Extensive folding variability between homologous chromosomes in mammalian cells.

Genetic variation and 3D chromatin structure have major roles in gene regulation. Due to challenges in mapping chromatin conformation with haplotype-specific resolution, the effects of genetic sequence variation on 3D genome structure and gene expression imbalance remain understudied. Here, we applied Genome Architecture Mapping (GAM) to a hybrid mouse embryonic stem cell (mESC) line with high density of single nucleotide polymorphisms (SNPs). GAM resolved haplotype-specific 3D genome structures with high sensitivity, revealing extensive allelic differences in chromatin compartments, topologically associating domains (TADs), long-range enhancer-promoter contacts, and CTCF loops. Architectural differences often coincide with allele-specific differences in gene expression, mediated by Polycomb repression. We show that histone genes are expressed with allelic imbalance in mESCs, are involved in haplotype-specific chromatin contact marked by H3K27me3, and are targets of Polycomb repression through conditional knockouts of Ezh2 or Ring1b. Our work reveals highly distinct 3D folding structures between homologous chromosomes, and highlights their intricate connections with allelic gene expression.

L-Glyceraldehyde Inhibits Neuroblastoma Cell Growth via a Multi-Modal Mechanism on Metabolism and Signaling.

Glyceraldehyde (GA) is a three-carbon monosaccharide that can be present in cells as a by-product of fructose metabolism. Bruno Mendel and Otto Warburg showed that the application of GA to cancer cells inhibits glycolysis and their growth. However, the molecular mechanism by which this occurred was not clarified. We describe a novel multi-modal mechanism by which the L-isomer of GA (L-GA) inhibits neuroblastoma cell growth. L-GA induces significant changes in the metabolic profile, promotes oxidative stress and hinders nucleotide biosynthesis. GC-MS and 13C-labeling was employed to measure the flow of carbon through glycolytic intermediates under L-GA treatment. It was found that L-GA is a potent inhibitor of glycolysis due to its proposed targeting of NAD(H)-dependent reactions. This results in growth inhibition, apoptosis and a redox crisis in neuroblastoma cells. It was confirmed that the redox mechanisms were modulated via L-GA by proteomic analysis. Analysis of nucleotide pools in L-GA-treated cells depicted a previously unreported observation, in which nucleotide biosynthesis is significantly inhibited. The inhibitory action of L-GA was partially relieved with the co-application of the antioxidant N-acetyl-cysteine. We present novel evidence for a simple sugar that inhibits cancer cell proliferation via dysregulating its fragile homeostatic environment.

Protective role of the HSP90 inhibitor, STA-9090, in lungs of SARS-CoV-2-infected Syrian golden hamsters.

INTRODUCTION: The emergence of new SARS-CoV-2 variants, capable of escaping the humoral immunity acquired by the available vaccines, together with waning immunity and vaccine hesitancy, challenges the efficacy of the vaccination strategy in fighting COVID-19. Improved therapeutic strategies are urgently needed to better intervene particularly in severe cases of the disease. They should aim at controlling the hyperinflammatory state generated on infection, reducing lung tissue pathology and inhibiting viral replication. Previous research has pointed to a possible role for the chaperone HSP90 in SARS-CoV-2 replication and COVID-19 pathogenesis. Pharmacological intervention through HSP90 inhibitors was shown to be beneficial in the treatment of inflammatory diseases, infections and reducing replication of diverse viruses. METHODS: In this study, we investigated the effects of the potent HSP90 inhibitor Ganetespib (STA-9090) in vitro on alveolar epithelial cells and alveolar macrophages to characterise its effects on cell activation and viral replication. Additionally, the Syrian hamster animal model was used to evaluate its efficacy in controlling systemic inflammation and viral burden after infection. RESULTS: In vitro, STA-9090 reduced viral replication on alveolar epithelial cells in a dose-dependent manner and lowered significantly the expression of proinflammatory genes, in both alveolar epithelial cells and alveolar macrophages. In vivo, although no reduction in viral load was observed, administration of STA-9090 led to an overall improvement of the clinical condition of infected animals, with reduced oedema formation and lung tissue pathology. CONCLUSION: Altogether, we show that HSP90 inhibition could serve as a potential treatment option for moderate and severe cases of COVID-19.

Sodium as an Important Regulator of Immunometabolism.

Salt sensitivity concerns blood pressure alterations after a change in salt intake (sodium chloride). The heart is a pump, and vessels are tubes; sodium can affect both. A high salt intake increases cardiac output, promotes vascular dysfunction and capillary rarefaction, and chronically leads to increased systemic vascular resistance. More recent findings suggest that sodium also acts as an important second messenger regulating energy metabolism and cellular functions. Besides endothelial cells and fibroblasts, sodium also affects innate and adaptive immunometabolism, immune cell function, and influences certain microbes and microbiota-derived metabolites. We propose the idea that the definition of salt sensitivity should be expanded beyond high blood pressure to cellular and molecular salt sensitivity.

Spatiotemporal, optogenetic control of gene expression in organoids.

Organoids derived from stem cells have become an increasingly important tool for studying human development and modeling disease. However, methods are still needed to control and study spatiotemporal patterns of gene expression in organoids. Here we combined optogenetics and gene perturbation technologies to activate or knock-down RNA of target genes in programmable spatiotemporal patterns. To illustrate the usefulness of our approach, we locally activated Sonic Hedgehog (SHH) signaling in an organoid model for human neurodevelopment. Spatial and single-cell transcriptomic analyses showed that this local induction was sufficient to generate stereotypically patterned organoids and revealed new insights into SHH's contribution to gene regulation in neurodevelopment. With this study, we propose optogenetic perturbations in combination with spatial transcriptomics as a powerful technology to reprogram and study cell fates and tissue patterning in organoids.

A comprehensive molecular profiling approach reveals metabolic alterations that steer bone tissue regeneration.

Bone regeneration after fracture is a complex process with high and dynamic energy demands. The impact of metabolism on bone healing progression and outcome, however, is so far understudied. Our comprehensive molecular profiling reveals that central metabolic pathways, such as glycolysis and the citric acid cycle, are differentially activated between rats with successful or compromised bone regeneration (young versus aged female Sprague-Dawley rats) early in the inflammatory phase of bone healing. We also found that the citric acid cycle intermediate succinate mediates individual cellular responses and plays a central role in successful bone healing. Succinate induces IL-1ß in macrophages, enhances vessel formation, increases mesenchymal stromal cell migration, and potentiates osteogenic differentiation and matrix formation in vitro. Taken together, metabolites-here particularly succinate-are shown to play central roles as signaling molecules during the onset of healing and in steering bone tissue regeneration.

Corrigendum: Exercise blood-drop metabolic profiling links metabolism with perceived exertion.

[This corrects the article DOI: 10.3389/fmolb.2022.1042231.].

Sodium perturbs mitochondrial respiration and induces dysfunctional Tregs.

FOXP3+ regulatory T cells (Tregs) are central for peripheral tolerance, and their deregulation is associated with autoimmunity. Dysfunctional autoimmune Tregs display pro-inflammatory features and altered mitochondrial metabolism, but contributing factors remain elusive. High salt (HS) has been identified to alter immune function and to promote autoimmunity. By investigating longitudinal transcriptional changes of human Tregs, we identified that HS induces metabolic reprogramming, recapitulating features of autoimmune Tregs. Mechanistically, extracellular HS raises intracellular Na+, perturbing mitochondrial respiration by interfering with the electron transport chain (ETC). Metabolic disturbance by a temporary HS encounter or complex III blockade rapidly induces a pro-inflammatory signature and FOXP3 downregulation, leading to long-term dysfunction in vitro and in vivo. The HS-induced effect could be reversed by inhibition of mitochondrial Na+/Ca2+ exchanger (NCLX). Our results indicate that salt could contribute to metabolic reprogramming and that short-term HS encounter perturb metabolic fitness and long-term function of human Tregs with important implications for autoimmunity.

Advancements in Pulsed Stable Isotope-Resolved Metabolomics.

The understanding of biochemical processes of metabolism is gained through the measurement of the concentration of intermediates and the rate of metabolite conversion. However, the measurement of metabolite concentrations does not give a full representation of this dynamic system. To understand the kinetics of metabolism, the system must be described and quantified in terms of metabolite flow as a function of time. In order to measure the metabolite flow, or more precisely the metabolic flux through a biological system, substrates of the cell are labelled with stable isotopes. The usage of these substrates by the cell leads to the incorporation of the isotopes into downstream intermediates.The most important metabolic pathways are encompassed in the central carbon metabolism (CCM). According to the Kyoto Encyclopedia of Genes and Genomes (KEGG), the central carbon metabolism "is the most basic aspect of life". It includes all metabolites and enzymatic reactions within: glycolysis and gluconeogenesis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), amino acids and nucleotide metabolic pathways. Some molecules are at the crossroad of metabolic pathways, interconnecting diverse metabolic and therefore functional outcomes. Labelling these nodal metabolites and analysing their isotopic composition allows the precise determination of the metabolic flow within the biochemical networks that they are in.Application of stable isotope labelled substrates allows the measurement of metabolic flux through a biochemical pathway. The rapid turnover of metabolites in pathways requires pulse-feeding cells with a labelled substrate. This method allows for the determination of different cell states. For example, the action of a drug from immediate impact until the compensatory response of the metabolic system (cell, organs, organisms). Pulsed labelling is an elegant way to analyse the action of small molecules and drugs and enables the analysis of regulatory metabolic processes in short time scales.

Quantifying the impact of gut microbiota on inflammation and hypertensive organ damage.

AIMS: Hypertension (HTN) can lead to heart and kidney damage. The gut microbiota has been linked to HTN, although it is difficult to estimate its significance due to the variety of other features known to influence HTN. In the present study, we used germ-free (GF) and colonized (COL) littermate mice to quantify the impact of microbial colonization on organ damage in HTN. METHODS AND RESULTS: 4-week-old male GF C57BL/6J littermates were randomized to remain GF or receive microbial colonization. HTN was induced by subcutaneous infusion with angiotensin (Ang) II (1.44 mg/kg/day) and 1% NaCl in the drinking water; sham-treated mice served as control. Renal damage was exacerbated in GF mice, whereas cardiac damage was more comparable between COL and GF, suggesting that the kidney is more sensitive to microbial influence. Multivariate analysis revealed a larger effect of HTN in GF mice. Serum metabolomics demonstrated that the colonization status influences circulating metabolites relevant to HTN. Importantly, GF mice were deficient in anti-inflammatory faecal short-chain fatty acids (SCFA). Flow cytometry showed that the microbiome has an impact on the induction of anti-hypertensive myeloid-derived suppressor cells and pro-inflammatory Th17 cells in HTN. In vitro inducibility of Th17 cells was significantly higher for cells isolated from GF than conventionally raised mice. CONCLUSION: The microbial colonization status of mice had potent effects on their phenotypic response to a hypertensive stimulus, and the kidney is a highly microbiota-susceptible target organ in HTN. The magnitude of the pathogenic response in GF mice underscores the role of the microbiome in mediating inflammation in HTN.

Inflammation in Children with CKD Linked to Gut Dysbiosis and Metabolite Imbalance.

BACKGROUND: CKD is characterized by a sustained proinflammatory response of the immune system, promoting hypertension and cardiovascular disease. The underlying mechanisms are incompletely understood but may be linked to gut dysbiosis. Dysbiosis has been described in adults with CKD; however, comorbidities limit CKD-specific conclusions. METHODS: We analyzed the fecal microbiome, metabolites, and immune phenotypes in 48 children (with normal kidney function, CKD stage G3-G4, G5 treated by hemodialysis [HD], or kidney transplantation) with a mean±SD age of 10.6±3.8 years. RESULTS: Serum TNF-α and sCD14 were stage-dependently elevated, indicating inflammation, gut barrier dysfunction, and endotoxemia. We observed compositional and functional alterations of the microbiome, including diminished production of short-chain fatty acids. Plasma metabolite analysis revealed a stage-dependent increase of tryptophan metabolites of bacterial origin. Serum from patients on HD activated the aryl hydrocarbon receptor and stimulated TNF-α production in monocytes, corresponding to a proinflammatory shift from classic to nonclassic and intermediate monocytes. Unsupervised analysis of T cells revealed a loss of mucosa-associated invariant T (MAIT) cells and regulatory T cell subtypes in patients on HD. CONCLUSIONS: Gut barrier dysfunction and microbial metabolite imbalance apparently mediate the proinflammatory immune phenotype, thereby driving the susceptibility to cardiovascular disease. The data highlight the importance of the microbiota-immune axis in CKD, irrespective of confounding comorbidities.

Investigating the role of GLUL as a survival factor in cellular adaptation to glutamine depletion via targeted stable isotope resolved metabolomics.

Cellular glutamine synthesis is thought to be an important resistance factor in protecting cells from nutrient deprivation and may also contribute to drug resistance. The application of ?targeted stable isotope resolved metabolomics" allowed to directly measure the activity of glutamine synthetase in the cell. With the help of this method, the fate of glutamine derived nitrogen within the biochemical network of the cells was traced. The application of stable isotope labelled substrates and analyses of isotope enrichment in metabolic intermediates allows the determination of metabolic activity and flux in biological systems. In our study we used stable isotope labelled substrates of glutamine synthetase to demonstrate its role in the starvation response of cancer cells. We applied 13C labelled glutamate and 15N labelled ammonium and determined the enrichment of both isotopes in glutamine and nucleotide species. Our results show that the metabolic compensatory pathways to overcome glutamine depletion depend on the ability to synthesise glutamine via glutamine synthetase. We demonstrate that the application of dual-isotope tracing can be used to address specific reactions within the biochemical network directly. Our study highlights the potential of concurrent isotope tracing methods in medical research.

Renal Deletion of LRRC8/VRAC Channels Induces Proximal Tubulopathy.

BACKGROUND: Volume-regulated anion channels (VRACs) are heterohexamers of LRRC8A with LRRC8B, -C, -D, or -E in various combinations. Depending on the subunit composition, these swelling-activated channels conduct chloride, amino acids, organic osmolytes, and drugs. Despite VRACs' role in cell volume regulation, and large osmolarity changes in the kidney, neither the localization nor the function of VRACs in the kidney is known. METHODS: Mice expressing epitope-tagged LRRC8 subunits were used to determine the renal localization of all VRAC subunits. Mice carrying constitutive deletions of Lrrc8b-e, or with inducible or cell-specific ablation of Lrrc8a, were analyzed to assess renal functions of VRACs. Analysis included histology, urine and serum parameters in different diuresis states, and metabolomics. RESULTS: The kidney expresses all five VRAC subunits with strikingly distinct localization. Whereas LRRC8C is exclusively found in vascular endothelium, all other subunits are found in the nephron. LRRC8E is specific for intercalated cells, whereas LRRC8A, LRRC8B, and LRRC8D are prominent in basolateral membranes of proximal tubules. Conditional deletion of LRRC8A in proximal but not distal tubules and constitutive deletion of LRRC8D cause proximal tubular injury, increased diuresis, and mild Fanconi-like symptoms. CONCLUSIONS: VRAC/LRRC8 channels are crucial for the function and integrity of proximal tubules, but not for more distal nephron segments despite their larger need for volume regulation. LRRC8A/D channels may be required for the basolateral exit of many organic compounds, including cellular metabolites, in proximal tubules. Proximal tubular injury likely results from combined accumulation of several transported molecules in the absence of VRAC channels.

HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins.

The biological role of RNA-binding proteins in the secretory pathway is not well established. Here, we describe that human HDLBP/Vigilin directly interacts with more than 80% of ER-localized mRNAs. PAR-CLIP analysis reveals that these transcripts represent high affinity HDLBP substrates and are specifically bound in their coding sequences (CDS), in contrast to CDS/3'UTR-bound cytosolic mRNAs. HDLBP crosslinks strongly to long CU-rich motifs, which frequently reside in CDS of ER-localized mRNAs and result in high affinity multivalent interactions. In addition to HDLBP-ncRNA interactome, quantification of HDLBP-proximal proteome confirms association with components of the translational apparatus and the signal recognition particle. Absence of HDLBP results in decreased translation efficiency of HDLBP target mRNAs, impaired protein synthesis and secretion in model cell lines, as well as decreased tumor growth in a lung cancer mouse model. These results highlight a general function for HDLBP in the translation of ER-localized mRNAs and its relevance for tumor progression.

Exercise blood-drop metabolic profiling links metabolism with perceived exertion.

Background: Assessing detailed metabolism in exercising persons minute-to-minute has not been possible. We developed a "drop-of-blood" platform to fulfill that need. Our study aimed not only to demonstrate the utility of our methodology, but also to give insights into unknown mechanisms and new directions. Methods: We developed a platform, based on gas chromatography and mass spectrometry, to assess metabolism from a blood-drop. We first observed a single volunteer who ran 13 km in 60 min. We particularly monitored relative perceived exertion (RPE). We observed that 2,3-bisphosphoglycerate peaked at RPE in this subject. We next expanded these findings to women and men volunteers who performed an RPE-based exercise protocol to RPE at Fi O 2 20.9% or Fi O 2 14.5% in random order. Results: At 6 km, our subject reached his maximum relative perceived exertion (RPE); however, he continued running, felt better, and finished his run. Lactate levels had stably increased by 2 km, ketoacids increased gradually until the run's end, while the hypoxia marker, 2,3 bisphosphoglycerate, peaked at maximum relative perceived exertion. In our normal volunteers, the changes in lactate, pyruvate, ß hydroxybutyrate and a hydroxybutyrate were not identical, but similar to our model proband runner. Conclusion: Glucose availability was not the limiting factor, as glucose availability increased towards exercise end in highly exerted subjects. Instead, the tricarboxylic acid→oxphos pathway, lactate clearance, and thus and the oxidative capacity appeared to be the defining elements in confronting maximal exertion. These ideas must be tested further in more definitive studies. Our preliminary work suggests that our single-drop methodology could be of great utility in studying exercise physiology.

Optimized Workflow for On-Line Derivatization for Targeted Metabolomics Approach by Gas Chromatography-Mass Spectrometry.

Using manual derivatization in gas chromatography-mass spectrometry samples have varying equilibration times before analysis which increases technical variability and limits the number of potential samples analyzed. By contrast, automated derivatization methods can derivatize and inject each sample in an identical manner. We present a fully automated (on-line) derivatization method used for targeted analysis of different matrices. We describe method optimization and compare results from using off-line and on-line derivatization protocols, including the robustness and reproducibility of the methods. Our final parameters for the derivatization process were 20 µL of methoxyamine (MeOx) in pyridine for 60 min at 30 °C followed by 80 µL N-Methyl-N-trimethylsilyltrifluoracetamide (MSTFA) for 30 min at 30 °C combined with 4 h of equilibration time. The repeatability test in plasma and liver revealed a median relative standard deviation (RSD) of 16% and 10%, respectively. Serum samples showed a consistent intra-batch median RSD of 20% with an inter-batch variability of 27% across three batches. The direct comparison of on-line versus off-line demonstrated that on-line was fit for purpose and improves repeatability with a measured median RSD of 11% compared to 17% using the same method off-line. In summary, we recommend that optimized on-line methods may improve results for metabolomics and should be used where available.

Inhibiting PHGDH with NCT-503 reroutes glucose-derived carbons into the TCA cycle, independently of its on-target effect.

The small-molecule inhibitor of phosphoglycerate dehydrogenase, NCT-503, reduces incorporation of glucose-derived carbons into serine in vitro. Here we describe an off-target effect of NCT-503 in neuroblastoma cell lines expressing divergent phosphoglycerate dehydrogenase (PHGDH) levels and single-cell clones with CRISPR-Cas9-directed PHGDH knockout or their respective wildtype controls. NCT-503 treatment strongly reduced synthesis of glucose-derived citrate in all cell models investigated compared to the inactive drug control and independent of PHGDH expression level. Incorporation of glucose-derived carbons entering the TCA cycle via pyruvate carboxylase was enhanced by NCT-503 treatment. The activity of citrate synthase was not altered by NCT-503 treatment. We also detected no change in the thermal stabilisation of citrate synthase in cellular thermal shift assays from NCT-503-treated cells. Thus, the direct cause of the observed off-target effect remains enigmatic. Our findings highlight off-target potential within a metabolic assessment of carbon usage in cells treated with the small-molecule inhibitor, NCT-503.

Salt Transiently Inhibits Mitochondrial Energetics in Mononuclear Phagocytes.

BACKGROUND: Dietary high salt (HS) is a leading risk factor for mortality and morbidity. Serum sodium transiently increases postprandially but can also accumulate at sites of inflammation affecting differentiation and function of innate and adaptive immune cells. Here, we focus on how changes in extracellular sodium, mimicking alterations in the circulation and tissues, affect the early metabolic, transcriptional, and functional adaption of human and murine mononuclear phagocytes. METHODS: Using Seahorse technology, pulsed stable isotope-resolved metabolomics, and enzyme activity assays, we characterize the central carbon metabolism and mitochondrial function of human and murine mononuclear phagocytes under HS in vitro. HS as well as pharmacological uncoupling of the electron transport chain under normal salt is used to analyze mitochondrial function on immune cell activation and function (as determined by Escherichiacoli killing and CD4+ T cell migration capacity). In 2 independent clinical studies, we analyze the effect of a HS diet during 2 weeks (URL: http://www.clinicaltrials.gov. Unique identifier: NCT02509962) and short-term salt challenge by a single meal (URL: http://www.clinicaltrials.gov. Unique identifier: NCT04175249) on mitochondrial function of human monocytes in vivo. RESULTS: Extracellular sodium was taken up into the intracellular compartment, followed by the inhibition of mitochondrial respiration in murine and human macrophages. Mechanistically, HS reduces mitochondrial membrane potential, electron transport chain complex II activity, oxygen consumption, and ATP production independently of the polarization status of macrophages. Subsequently, cell activation is altered with improved bactericidal function in HS-treated M1-like macrophages and diminished CD4+ T cell migration in HS-treated M2-like macrophages. Pharmacological uncoupling of the electron transport chain under normal salt phenocopies HS-induced transcriptional changes and bactericidal function of human and murine mononuclear phagocytes. Clinically, also in vivo, rise in plasma sodium concentration within the physiological range reversibly reduces mitochondrial function in human monocytes. In both a 14-day and single meal HS challenge, healthy volunteers displayed a plasma sodium increase of [Formula: see text] and [Formula: see text] respectively, that correlated with decreased monocytic mitochondrial oxygen consumption. CONCLUSIONS: Our data identify the disturbance of mitochondrial respiration as the initial step by which HS mechanistically influences immune cell function. Although these functional changes might help to resolve bacterial infections, a shift toward proinflammation could accelerate inflammatory cardiovascular disease.

Rbm10 facilitates heterochromatin assembly via the Clr6 HDAC complex.

Splicing factors have recently been shown to be involved in heterochromatin formation, but their role in controlling heterochromatin structure and function remains poorly understood. In this study, we identified a fission yeast homologue of human splicing factor RBM10, which has been linked to TARP syndrome. Overexpression of Rbm10 in fission yeast leads to strong global intron retention. Rbm10 also interacts with splicing factors in a pattern resembling that of human RBM10, suggesting that the function of Rbm10 as a splicing regulator is conserved. Surprisingly, our deep-sequencing data showed that deletion of Rbm10 caused only minor effect on genome-wide gene expression and splicing. However, the mutant displays severe heterochromatin defects. Further analyses indicated that the heterochromatin defects in the mutant did not result from mis-splicing of heterochromatin factors. Our proteomic data revealed that Rbm10 associates with the histone deacetylase Clr6 complex and chromatin remodelers known to be important for heterochromatin silencing. Deletion of Rbm10 results in significant reduction of Clr6 in heterochromatin. Our work together with previous findings further suggests that different splicing subunits may play distinct roles in heterochromatin regulation.