MinD-RNase E interplay controls localization of polar mRNAs in E. coli.

The E. coli transcriptome at the cell's poles (polar transcriptome) is unique compared to the membrane and cytosol. Several factors have been suggested to mediate mRNA localization to the membrane, but the mechanism underlying polar localization of mRNAs remains unknown.

ChREBP is activated by reductive stress and mediates GCKR-associated metabolic traits.

Common genetic variants in glucokinase regulator (GCKR), which encodes GKRP, a regulator of hepatic glucokinase (GCK), influence multiple metabolic traits in genome-wide association studies (GWASs), making GCKR one of the most pleiotropic GWAS loci in the genome. It is unclear why. Prior work has demonstrated that GCKR influences the hepatic cytosolic NADH/NAD ratio, also referred to as reductive stress.

Regulation of major bacterial survival strategies by transcripts sequestration in a membraneless organelle.

TmaR, the only known pole-localizer protein in Escherichia coli, was shown to cluster at the cell poles and control localization and activity of the major sugar regulator in a tyrosine phosphorylation-dependent manner. Here, we show that TmaR assembles by phase separation (PS) via heterotypic interactions with RNA in vivo and in vitro. An unbiased automated mutant screen combined with directed mutagenesis and genetic manipulations uncovered the importance of a predicted nucleic-acid-binding domain, a disordered region, and charged patches, one containing the phosphorylated tyrosine, for TmaR condensation.

Heterotypic phase separation of Hfq is linked to its roles as an RNA chaperone.

Hfq, an Sm-like protein and the major RNA chaperone in E. coli, has been shown to distribute non-uniformly along a helical path under normal growth conditions and to relocate to the cell poles under certain stress conditions. We have previously shown that Hfq relocation to the poles is accompanied by polar accumulation of most small RNAs (sRNAs).

Combinatorial GxGxE CRISPR screen identifies SLC25A39 in mitochondrial glutathione transport linking iron homeostasis to OXPHOS.

The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes. The family is highly redundant and their transport activities coupled to metabolic state. Here, we use a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states - glucose, galactose, OXPHOS inhibition, and absence of pyruvate - designed to unmask the inter-dependence of these genes.

Wisdom of the crowds: A suggested polygenic plan for small-RNA-mediated regulation in bacteria.

The omnigenic/polygenic theory, which states that complex traits are not shaped by single/few genes, but by situation-specific large networks, offers an explanation for a major enigma in microbiology: deletion of specific small RNAs (sRNAs) playing key roles in various aspects of bacterial physiology, including virulence and antibiotic resistance, results in surprisingly subtle phenotypes. A recent study uncovered polar accumulation of most sRNAs upon osmotic stress, the majority not known to be involved in the applied stress. Here we show that cells deleted for a handful of pole-enriched sRNAs exhibit fitness defect in several stress conditions, as opposed to single, double, or triple sRNA-knockouts, implying that regulation by sRNA relies on sets of genes.

Hypoxia ameliorates brain hyperoxia and NAD deficiency in a murine model of Leigh syndrome.

Leigh syndrome is a severe mitochondrial neurodegenerative disease with no effective treatment. In the Ndufs4 mouse model of Leigh syndrome, continuously breathing 11% O (hypoxia) prevents neurodegeneration and leads to a dramatic extension (~5-fold) in lifespan. We investigated the effect of hypoxia on the brain metabolism of Ndufs4 mice by studying blood gas tensions and metabolite levels in simultaneously sampled arterial and cerebral internal jugular venous (IJV) blood.

Evolutionary divergence reveals the molecular basis of EMRE dependence of the human MCU.

The mitochondrial calcium uniporter (MCU) is a calcium-activated calcium channel critical for signaling and bioenergetics. MCU, the pore-forming subunit of the uniporter, contains two transmembrane domains and is found in all major eukaryotic taxa. In amoeba and fungi, MCU homologs are sufficient to form a functional calcium channel, whereas human MCU exhibits a strict requirement for the metazoan protein essential MCU regulator (EMRE) for conductance.

Hepatic NADH reductive stress underlies common variation in metabolic traits.

The cellular NADH/NAD ratio is fundamental to biochemistry, but the extent to which it reflects versus drives metabolic physiology in vivo is poorly understood. Here we report the in vivo application of Lactobacillus brevis (Lb)NOX, a bacterial water-forming NADH oxidase, to assess the metabolic consequences of directly lowering the hepatic cytosolic NADH/NAD ratio in mice. By combining this genetic tool with metabolomics, we identify circulating α-hydroxybutyrate levels as a robust marker of an elevated hepatic cytosolic NADH/NAD ratio, also known as reductive stress.

Multi-site reproducibility of a human immunophenotyping assay in whole blood and peripheral blood mononuclear cells preparations using CyTOF technology coupled with Maxpar Pathsetter, an automated data analysis system.

High-dimensional mass cytometry data potentially enable a comprehensive characterization of immune cells. In order to positively affect clinical trials and translational clinical research, this advanced technology needs to demonstrate a high reproducibility of results across multiple sites for both peripheral blood mononuclear cells (PBMC) and whole blood preparations. A dry 30-marker broad immunophenotyping panel and customized automated analysis software were recently engineered and are commercially available as the Fluidigm® Maxpar® Direct™ Immune Profiling Assay™.

Leigh Syndrome Mouse Model Can Be Rescued by Interventions that Normalize Brain Hyperoxia, but Not HIF Activation.

Leigh syndrome is a devastating mitochondrial disease for which there are no proven therapies. We previously showed that breathing chronic, continuous hypoxia can prevent and even reverse neurological disease in the Ndufs4 knockout (KO) mouse model of complex I (CI) deficiency and Leigh syndrome. Here, we show that genetic activation of the hypoxia-inducible factor transcriptional program via any of four different strategies is insufficient to rescue disease.

Exploring the In Vivo Role of the Mitochondrial Calcium Uniporter in Brown Fat Bioenergetics.

The mitochondrial calcium uniporter has been proposed to coordinate the organelle's energetics with calcium signaling. Uniporter current has previously been reported to be extremely high in brown adipose tissue (BAT), yet it remains unknown how the uniporter contributes to BAT physiology. Here, we report the generation and characterization of a mouse model lacking Mcu, the pore forming subunit of the uniporter, specifically in BAT (BAT-Mcu-KO).

Impaired hypoxic pulmonary vasoconstriction in a mouse model of Leigh syndrome.

Hypoxic pulmonary vasoconstriction (HPV) is a physiological vasomotor response that maintains systemic oxygenation by matching perfusion to ventilation during alveolar hypoxia. Although mitochondria appear to play an essential role in HPV, the impact of mitochondrial dysfunction on HPV remains incompletely defined. Mice lacking the mitochondrial complex I (CI) subunit Ndufs4 ( Ndufs4) develop a fatal progressive encephalopathy and serve as a model for Leigh syndrome, the most common mitochondrial disease in children.

Modulation of allergic responses by mitochondrial STAT3 inhibitors.

Background: Recently, we have shown that mast cell mitochondrial STAT3 could serve as a new target for the regulation of the allergic response as it plays an essential role in immunologically mediated degranulation of mast cells. In the present work, we explored how two recently developed mitochondrial STAT3 inhibitors (Mitocur-1 and Mitocur-3) modulate the allergic response.

Methods: Experiments were performed both in vitro in cultured human/mouse mast cells and with rat basophilic leukemia (RBL) cells and also in vivo in mice.

Cardiovascular homeostasis dependence on MICU2, a regulatory subunit of the mitochondrial calcium uniporter.

Comparative analyses of transcriptional profiles from humans and mice with cardiovascular pathologies revealed consistently elevated expression of , a regulatory subunit of the mitochondrial calcium uniporter complex. To determine if expression was cardioprotective, we produced and characterized mice. Mutant mice had left atrial enlargement and cardiomyocytes had delayed sarcomere relaxation and cytosolic calcium reuptake kinetics, indicating diastolic dysfunction.

Hypoxia treatment reverses neurodegenerative disease in a mouse model of Leigh syndrome.

The most common pediatric mitochondrial disease is Leigh syndrome, an episodic, subacute neurodegeneration that can lead to death within the first few years of life, for which there are no proven general therapies. Mice lacking the complex I subunit, Ndufs4, develop a fatal progressive encephalopathy resembling Leigh syndrome and die at ≈60 d of age. We previously reported that continuously breathing normobaric 11% O from an early age prevents neurological disease and dramatically improves survival in these mice.

Mitochondrial dysfunction remodels one-carbon metabolism in human cells.

Mitochondrial dysfunction is associated with a spectrum of human disorders, ranging from rare, inborn errors of metabolism to common, age-associated diseases such as neurodegeneration. How these lesions give rise to diverse pathology is not well understood, partly because their proximal consequences have not been well-studied in mammalian cells. Here we provide two lines of evidence that mitochondrial respiratory chain dysfunction leads to alterations in one-carbon metabolism pathways.

Tetramers reveal IL-17-secreting CD4+ T cells that are specific for U1-70 in lupus and mixed connective tissue disease.

Antigen-specific CD4(+) T cells are implicated in the autoimmune disease systemic lupus erythematosus (SLE), but little is known about the peptide antigens that they recognize and their precise function in disease. We generated a series of MHC class II tetramers of I-E(k)-containing peptides from the spliceosomal protein U1-70 that specifically stain distinct CD4(+) T-cell populations in MRL/lpr mice. The T-cell populations recognize an epitope differing only by the presence or absence of a single phosphate residue at position serine(140).

EMRE is an essential component of the mitochondrial calcium uniporter complex.

The mitochondrial uniporter is a highly selective calcium channel in the organelle's inner membrane. Its molecular components include the EF-hand-containing calcium-binding proteins mitochondrial calcium uptake 1 (MICU1) and MICU2 and the pore-forming subunit mitochondrial calcium uniporter (MCU). We sought to achieve a full molecular characterization of the uniporter holocomplex (uniplex).

Significance analysis of xMap cytokine bead arrays.

Highly multiplexed assays using antibody coated, fluorescent (xMap) beads are widely used to measure quantities of soluble analytes, such as cytokines and antibodies in clinical and other studies. Current analyses of these assays use methods based on standard curves that have limitations in detecting low or high abundance analytes. Here we describe SAxCyB (Significance Analysis of xMap Cytokine Beads), a method that uses fluorescence measurements of individual beads to find significant differences between experimental conditions.

Split immunity: immune inhibition of rat gliomas by subcutaneous exposure to unmodified live tumor cells.

Gliomas that grow uninhibited in the brain almost never metastasize outside the CNS. The rare occurrences of extracranial metastasis are usually associated with a suppressed immune system. This observation raises the possibility that some gliomas might not grow outside the CNS due to an inherent immune response, We report in this study that the highly malignant F98 Fischer rat undifferentiated glioma, which grows aggressively in the brain, spontaneously regresses when injected live s.

Mutations in MTFMT underlie a human disorder of formylation causing impaired mitochondrial translation.

The metazoan mitochondrial translation machinery is unusual in having a single tRNA(Met) that fulfills the dual role of the initiator and elongator tRNA(Met). A portion of the Met-tRNA(Met) pool is formylated by mitochondrial methionyl-tRNA formyltransferase (MTFMT) to generate N-formylmethionine-tRNA(Met) (fMet-tRNA(met)), which is used for translation initiation; however, the requirement of formylation for initiation in human mitochondria is still under debate. Using targeted sequencing of the mtDNA and nuclear exons encoding the mitochondrial proteome (MitoExome), we identified compound heterozygous mutations in MTFMT in two unrelated children presenting with Leigh syndrome and combined OXPHOS deficiency.

Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter.

Mitochondria from diverse organisms are capable of transporting large amounts of Ca(2+) via a ruthenium-red-sensitive, membrane-potential-dependent mechanism called the uniporter. Although the uniporter's biophysical properties have been studied extensively, its molecular composition remains elusive. We recently used comparative proteomics to identify MICU1 (also known as CBARA1), an EF-hand-containing protein that serves as a putative regulator of the uniporter.

High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency.

Discovering the molecular basis of mitochondrial respiratory chain disease is challenging given the large number of both mitochondrial and nuclear genes that are involved. We report a strategy of focused candidate gene prediction, high-throughput sequencing and experimental validation to uncover the molecular basis of mitochondrial complex I disorders. We created seven pools of DNA from a cohort of 103 cases and 42 healthy controls and then performed deep sequencing of 103 candidate genes to identify 151 rare variants that were predicted to affect protein function.