Regulation of plant root angle is critical for obtaining nutrients and water and is an important trait for plant breeding. A plant's final, long-term root angle is the net result of a complex series of decisions made by a root tip in response to changes in nutrient availability, impediments, the gravity vector and other stimuli. When a root tip is displaced from the gravity vector, the short-term process of gravitropism results in rapid reorientation of the root toward the vertical. Here, we explore both short- and long-term regulation of root growth angle, using natural variation in tomato to identify shared and separate genetic features of the two responses. Mapping of expression quantitative trait loci mapping and leveraging natural variation between and within species including Arabidopsis suggest a role for PURPLE ACID PHOSPHATASE 27 and CELL DIVISION CYCLE 73 in determining root angle.

Cellular mosaicism due to monoallelic autosomal expression (MAE), with cell selection during development, is becoming increasingly recognized as prevalent in mammals, leading to interest in understanding its extent and mechanism(s). We report here use of clonal cell lines derived from the CNS of adult female [Formula: see text] hybrid (C57BL/6 X JF1) mice to characterize MAE as neural stem cells (nscs) differentiate to astrocyte-like cells (asls). We found that different subsets of genes show MAE in the two populations of cells; in each case, there is strong enrichment for genes specific to the respective developmental state. Genes that exhibit MAE are 22% of nsc-specific genes and 26% of asl-specific genes. Moreover, the promoters of genes with MAE have reduced CpG dinucleotides but increased CpG differences between the two parental mouse strains. Extending the study of variability to wild populations of mice, we found evidence for balancing selection as a contributing force in evolution of those genes showing developmental specificity (i.e., expressed in either nsc or asl), not just for genes showing MAE. Furthermore, we found that genes showing skewed allelic expression (SKE) were similarly enriched among cell type-specific genes and also showed a heightened probability of balancing selection. Thus, developmental stage-specific genes and genes with MAE or SKE seem to make up overlapping classes subject to selection for increased diversity. The implications of these results for development and evolution are discussed in the context of a model with stochastic epigenetic modifications taking place only during a relatively brief developmental window.

Pathogens, particularly human herpesviruses (HHVs), are implicated as triggers of disease onset/progression in multiple sclerosis (MS) and other neuroinflammatory disorders. However, the time between viral acquisition in childhood and disease onset in adulthood complicates the study of this association. Using nonhuman primates, we demonstrate that intranasal inoculations with HHV-6A and HHV-6B accelerate an MS-like neuroinflammatory disease, experimental autoimmune encephalomyelitis (EAE). Although animals inoculated intranasally with HHV-6 (virus/EAE marmosets) were asymptomatic, they exhibited significantly accelerated clinical EAE compared with control animals. Expansion of a proinflammatory CD8 subset correlated with post-EAE survival in virus/EAE marmosets, suggesting that a peripheral (viral?) antigen-driven expansion may have occurred post-EAE induction. HHV-6 viral antigen in virus/EAE marmosets was markedly elevated and concentrated in brain lesions, similar to previously reported localizations of HHV-6 in MS brain lesions. Collectively, we demonstrate that asymptomatic intranasal viral acquisition accelerates subsequent neuroinflammation in a nonhuman primate model of MS.

In vertebrate lens, lens epithelial cells cover the anterior half of the lens fiber core. Lens epithelial cells proliferate, move posteriorly and start to differentiate into lens fiber cells at the lens equator. Although FGF signaling promotes this equatorial commencement of lens fiber differentiation, the underlying mechanism is not fully understood. Here, we show that lens epithelial cells abnormally enter lens fiber differentiation without passing through the equator in zebrafish vps45 mutants. VPS45 belongs to the Sec1/Munc18-like protein family and promotes endosome trafficking, which differentially modulates signal transduction. Ectopic lens fiber differentiation in vps45 mutants does not depend on FGF, but is mediated through activation of TGFβ signaling and inhibition of canonical Wnt signaling. Thus, VPS45 normally suppresses lens fiber differentiation in the anterior region of lens epithelium by modulating TGFβ and canonical Wnt signaling pathways. These data indicate a novel role of endosome trafficking to ensure equator-dependent commencement of lens fiber differentiation.

Stress granules (SGs) are transient membraneless organelles of nontranslating mRNA-protein complexes (mRNPs) that form during stress. In this study, we used multiple single-molecule FISH probes for particular mRNAs to examine their SG recruitment and spatial organization. Ribosome runoff is required for SG entry, as long open reading frame (ORF) mRNAs are delayed in SG accumulation, indicating that the SG transcriptome changes over time. Moreover, mRNAs are ∼20× compacted from an expected linear length when translating and compact ∼2-fold further in a stepwise manner beginning at the 5' end during ribosome runoff. Surprisingly, the 5' and 3' ends of the examined mRNAs were separated when translating, but in nontranslating conditions the ends of long ORF mRNAs become close, suggesting that the closed-loop model of mRNPs preferentially forms on nontranslating mRNAs. Compaction of ribosome-free mRNAs is ATP independent, consistent with compaction occurring through RNA structure formation. These results suggest that translation inhibition triggers an mRNP reorganization that brings ends closer, which has implications for the regulation of mRNA stability and translation by 3' UTR elements and the poly(A) tail.

Occidiofungin is produced by the soil bacterium Burkolderia contaminans MS14 and is structurally similar or identical to the burkholdines, xylocandins, and cepacidines. This study identified the primary cellular target of occidiofungin, which was determined to be actin. The modification of occidiofungin with a functional alkyne group enabled affinity purification assays and localization studies in yeast. Occidiofungin has a subtle effect on actin dynamics that triggers apoptotic cell death. We demonstrate the highly specific localization of occidiofungin to cellular regions rich in actin in yeast and the binding of occidiofungin to purified actin in vitro Furthermore, a disruption of actin-mediated cellular processes, such as endocytosis, nuclear segregation, and hyphal formation, was observed. All of these processes require the formation of stable actin cables, which are disrupted following the addition of a subinhibitory concentration of occidiofungin. We were also able to demonstrate the effectiveness of occidiofungin in treating a vulvovaginal yeast infection in a murine model. The results of this study are important for the development of an efficacious novel class of actin binding drugs that may fill the existing gap in treatment options for fungal infections or different types of cancer.

Sirtuin 3 (SIRT3) deacetylates and activates several mitochondrial fatty acid oxidation enzymes in the liver. Here, we investigated whether the protein acetylase GCN5 general control of amino acid synthesis 5-like 1 (GCN5L1), previously shown to oppose SIRT3 activity, is involved in the regulation of hepatic fatty acid oxidation. We show that GCN5L1 abundance is significantly up-regulated in response to an acute high-fat diet (HFD). Transgenic GCN5L1 overexpression in the mouse liver increased protein acetylation levels, and proteomic detection of specific lysine residues identified numerous sites that are co-regulated by GCN5L1 and SIRT3. We analyzed several fatty acid oxidation proteins identified by the proteomic screen and found that hyperacetylation of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit α (HADHA) correlates with increased GCN5L1 levels. Stable GCN5L1 knockdown in HepG2 cells reduced HADHA acetylation and increased activities of fatty acid oxidation enzymes. Mice with a liver-specific deletion of GCN5L1 were protected from hepatic lipid accumulation following a chronic HFD and did not exhibit hyperacetylation of HADHA compared with WT controls. Finally, we found that GCN5L1-knockout mice lack HADHA that is hyperacetylated at three specific lysine residues (Lys-350, Lys-383, and Lys-406) and that acetylation at these sites is significantly associated with increased HADHA activity. We conclude that GCN5L1-mediated regulation of mitochondrial protein acetylation plays a role in hepatic metabolic homeostasis.

An essential characteristic of sleep is heightened arousal threshold, with decreased behavioral response to external stimuli. The molecular and cellular mechanisms underlying arousal threshold changes during sleep are not fully understood. We report that loss of UNC-7 or UNC-9 innexin function dramatically reduced sleep and decreased arousal threshold during developmentally timed sleep in Caenorhabditiselegans UNC-7 function was required in premotor interneurons and UNC-9 function was required in motor neurons in this paradigm. Simultaneous transient overexpression of UNC-7 and UNC-9 was sufficient to induce anachronistic sleep in adult animals. Moreover, loss of UNC-7 or UNC-9 suppressed the increased sleep of EGL-4 gain-of-function animals, which have increased cyclic-GMP-dependent protein kinase activity. These results suggest C. elegans gap junctions may act downstream of previously identified sleep regulators. In other paradigms, the NCA cation channels act upstream of gap junctions. Consistent with this, diminished NCA channel activity in C. elegans robustly increased arousal thresholds during sleep bouts in L4-to-adult developmentally timed sleep. Total time in sleep bouts was only modestly increased in animals lacking NCA channel auxiliary subunit UNC-79, whereas increased channel activity dramatically decreased sleep. Loss of EGL-4 or innexin proteins suppressed UNC-79 loss-of-function sleep and arousal defects. In Drosophila, the ion channel narrow abdomen, an ortholog of the C. elegans NCA channels, drive the pigment dispersing factor (PDF) neuropeptide release, regulating circadian behavior. However, in C. elegans, we found that loss of the PDF receptor PDFR-1 did not suppress gain-of-function sleep defects, suggesting an alternative downstream pathway. This study emphasizes the conservation and importance of neuronal activity modulation during sleep, and unequivocally demonstrates that gap junction function is critical for normal sleep.

We previously demonstrated that administering 2-(2-benzofuranyl)-2-imidazolin (2-BFI), an imidazoline I2 receptor agonist, immediately after ischemia onset can protect the brain from ischemic insult. However, immediate administration after stroke is difficult to realize in the clinic. Thus, the therapeutic time window of 2-BFI should be determined. Sprague-Dawley rats provided by Wenzhou Medical University in China received right middle cerebral artery occlusion for 120 minutes, and were treated with 2-BFI (3 mg/kg) through the caudal vein at 0, 1, 3, 5, 7, and 9 hours after reperfusion. Neurological function was assessed using the Longa's method. Infarct volume was measured by 2,3,5-triphenyltetrazolium chloride assay. Morphological changes in the cortical penumbra were observed by hematoxylin-eosin staining under transmission electron microscopy . The apoptosis levels in the ipsilateral cortex were examined with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The protein expression of Bcl-2 and BAX was detected using immunohistochemistry. We found the following: Treatment with 2-BFI within 5 hours after reperfusion obviously improved neurological function. Administering 2-BFI within 9 hours after ischemia/reperfusion decreased infarct volume and alleviated apoptosis. 2-BFI administration at different time points after reperfusion alleviated the pathological damage of the ischemic penumbra and reduced the number of apoptotic neurons, but the protective effect was more obvious when administered within 5 hours. Administration of 2-BFI within 5 hours after reperfusion remarkably increased Bcl-2 expression and decreased BAX expression. To conclude, 2-BFI shows potent neuroprotective effects when administered within 5 hours after reperfusion, seemingly by up-regulating Bcl-2 and down-regulating BAX expression. The time window provided clinical potential for ischemic stroke by 2-BFI.

An aging-induced decrease in Schwann cell viability can affect regeneration following peripheral nerve injury in mammals. It is therefore necessary to investigate possible age-related changes in gene expression that may affect the biological function of peripheral nerves. Ten 1-week-old and ten 12-month-old healthy male Sprague-Dawley rats were divided into young (1 week old) and adult (12 months old) groups according to their ages. mRNA expression in the sciatic nerve was compared between young and adult rats using next-generation sequencing (NGS) and bioinformatics (n = 4/group). The 18 groups of differentially expressed mRNA (DEmRNAs) were also tested by quantitative reverse transcription polymerase chain reaction (n = 6/group). Results revealed that (1) compared with young rats, adult rats had 3608 groups of DEmRNAs. Of these, 2684 were groups of upregulated genes, and 924 were groups of downregulated genes. Their functions mainly involved cell viability, proliferation, differentiation, regeneration, and myelination. (2) The gene with the most obvious increase of all DEmRNAs in adult rats was Thrsp (log2FC = 9.01, P < 0.05), and the gene with the most obvious reduction was Col2a1 (log2FC = -8.89, P < 0.05). (3) Gene Ontology analysis showed that DEmRNAs were mainly concentrated in oligosaccharide binding, nucleotide-binding oligomerization domain containing one signaling pathway, and peptide-transporting ATPase activity. (4) Analysis using the Kyoto Encyclopedia of Genes and Genomes showed that, with increased age, DEmRNAs were mainly enriched in steroid biosynthesis, Staphylococcus aureus infection, and graft-versus-host disease. (5) Spearman's correlation coefficient method for evaluating NGS accuracy showed that the NGS results and quantitative reverse transcription polymerase chain reaction results were positively correlated (rs = 0.74, P < 0.05). These findings confirm a difference in sciatic nerve gene expression between adult and young rats, suggesting that, in peripheral nerves, cells and the microenvironment change with age, thus influencing the function and repair of peripheral nerves.

BACKGROUND Down syndrome (DS) is the most frequent genetic mental disability. Individuals with DS experience a variety of physical, motor, and functional challenges throughout the lifespan. However, the inter-relatedness between these domains is relatively unexplored in children with DS. This study aimed to determine which physical and motor characteristics contribute to functional performance in children and adolescents with DS. It also investigated the relationship between physical, motor, and functional domains. MATERIAL AND METHODS We enrolled 44 children and adolescents with DS, ages 3-18 years, in this cross-sectional study. The participants were assessed for functional skills (PEDI-CAT), gross motor skills (GMFM-88), balance (PBS), fine motor skills (Nine-hole peg test), grip strength (hand-held Jamar dynamometer), and body mass index (BMI). Descriptive statistics, Pearson's correlation, and stepwise linear regression were employed for statistical analysis. RESULTS Fine motor skills and grip strength were found to be significant predictors of functional performance. All measures, except BMI, were significantly correlated with each other. The participants scored below standard values in all 4 domains of PEDI-CAT, with the social/cognitive skills being most impaired, while mobility proficiency was found to be participants' strongest asset. CONCLUSIONS This study demonstrated fine motor skills and grip strength to be predictors of functional performance in children and adolescents with DS. It also showed a high level of inter-relatedness between the variables of physical, motor, and functional domains in this population.

Somatic mutations during stem cell division are responsible for several cancers. In principle, a similar process could occur during the intense cell proliferation accompanying human brain development, leading to the accumulation of regionally distributed foci of mutations. Using dual platform >5000-fold depth sequencing of 102 genes in 173 adult human brain samples, we detect and validate somatic mutations in 27 of 54 brains. Using a mathematical model of neurodevelopment and approximate Bayesian inference, we predict that macroscopic islands of pathologically mutated neurons are likely to be common in the general population. The detected mutation spectrum also includes DNMT3A and TET2 which are likely to have originated from blood cell lineages. Together, these findings establish developmental mutagenesis as a potential mechanism for neurodegenerative disorders, and provide a novel mechanism for the regional onset and focal pathology in sporadic cases.

Late infantile neuronal ceroid lipofuscinosis (CLN2 disease) is a rare lysosomal storage disorder caused by a monogenetic deficiency of tripeptidyl peptidase-1 (TPP1). Despite knowledge that lipofuscin is the hallmark disease product, the relevant TPP1 substrate and its role in neuronal physiology/pathology is unknown. We hypothesized that untargeted metabolite profiling of cerebrospinal fluid (CSF) could be used as an effective tool to identify disease-associated metabolic disruptions in CLN2 disease, offering the potential to identify biomarkers that inform on disease severity and progression. Accordingly, a mass spectrometry-based untargeted metabolite profiling approach was employed to differentiate CSF from normal vs. CLN2 deficient individuals. Of 1,433 metabolite features surveyed, 29 linearly correlated with currently employed disease severity scores. With tandem mass spectrometry 8 distinct metabolite identities were structurally confirmed based on retention time and fragmentation pattern matches, vs. standards. These putative CLN2 biomarkers include 7 acetylated species - all attenuated in CLN2 compared to controls. Because acetate is the major bioenergetic fuel for support of mitochondrial respiration, deficient acetylated species in CSF suggests a brain energy defect that may drive neurodegeneration. Targeted analysis of these metabolites in CSF of CLN2 patients offers a powerful new approach for monitoring CLN2 disease progression and response to therapy.

Salmonella enterica ser. Typhimurium monophasic variant 4,[5],12:i:- has been associated with food-borne epidemics worldwide and swine appeared to be the main reservoir in most of the countries of isolation. However, the monomorphic nature of this serovar has, so far, hindered identification of the source due to expansion of clonal lineages in multiple hosts and food producing systems. Since geographically structured genetic signals can shape bacterial populations, identification of biogeographical markers in S. 1,4,[5],12:i:- genomes can contribute to improving source attribution. In this study, the phylogeographical structure of 148 geographically and temporally related Italian S. 1,4,[5],12:i:- has been investigated. The Italian isolates belong to a large population of clonal S. Typhimurium/1,4,[5],12:i:- isolates collected worldwide in two decades showing up to 2.5% of allele differences. Phylogenetic reconstruction revealed that isolates from the same geographical origin form highly supported monophyletic groups, suggesting discrete geographical segregation. These monophyletic groups are characterized by the gene content of a large sopE-containing prophage. Within this prophage, genome-wide comparison identified several genes overrepresented in strains of Italian origin. This suggests that certain lineages may be characterized by the acquisition of specific accessory genetic markers useful for improving identification of the source in ongoing epidemics.

Mitochondrial morphology is remodeled by continuous dynamic cycles of fission and fusion. Emerging data have shown that the disturbance of balance between mitochondrial fission and fusion is involved in the progression of several types of neoplasms. However, the status of mitochondrial dynamics and its potential biological roles in breast cancer (BC), particularly in triple negative BC (TNBC) are not fully clear. Here, we reported that the mitochondrial fission was significantly increased in BC tissues, especially in the TNBC tissues, when compared with that in the corresponding peritumor tissues. Meanwhile, our data showed that Drp1 was upregulated, while Mfn1 was downregulated in TNBC. Moreover, elevated mitochondrial fission was associated with poorer prognosis in TNBC patients. Mitochondrial fission promoted the survival of TNBC cells both in vitro and in vivo. Furthermore, we identified a positive feedback loop between mitochondrial fission and Notch signaling pathway in TNBC cells, as proved by the experimental evidence that the activation of Notch signaling enhanced Drp1-mediated mitochondrial fission and Drp1-mediated mitochondrial fission in turn promoted the activation of Notch signaling, which ultimately promoted the cell survival of TNBC via increasing survivin expression level. Inhibition of either Notch1 or Drp1 significantly impaired the activation of the other, leading to the suppression of TNBC cell survival and proliferation. Collectively, our data reveal a novel mechanism that the positive feedback loop between mitochondrial fission and Notch signaling promotes the survival, proliferation and apoptotic resistance of TNBC cells via increasing survivin expression and thus favors cancer progression.

Specific RNA structures control numerous metabolic processes that impact human health, and yet efforts to target RNA structures de novo have been limited. In eukaryotes, the self-splicing group II intron is a mitochondrial RNA tertiary structure that is absent in vertebrates but essential for respiration in plants, fungi and yeast. Here we show that this RNA can be targeted through a process of high-throughput in vitro screening, SAR and lead optimization, resulting in high-affinity compounds that specifically inhibit group IIB intron splicing in vitro and in vivo and lack toxicity in human cells. The compounds are potent growth inhibitors of the pathogen Candida parapsilosis, displaying antifungal activity comparable to that of amphotericin B. These studies demonstrate that RNA tertiary structures can be successfully targeted de novo, resulting in pharmacologically valuable compounds.

PHD finger protein 19 (PHF19), a critical component of the polycomb repressive complex 2 (PRC2), is crucial for maintaining the repressive transcriptional activity of several developmental regulatory genes and plays essential roles in various biological processes. Abnormal expression of PHF19 causes dysplasia or serious diseases, including chronic myeloid disorders and tumors. However, the biological functions and molecular mechanisms of PHF19 in glioblastoma (GBM) remain unclear. Here, we demonstrated that PHF19 expression was positively associated with GBM progression, including cell proliferation, migration, invasion, chemosensitivity, and tumorigenesis. Using XAV-939, a Wnt/β-catenin inhibitor, we found that the effects of PHF19 on GBM cells were β-catenin-dependent. We also demonstrated that PHF19 expression was positively correlated with cytoplasmic β-catenin expression. PHF19 stabilized β-catenin by inhibiting the transcription of seven in absentia homolog 1 (SIAH1), an E3 ubiquitin ligase of β-catenin, through direct binding to the SIAH1 promoter region. Taken together, our results revealed the novel PHF19-SIAH1-β-catenin axis as a potential and promising therapeutic target.

Menaquinone (vitamin K2) shuttles electrons between membrane-bound respiratory complexes under microaerophilic conditions. In photosynthetic eukaryotes and cyanobacteria, phylloquinone (vitamin K1) participates in photosystem I function. Here we elucidate the evolutionary history of vitamin K metabolism in algae and plants. We show that Chlamydiales intracellular pathogens made major genetic contributions to the synthesis of the naphthoyl ring core and the isoprenoid side-chain of these quinones. Production of the core in extremophilic red algae is under control of a menaquinone (Men) gene cluster consisting of 7 genes that putatively originated via lateral gene transfer (LGT) from a chlamydial donor to the plastid genome. In other green and red algae, functionally related nuclear genes also originated via LGT from a non-cyanobacterial, albeit unidentified source. In addition, we show that 3-4 of the 9 required steps for synthesis of the isoprenoid side chains are under control of genes of chlamydial origin. These results are discussed in the light of the hypoxic response experienced by the cyanobacterial endosymbiont when it gained access to the eukaryotic cytosol.

Non-alcoholic fatty liver disease is a spectrum of liver diseases ranging from steatosis only to non-alcoholic steatohepatitis (NASH). The latter is characterized by hepatic inflammation, which increases the risk of cardiovascular disease. It is poorly understood which factors contribute to the onset of hepatic inflammation characterizing the progression from steatosis to NASH. Previously, we demonstrated increased advanced glycation endproducts (AGEs) in the livers of NASH patients. We hypothesise that AGEs play a key role in NASH development by activating their proinflammatory receptor, RAGE. RAGE-deficient mice and wildtype littermates, both on Ldlr-/- background, were fed a Western type diet (WTD) for 3 or 12 weeks. Flow cytometry, histology, gene expression and AGE measurements were performed to evaluate the effects of RAGE deficiency. RAGE-deficient mice displayed reduced weight gain and visceral fat expansion compared to control mice. No difference in adipose tissue inflammation was observed between groups. RAGE deficiency did not affect WTD-induced monocytosis, circulating lipids or hepatic steatosis. WTD-induced hepatic neutrophil and macrophage accumulation and atherosclerotic plaque development was comparable between control and RAGE-deficient mice. No difference in AGE levels was observed. RAGE does not seem to play a major role in the development of NASH or atherosclerosis in a hyperlipidemic mouse model.

Invariant natural killer T cells (iNKT cells) are activated by lipid antigens presented by CD1d, but the pathway leading to lipid antigen presentation remains incompletely characterized. Here we show a whole-genome siRNA screen to elucidate the CD1d presentation pathway. A majority of gene knockdowns that diminish antigen presentation reduced formation of glycolipid-CD1d complexes on the cell surface, including members of the HOPS and ESCRT complexes, genes affecting cytoskeletal rearrangement, and ABC family transporters. We validated the role in vivo for the multidrug resistance protein 1 (Mrp1) in CD1d antigen presentation. Mrp1 deficiency reduces surface clustering of CD1d, which decreased iNKT cell activation. Infected Mrp1 knockout mice show decreased iNKT cell responses to antigens from Streptococcus pneumoniae and were associated with increased mortality. Our results highlight the unique cellular events involved in lipid antigen presentation and show how modification of this pathway can lead to lethal infection.