. The microbiome variance reported here has implications for experimental reproducibility, and as a consequence, experimental design and the interpretation of research outcomes across wide range of contexts

Synthesis and mass spectrometric analysis of disaccharides from methanolysis of heparan sulfate

The quantification of heparan sulfate (HS) in biological matrices, e.g., urine, cerebrospinal fluid, tissue samples etc., is of great importance for the diagnosis and prognosis of several of the mucopolysaccharidosis (MPS) disorders, which are lysosomal storage diseases of impaired glycosaminoglycan metabolism. The development of suitable assays for this purpose is challenging due to the high molecular weight and complexity of HS. Recent efforts towards this goal include the acid catalysed methanolysis of HS, which desulfates the polymer and results in the formation of disaccharide cleavage products which can be detected and quantified by LC-MS/MS. We have synthesized a library of 12 HS-derived disaccharides as methanolysis standards via the stereoselective 1,2-cis glycosylation of suitably protected GlcA and IdoA acceptors with a 2-deoxy-2-azido thioglucoside donor. This facilitated identification of the major peaks in the LC-MS/MS chromatograms, and potentially will allow the monitoring of specific metabolites as surrogate markers for genotype. This work also paves the way towards a fully quantitative LC-MS/MS assay for HS via the preparation of a suitably labelled derivative

Unravelling Prostate Cancer Heterogeneity Using Spatial Approaches to Lipidomics and Transcriptomics

Due to advances in the detection and management of prostate cancer over the past 20 years, most cases of localised disease are now potentially curable by surgery or radiotherapy, or amenable to active surveillance without treatment. However, this has given rise to a new dilemma for disease management; the inability to distinguish indolent from lethal, aggressive forms of prostate cancer, leading to substantial overtreatment of some patients and delayed intervention for others. Driving this uncertainty is the critical deficit of novel targets for systemic therapy and of validated biomarkers that can inform treatment decision-making and to select and monitor therapy. In part, this lack of progress reflects the inherent challenge of undertaking target and biomarker discovery in clinical prostate tumours, which are cellularly heterogeneous and multifocal, necessitating the use of spatial analytical approaches. In this review, the principles of mass spectrometry-based lipid imaging and complementary gene-based spatial omics technologies, their application to prostate cancer and recent advancements in these technologies are considered. We put in perspective studies that describe spatially-resolved lipid maps and metabolic genes that are associated with prostate tumours compared to benign tissue and increased risk of disease progression, with the aim of evaluating the future implementation of spatial lipidomics and complementary transcriptomics for prognostication, target identification and treatment decision-making for prostate cancer

Eukaryotic elongation factor 2 kinase upregulates the expression of proteins implicated in cell migration and cancer cell metastasis

Eukaryotic elongation factor 2 kinase (eEF2K) negatively regulates the elongation phase of mRNA translation and hence protein synthesis. Increasing evidence indicates that eEF2K plays an important role in the survival and migration of cancer cells and in tumor progression. As demonstrated by two-dimensional wound-healing and three-dimensional transwell invasion assays, knocking down or inhibiting eEF2K in cancer cells impairs migration and invasion of cancer cells. Conversely, exogenous expression of eEF2K or knocking down eEF2 (the substrate of eEF2K) accelerates wound healing and invasion. Importantly, using LC-HDMSE analysis, we identify 150 proteins whose expression is decreased and 73 proteins which are increased upon knocking down eEF2K in human lung carcinoma cells. Of interest, 34 downregulated proteins are integrins and other proteins implicated in cell migration, suggesting that inhibiting eEF2K may help prevent cancer cell mobility and metastasis. Interestingly, eEF2K promotes the association of integrin mRNAs with polysomes, providing a mechanism by which eEF2K may enhance their cellular levels. Consistent with this, genetic knock down or pharmacological inhibition of eEF2K reduces the protein expression levels of integrins. Notably, pharmacological or genetic inhibition of eEF2K almost completely blocked tumor growth and effectively prevented the spread of tumor cells in vivo. High levels of eEF2K expression were associated with invasive carcinoma and metastatic tumors. These data provide the evidence that eEF2K is a new potential therapeutic target for preventing tumor metastasis.</p

Increased Alveolar Heparan Sulphate and Reduced Pulmonary Surfactant Amount and Function in the Mucopolysaccharidosis IIIA Mouse

Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disease with significant neurological and skeletal pathologies. Respiratory dysfunction is a secondary pathology contributing to mortality in MPS IIIA patients. Pulmonary surfactant is crucial to optimal lung function and has not been investigated in MPS IIIA. We measured heparan sulphate (HS), lipids and surfactant proteins (SP) in pulmonary tissue and bronchoalveolar lavage fluid (BALF), and surfactant activity in healthy and diseased mice (20 weeks of age). Heparan sulphate, ganglioside GM3 and bis(monoacylglycero)phosphate (BMP) were increased in MPS IIIA lung tissue. There was an increase in HS and a decrease in BMP and cholesteryl esters (CE) in MPS IIIA BALF. Phospholipid composition remained unchanged, but BALF total phospholipids were reduced (49.70%) in MPS IIIA. There was a reduction in SP-A, -C and -D mRNA, SP-D protein in tissue and SP-A, -C and -D protein in BALF of MPS IIIA mice. Captive bubble surfactometry showed an increase in minimum and maximum surface tension and percent surface area compression, as well as a higher compressibility and hysteresis in MPS IIIA surfactant upon dynamic cycling. Collectively these biochemical and biophysical changes in alveolar surfactant are likely to be detrimental to lung function in MPS III

Reciprocal signaling between mTORC1 and MNK2 controls cell growth and oncogenesis

eIF4E plays key roles in protein synthesis and tumorigenesis. It is phosphorylated by the kinases MNK1 and MNK2. Binding of MNKs to eIF4G enhances their ability to phosphorylate eIF4E. Here, we show that mTORC1, a key regulator of mRNA translation and oncogenesis, directly phosphorylates MNK2 on Ser74. This suppresses MNK2 activity and impairs binding of MNK2 to eIF4G. These effects provide a novel mechanism by which mTORC1 signaling impairs the function of MNK2 and thereby decreases eIF4E phosphorylation. MNK2[S74A] knock-in cells show enhanced phosphorylation of eIF4E and S6K1 (i.e., increased mTORC1 signaling), enlarged cell size, and increased invasive and transformative capacities. MNK2[Ser74] phosphorylation was inversely correlated with disease progression in human prostate tumors. MNK inhibition exerted anti-proliferative effects in prostate cancer cells in vitro. These findings define a novel feedback loop whereby mTORC1 represses MNK2 activity and oncogenic signaling through eIF4E phosphorylation, allowing reciprocal regulation of these two oncogenic pathways

Compromised transcription-mRNA export factor THOC2 causes R-loop accumulation, DNA damage and adverse neurodevelopment

Abstract We implicated the X-chromosome THOC2 gene, which encodes the largest subunit of the highly-conserved TREX (Transcription-Export) complex, in a clinically complex neurodevelopmental disorder with intellectual disability as the core phenotype. To study the molecular pathology of this essential eukaryotic gene, we generated a mouse model based on a hypomorphic Thoc2 exon 37–38 deletion variant of a patient with ID, speech delay, hypotonia, and microcephaly. The Thoc2 exon 37–38 deletion male (Thoc2 Δ/Y ) mice recapitulate the core phenotypes of THOC2 syndrome including smaller size and weight, and significant deficits in spatial learning, working memory and sensorimotor functions. The Thoc2 Δ/Y mouse brain development is significantly impacted by compromised THOC2/TREX function resulting in R-loop accumulation, DNA damage and consequent cell death. Overall, we suggest that perturbed R-loop homeostasis, in stem cells and/or differentiated cells in mice and the patient, and DNA damage-associated functional alterations are at the root of THOC2 syndrome