Since antibiotic resistance is a major threat to global health, recent observations that the traditional test of minimum inhibitory concentration (MIC) is not informative enough to guide effective ...antibiotic treatment are alarming. Bacterial heteroresistance, in which seemingly susceptible isogenic bacterial populations contain resistant sub-populations, underlies much of this challenge. To close this gap, here we developed a droplet-based digital MIC screen that constitutes a practical analytical platform for quantifying the single-cell distribution of phenotypic responses to antibiotics, as well as for measuring inoculum effect with high accuracy. We found that antibiotic efficacy is determined by the amount of antibiotic used per bacterial colony forming unit (CFU), not by the absolute antibiotic concentration, as shown by the treatment of beta-lactamase-carrying Escherichia coli with cefotaxime. We also noted that cells exhibited a pronounced clustering phenotype when exposed to near-inhibitory amounts of cefotaxime. Overall, our method facilitates research into the interplay between heteroresistance and antibiotic efficacy, as well as research into the origin and stimulation of heterogeneity by exposure to antibiotics. Due to the absolute bacteria quantification in this digital assay, our method provides a platform for developing reference MIC assays that are robust against inoculum-density variations.
Despite the crucial roles of phytohormones in plant development, comparison of the exact distribution profiles of different hormones within plant meristems has thus far remained scarce. Vascular ...cambium, a wide lateral meristem with an extensive developmental zonation, provides an optimal system for hormonal and genetic profiling. By taking advantage of this spatial resolution, we show here that two major phytohormones, cytokinin and auxin, display different yet partially overlapping distribution profiles across the cambium. In contrast to auxin, which has its highest concentration in the actively dividing cambial cells, cytokinins peak in the developing phloem tissue of a Populus trichocarpa stem. Gene expression patterns of cytokinin biosynthetic and signaling genes coincided with this hormonal gradient. To explore the functional significance of cytokinin signaling for cambial development, we engineered transgenic Populus tremula × tremuloides trees with an elevated cytokinin biosynthesis level. Confirming that cytokinins function as major regulators of cambial activity, these trees displayed stimulated cambial cell division activity resulting in dramatically increased (up to 80% in dry weight) production of the lignocellulosic trunk biomass. To connect the increased growth to hormonal status, we analyzed the hormone distribution and genome-wide gene expression profiles in unprecedentedly high resolution across the cambial zone. Interestingly, in addition to showing an elevated cambial cytokinin content and signaling level, the cambial auxin concentration and auxin-responsive gene expression were also increased in the transgenic trees. Our results indicate that cytokinin signaling specifies meristematic activity through a graded distribution that influences the amplitude of the cambial auxin gradient.
•Gene expression was profiled globally across the cambium in high resolution•Auxin and cytokinin display distinct distribution profiles across the cambium•Increased cytokinin content and signaling level stimulate cambial cell divisions•Elevation of cytokinin content leads to an increased cambial auxin concentration
A new report explores how two major phytohormones, cytokinin and auxin, contribute to the control of tree trunk growth. Immanen et al. show that by boosting cytokinin biosynthesis, they can both increase auxin level and stimulate lignocellulosic biomass production. Both hormones represent optimal targets for tree breeding and forest biotechnology.
In Swiss-type cheeses, characteristic nut-like and sweet flavor develops during the cheese ripening due to the metabolic activities of cheese microbiota. Temperature changes during warm and cold room ...ripening, and duration of ripening can significantly change the gene expression of the cheese microbiota, which can affect the flavor formation. In this study, a metagenomic and metatranscriptomic analysis of Swiss-type Maasdam cheese was performed on samples obtained during ripening in the warm and cold rooms. We reconstructed four different bacterial genomes (Lactococcus lactis, Lactobacillus rhamnosus, Lactobacillus helveticus, and Propionibacterium freudenreichii subsp. shermanii strain JS) from the Maasdam cheese to near completeness. Based on the DNA and RNA mean coverage, Lc. lactis strongly dominated (~80–90%) within the cheese microbial community. Genome annotation showed the potential for the presence of several flavor forming pathways in these species, such as production of methanethiol, free fatty acids, acetoin, diacetyl, acetate, ethanol, and propionate. Using the metatranscriptomic data, we showed that, with the exception of Lc. lactis, the central metabolism of the microbiota was downregulated during cold room ripening suggesting that fewer flavor compounds such as acetoin and propionate were produced. In contrast, Lc. lactis genes related to the central metabolism, including the vitamin biosynthesis and homolactic fermentation, were upregulated during cold room ripening.
•We performed metagenomic and metatranscriptomic analysis of Maasdam cheese ripening.•Lc. lactis dramatically dominated the Maasdam cheese microbiota.•All lactic acid bacteria play a role in degradation of casein.•Cold room ripening caused downregulation of central metabolism genes in general.•Lc. lactis central metabolism genes were upregulated in cold room ripening.
Multiple sclerosis (MS) is an inflammatory autoimmune disorder of the central nervous system and the leading cause of progressive neurological disability in young adults. It decreases the patient's ...lifespan by about 10 years and affects women more than men. No medication entirely restricts or reverses neurological degradation. However, early diagnosis and treatment increase the possibility of a better outcome. To identify new MS biomarkers, we tested the expression of six potential markers (P2X4, P2X7, CXCR4, RGS1, RGS16 and VAV1) using qPCR in peripheral blood mononuclear cells (PBMC) of MS patients treated with interferon β (IFNβ), with glatiramer acetate (GA) or untreated. We showed that P2X7 and VAV1 are significantly induced in MS patients. In contrast, the expression of P2X4, CXCR4, RGS1 and RGS16 was not significantly modified by MS in PBMC. P2X7 and VAV1 are essentially induced in female patients, suggesting these markers are connected to sex‐specific mechanisms. Strikingly, VAV1 expression is higher in healthy women than healthy men and IFNβ treatment of MS reduced VAV1 expression in female MS patients while it up‐regulated VAV1 in male MS patients. Our data point to the differential, sex‐dependent value of MS markers and treatment effects. Although rgs16 expression in PBMC was not a valid MS marker in patients, the strong upregulation of P2X4 and P2X7 induced in the spinal cord of WT mice by EAE was abrogated in rgs16KO mice suggesting that rgs16 is required for P2X4 and P2X7 induction by neurological diseases.
Alternative oxidase (AOX) is a non‐mammalian enzyme that can bypass blockade of the complex III‐IV segment of the respiratory chain (RC). We crossed a Ciona intestinalis AOX transgene into RC complex ...III (cIII)‐deficient Bcs1lp.S78G knock‐in mice, displaying multiple visceral manifestations and premature death. The homozygotes expressing AOX were viable, and their median survival was extended from 210 to 590 days due to permanent prevention of lethal cardiomyopathy. AOX also prevented renal tubular atrophy and cerebral astrogliosis, but not liver disease, growth restriction, or lipodystrophy, suggesting distinct tissue‐specific pathogenetic mechanisms. Assessment of reactive oxygen species (ROS) production and damage suggested that ROS were not instrumental in the rescue. Cardiac mitochondrial ultrastructure, mitochondrial respiration, and pathological transcriptome and metabolome alterations were essentially normalized by AOX, showing that the restored electron flow upstream of cIII was sufficient to prevent cardiac energetic crisis and detrimental decompensation. These findings demonstrate the value of AOX, both as a mechanistic tool and a potential therapeutic strategy, for cIII deficiencies.
Synopsis
Bcs1l mutant mice show respiratory chain cIII dysfunction resulting in poor electron and proton transfer, and quinol oxidation. This study makes use of alternative oxidase (AOX), an enzyme that shunts electrons from quinols directly to oxygen, thus restoring electron flow upstream of cIII.
Broadly expressed transgenic AOX prevents lethal cardiomyopathy in Bcs1lp.S78G knock‐in mice and extends their median survival from 210 to 590 days.
Restoration of electron flow upstream of cIII by AOX is sufficient to prevent the cardiac decompensation.
AOX prevents pathological changes in the expression of central transcriptional regulators of substrate utilization and metabolic stress in the heart.
Analyses of reactive oxygen species (ROS) production and damage suggest that ROS are not instrumental in the rescue.
Bcs1l mutant mice show respiratory chain cIII dysfunction resulting in poor electron and proton transfer, and quinol oxidation. This study makes use of alternative oxidase (AOX), an enzyme that shunts electrons from quinols directly to oxygen, thus restoring electron flow upstream of cIII.
A decline in stem cell function impairs tissue regeneration during ageing, but the role of the stem-cell-supporting niche in ageing is not well understood. The small intestine is maintained by ...actively cycling intestinal stem cells that are regulated by the Paneth cell niche
. Here we show that the regenerative potential of human and mouse intestinal epithelium diminishes with age owing to defects in both stem cells and their niche. The functional decline was caused by a decrease in stemness-maintaining Wnt signalling due to production of Notum, an extracellular Wnt inhibitor, in aged Paneth cells. Mechanistically, high activity of mammalian target of rapamycin complex 1 (mTORC1) in aged Paneth cells inhibits activity of peroxisome proliferator activated receptor α (PPAR-α)
, and lowered PPAR-α activity increased Notum expression. Genetic targeting of Notum or Wnt supplementation restored function of aged intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the regenerative capacity of aged stem cells and promoted recovery from chemotherapy-induced damage. Our results reveal a role of the stem cell niche in ageing and demonstrate that targeting of Notum can promote regeneration of aged tissues.
Previous studies have reported that chromosome synteny in Lepidoptera has been well conserved, yet the number of haploid chromosomes varies widely from 5 to 223. Here we report the genome (393 Mb) of ...the Glanville fritillary butterfly (Melitaea cinxia; Nymphalidae), a widely recognized model species in metapopulation biology and eco-evolutionary research, which has the putative ancestral karyotype of n=31. Using a phylogenetic analyses of Nymphalidae and of other Lepidoptera, combined with orthologue-level comparisons of chromosomes, we conclude that the ancestral lepidopteran karyotype has been n=31 for at least 140 My. We show that fusion chromosomes have retained the ancestral chromosome segments and very few rearrangements have occurred across the fusion sites. The same, shortest ancestral chromosomes have independently participated in fusion events in species with smaller karyotypes. The short chromosomes have higher rearrangement rate than long ones. These characteristics highlight distinctive features of the evolutionary dynamics of butterflies and moths.
Water-in-oil droplets allow performing massive experimental parallelization and high-throughput studies, such as single-cell experiments. However, analyzing such vast arrays of droplets usually ...requires advanced expertise and sophisticated workflow tools, which limits accessibility for a wider user base in the fields of chemistry and biology. Thus, there is a need for more user-friendly tools for droplet analysis. In this article, we deliver a set of analytical pipelines for user-friendly analysis of typical scenarios in droplet experiments. We built pipelines that combine various open-source image-analysis software with a custom-developed data processing tool called “EasyFlow”. Our pipelines are applicable to the typical experimental scenarios that users encounter when working with droplets: i) mono- and polydisperse droplets, ii) brightfield and fluorescent images, iii) droplet and object detection, iv) signal profile of droplets and objects (e.g., fluorescence).
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•Image-analysis software are combined with data visualization tool EasyFlow for user-friendly analysis of droplet arrays.•EasyFlow simplifies building analysis and visualization pipelines for droplet arrays.•EasyFlow analyses your droplet data based on their size, signal and labels.
Cell-free RNAs have the potential to act as a means of gene expression regulation between cells and are therefore used as diagnostic markers describing the state of tissue environment. The origin and ...functions of such RNAs in human ovarian follicle, the environment of oocyte maturation, are unclear. The current study investigates the difference in the microRNA profiles of fertile women and polycystic ovary syndrome (PCOS) patients in three compartments from the same preovulatory follicle: mural granulosa cells (MGC), cell-free follicular fluid (FF), and extracellular vesicles (EV) of the FF by small RNA sequencing. In silico analysis was used for the prediction and over-representation of targeted pathways for the detected microRNAs. PCOS follicles were distinguished from normal tissue by the differential expression of 30 microRNAs in MGC and 10 microRNAs in FF (FDR < 0.1) that commonly regulate cytokine signaling pathways. The concentration of EV-s was higher in the FF of PCOS patients (
= 0.04) containing eight differentially expressed microRNAs (
< 0.05). In addition, we present the microRNA profiles of MGC, FF, and EV in the fertile follicle and demonstrate that microRNAs loaded into EVs target mRNAs of distinct signaling pathways in comparison to microRNAs in FF. To conclude, the three follicular compartments play distinct roles in the signaling disturbances associated with PCOS.
ABSTRACT
Biosynthetic precursors of NAD+ can replenish a decreased cellular NAD+ pool and, supposedly viasirtuin (SIRT) deacetylases, improve mitochondrial function. We found decreased hepatic NAD+ ...concentration and downregulated biosynthesis in Bcs1lpS78G knock‐in mice with respiratory chain complex III deficiency and mito chondrial hepatopathy. Aiming at ameliorating disease progression via NAD+ repletion and improved mitochon drial function, we fed these mice nicotinamide riboside (NR), a NAD+ precursor. A targeted metabolomics verified successful administration and suggested enhanced NAD+ biosynthesis in the treated mice, although hepatic NAD+ concentration was unchanged at the end point. In contrast to our expectations, NR did not improve the hepatopathy, hepatic mitochondrial respiration, or survival of Bcs1lpS78G mice. We linked this lack of therapeutic effect to NAD+‐independent activation of SIRT‐1 and ‐3 via AMPK and cAMP signaling related to the starvation‐like metabolic state of Bcs1lp.S78G mice. In summary, we describe an unusual metabolic state with NAD+ depletion accompanied by energy deprivation signals, uncompromised SIRT function, and upregulated oxidative me tabolism. Our study highlights that the knowledge of the underlying complex metabolic alterations is critical when designing therapies for mitochondrial dysfunction.—Purhonen, J., Rajendran, J., Tegelberg, S., Smolander, O.‐P., Pirinen, E., Kallijärvi, J., Fellman, V. NAD+ repletion produces no therapeutic effect in mice with respiratory chain complex III deficiency and chronic energy deprivation. FASEB J. 32, 5913–5926 (2018). www.fasebj.org
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