Teaching genomics to life science undergraduates using cloud computing platforms with open datasets

The final year of a biochemistry degree is usually a time to experience research. However, laboratory-based research projects were not possible during COVID-19. Instead, we used open datasets to provide computational research projects in metagenomics to biochemistry undergraduates (80 students with limited computing experience). We aimed to give the students a chance to explore any dataset, rather than use a small number of artificial datasets (~60 published datasets were used). To achieve this, we utilized Google Colaboratory (Colab), a virtual computing environment. Colab was used as a framework to retrieve raw sequencing data (analyzed with QIIME2) and generate visualizations. Setting up the environment requires no prior experience; all students have the same drive structure and notebooks can be shared (for synchronous sessions). We also used the platform to combine multiple datasets, perform a meta-analysis, and allowed the students to analyze large datasets with 1000s of subjects and factors. Projects that required increased computational resources were integrated with Google Cloud Compute. In future, all research projects can include some aspects of reanalyzing public data, providing students with data science experience. Colab is also an excellent environment in which to develop data skills in multiple languages (e.g., Perl, Python, Julia)

A non-transcriptional role for the glucocorticoid receptor in mediating the cell stress response

This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.The glucocorticoid receptor (GR) is essential for the stress response in mammals. We investigated potential non-transcriptional roles of GR in cellular stress response using fission yeast as a model.We surprisingly discovered marked heat stress resistance in yeast ectopically expressing human GR, which required expression of both the N-terminal transactivation domain, and the C-terminal ligand binding domain, but not the DNA-binding domain of the GR. This effect was not affected by GR ligand exposure, and occurred without significant GR nuclear accumulation. Mechanistically, the GR survival effect required Hsp104, and, indeed, GR expression increased Hsp104 expression. Proteomic analysis revealed GR binding to translasome components, including eIF3, a known partner for Sty1, a pattern of protein interaction which we confirmed using yeast two-hybrid studies.Taken together, we find evidence for a novel pathway conferring stress resistance in yeast that can be activated by the human GR, acting by protein-protein mechanisms in the cytoplasm. This suggests that in organisms where GR is natively expressed, GR likely contributes to stress responses through non-transcriptional mechanisms in addition to its well-established transcriptional responses

The clock gene <i>Bmal1</i> inhibits macrophage motility, phagocytosis, and impairs defense against pneumonia

The circadian clock regulates many aspects of immunity. Bacterial infections are affected by time of day, but the mechanisms involved remain undefined. Here we show that loss of the core clock protein BMAL1 in macrophages confers protection against pneumococcal pneumonia. Infected mice show both reduced weight loss and lower bacterial burden in circulating blood. In vivo studies of macrophage phagocytosis reveal increased bacterial ingestion following Bmal1 deletion, which was also seen in vitro. BMAL1−/− macrophages exhibited marked differences in actin cytoskeletal organization, a phosphoproteome enriched for cytoskeletal changes, with reduced phosphocofilin and increased active RhoA. Further analysis of the BMAL1−/− macrophages identified altered cell morphology and increased motility. Mechanistically, BMAL1 regulated a network of cell movement genes, 148 of which were within 100 kb of high-confidence BMAL1 binding sites. Links to RhoA function were identified, with 29 genes impacting RhoA expression or activation. RhoA inhibition restored the phagocytic phenotype to that seen in control macrophages. In summary, we identify a surprising gain of antibacterial function due to loss of BMAL1 in macrophages, associated with a RhoA-dependent cytoskeletal change, an increase in cell motility, and gain of phagocytic function

Cardiac mitochondrial function depends on BUD23 mediated ribosome programming.

Efficient mitochondrial function is required in tissues with high energy demand such as the heart, and mitochondrial dysfunction is associated with cardiovascular disease. Expression of mitochondrial proteins is tightly regulated in response to internal and external stimuli. Here we identify a novel mechanism regulating mitochondrial content and function, through BUD23-dependent ribosome generation. BUD23 was required for ribosome maturation, normal 18S/28S stoichiometry and modulated the translation of mitochondrial transcripts in human A549 cells. Deletion of Bud23 in murine cardiomyocytes reduced mitochondrial content and function, leading to severe cardiomyopathy and death. We discovered that BUD23 selectively promotes ribosomal interaction with low GC-content 5'UTRs. Taken together we identify a critical role for BUD23 in bioenergetics gene expression, by promoting efficient translation of mRNA transcripts with low 5'UTR GC content. BUD23 emerges as essential to mouse development, and to postnatal cardiac function

The Methyltransferase WBSCR22/Merm1 Enhances Glucocorticoid Receptor Function and Is Regulated in Lung Inflammation and Cancer

Glucocorticoids (GC) regulate cell fate and immune function. We identified the metastasis-promoting methyltransferase, metastasis-related methyltransferase 1 (WBSCR22/Merm1) as a novel glucocorticoid receptor (GR) regulator relevant to human disease. Merm1 binds the GR co-activator GRIP1 but not GR. Loss of Merm1 impaired both GR transactivation and transrepression by reducing GR recruitment to its binding sites. This was accompanied by loss of GR-dependent H3K4Me3 at a well characterized promoter. Inflammation promotes GC resistance, in part through the actions of TNFα and IFNγ. These cytokines suppressed Merm1 protein expression by driving ubiquitination of two conserved lysine residues. Restoration of Merm1 expression rescued GR transactivation. Cytokine suppression of Merm1 and of GR function was also seen in human lung explants. In addition, striking loss of Merm1 protein was observed in both inflammatory and neoplastic human lung pathologies. In conclusion, Merm1 is a novel regulator of chromatin structure affecting GR recruitment and function, contributing to loss of GC sensitivity in inflammation, with suppressed expression in pulmonary disease

AKR1D1 knockout mice develop a sex-dependent metabolic phenotype

Steroid 5β-reductase (AKR1D1) plays important role in hepatic bile acid synthesis and glucocorticoid clearance. Bile acids and glucocorticoids are potent metabolic regulators, but whether AKR1D1 controls metabolic phenotype in vivo is unknown. Akr1d1–/– mice were generated on a C57BL/6 background. Liquid chromatography/mass spectrometry, metabolomic and transcriptomic approaches were used to determine effects on glucocorticoid and bile acid homeostasis. Metabolic phenotypes including body weight and composition, lipid homeostasis, glucose tolerance and insulin tolerance were evaluated. Molecular changes were assessed by RNA-Seq and Western blotting. Male Akr1d1–/– mice were challenged with a high fat diet (60% kcal from fat) for 20 weeks. Akr1d1–/– mice had a sex-specific metabolic phenotype. At 30 weeks of age, male, but not female, Akr1d1–/– mice were more insulin tolerant and had reduced lipid accumulation in the liver and adipose tissue yet had hypertriglyceridemia and increased intramuscular triacylglycerol. This phenotype was associated with sexually dimorphic changes in bile acid metabolism and composition but without overt effects on circulating glucocorticoid levels or glucocorticoid-regulated gene expression in the liver. Male Akr1d1–/– mice were not protected against diet-induced obesity and insulin resistance. In conclusion, this study shows that AKR1D1 controls bile acid homeostasis in vivo and that altering its activity can affect insulin tolerance and lipid homeostasis in a sex-dependent manner.</p

REVERBa couples the circadian clock to hepatic glucocorticoid action.

The glucocorticoid receptor (GR) is a major drug target in inflammatory disease. However, chronic glucocorticoid (GC) treatment leads to disordered energy metabolism, including increased weight gain, adiposity, and hepatosteatosis - all programs modulated by the circadian clock. We demonstrated that while antiinflammatory GC actions were maintained irrespective of dosing time, the liver was significantly more GC sensitive during the day. Temporal segregation of GC action was underpinned by a physical interaction of GR with the circadian transcription factor REVERBa and co-binding with liver-specific hepatocyte nuclear transcription factors (HNFs) on chromatin. REVERBa promoted efficient GR recruitment to chromatin during the day, acting in part by maintaining histone acetylation, with REVERBa-dependent GC responses providing segregation of carbohydrate and lipid metabolism. Importantly, deletion of Reverba inverted circadian liver GC sensitivity and protected mice from hepatosteatosis induced by chronic GC administration. Our results reveal a mechanism by which the circadian clock acts through REVERBa in liver on elements bound by HNF4A/HNF6 to direct GR action on energy metabolism

Investigations into the cardioprotective properties of resveratrol

In this study the ability of resveratrol to inhibit the activation of the monocyte respiratory burst was investigated.;Differentiated U937 (dU937) cells were pre-treated with resveratrol before stimulation with f-met-leu-phe (fMLP), phorbol 12-myristate-13-acetate (PMA) or arachidonic acid (AA). The extra-cellular and total production of reactive oxygen species (ROS) were measured by isoluminol and luminol chemiluminescence (CL). Superoxide production was measured using lucigenin. Resveratrol was found to inhibit ROS production induced by all three stimuli. Measurement of ROS production was also confirmed using with two used, 2',7'-dichlorofluorescein (DCF) and dihydrorhodamine (DHR). Again resveratrol inhibited both responses. There were significant between the inhibitory effects of resveratrol on peroxidase-dependent (isoluminol, luminol, DCF and DHR) and independent (lucigenin- ROS measuring principles. Moreover, resveratrol was found to be oxidised by the horseradish peroxidase/hydrogen peroxide system.;The cell signal transduction pathways activated by fMLP, PMA and AA were investigated. Only fMLP was found to activate phosphatidylinositol-3-kinase (PI3K) and Akt, using specific inhibitors of both kinases. Resveratrol inhibited PI3K activity with little direct effect on other kinases shown to regulate the respiratory burst, including Akt, extra-cellular regulated protein kinase (ERK); and protein kinase C (PKC). Akt and ERK were found to be activated by fMLP.;In conclusion, resveratrol was found to be a potent inhibitor of ROS production, particularly if a peroxidase-dependent measuring principle was used. Resveratrol can be oxidised by peroxidases, which inhibit the oxidation of the redox probe. Use of a detection method that did not require peroxidase revealed that resveratrol was still a potent inhibitor of fMLP-induced ROS. Moreover, this inhibitory dose of resveratrol correlated with its ability to inhibit the PI3K-Akt pathway, one of the major regulatory pathways of fMLP-induced ROS production. Modulation of these cell signalling intermediates by resveratrol might represent an important anti-inflammatory pathway and further add to its potential cardioprotective properties

Protein Kinase C Epsilon Contributes to NADPH Oxidase Activation in a Pre-Eclampsia Lymphoblast Cell Model

Pre-eclampsia is a pregnancy-specific disorder characterised by hypertension and proteinuria, which in severe cases results in multi-system disturbances. The maternal syndrome is associated with a pro-inflammatory state, consisting of leukocyte activation, which is thought to contribute to the widespread endothelial dysfunction. We previously showed increased activation of NADPH oxidase in pre-eclampsia, in both neutrophils and B-lymphoblast cell lines (B-LCLs). In this study, the mechanism by which NADPH oxidase activity is increased in pre-eclampsia was further investigated. NADPH oxidase activity was found to be increased in phorbol-12-myristate-13-acetate (PMA) stimulated B-LCLs isolated from women with pre-eclampsia. This correlated with an increase in protein kinase C (PKC) substrate phosphorylation, p47-phox phosphorylation (a regulatory component of NADPH oxidase) and p47-phox directed-kinase activity. Using ion exchange and hydroxyapatite chromatography we identified a major peak of PMA regulated p47-phox kinase activity. Chromatography fractions were probed for PKC isoforms. We found the major peak of p47-phox kinase activity could not be separated from the elution profile of PKC epsilon. Using a peptide inhibitor of PKC epsilon, PMA-induced reactive oxygen species (ROS) production could be reduced to that of a normal B-LCL. These data suggest a pro-inflammatory role for PKC epsilon in the pathogenesis of pre-eclampsia

An improved method for quantitative ChIP studies of nuclear receptor function

Chromatin immunoprecipitation (ChIP) is a valuable tool for the endocrine researcher, providing a means to measure the recruitment of hormone-activated nuclear receptors, for example. However, the technique can be challenging to perform and has multiple experimental steps, risking introduction of error at each. The data produced can be challenging to interpret; several different methods are commonly used for normalising data and thus comparing between conditions. Absolute, sensitive quantification of protein-bound DNA is important for correct interpretation of the data. In addition, such quantification can help the investigator in troubleshooting experiments. Here, we outline a ChIP strategy combining droplet digital PCR for accurate quantification with an internal spike-in control for normalisation. This combination strengthens the reliability of ChIP data and allows the operator to optimise their protocol with greater confidence