29 research outputs found
Tolerance of human embryonic stem cell derived islet progenitor cells to vitrification-relevant solutions
We have previously shown that human embryonic stem cell derived islet progenitors (hESC-IPs), encapsulated inside an immunoprotective device, mature in vivo and ameliorate diabetes in mice. The ability to cryopreserve hESC-IPs preloaded in these devices would enhance consistency and portability, but traditional ‘slow freezing’ methods did not work well for cells encapsulated in the device. Vitrification is an attractive alternative cryopreservation approach. To assess the tolerance of hESC-IPs to vitrification relevant conditions, we here are reporting cell survival following excursions in tonicity, exposure to fifteen 40% v/v combinations of 4 cryoprotectants, and varied methods for addition and elution. We find that 78% survival is achieved using a protocol in which cells are abruptly (in one step) exposed to a solution containing 10% v/v each dimethyl sulfoxide, propylene glycol, ethylene glycol, and glycerol on ice, and eluted step-wise with DPBS + 0.5 M sucrose at 37 °C. Importantly, the hESC-IPs also maintain expression of the critical islet progenitor markers PDX-1, NKX6.1, NGN3 and NEURO-D1. Thus, hESC-IPs exhibit robust tolerance to exposure to vitrification solutions in relevant conditions
SHH1, a Homeodomain Protein Required for DNA Methylation, As Well As RDR2, RDM4, and Chromatin Remodeling Factors, Associate with RNA Polymerase IV
DNA methylation is an evolutionarily conserved epigenetic modification that is critical for gene silencing and the maintenance of genome integrity. In Arabidopsis thaliana, the de novo DNA methyltransferase, DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), is targeted to specific genomic loci by 24 nt small interfering RNAs (siRNAs) through a pathway termed RNA–directed DNA methylation (RdDM). Biogenesis of the targeting siRNAs is thought to be initiated by the activity of the plant-specific RNA polymerase IV (Pol-IV). However, the mechanism through which Pol-IV is targeted to specific genomic loci and whether factors other than the core Pol-IV machinery are required for Pol-IV activity remain unknown. Through the affinity purification of NUCLEAR RNA POLYMERASE D1 (NRPD1), the largest subunit of the Pol-IV polymerase, we found that several previously identified RdDM components co-purify with Pol-IV, namely RNA–DEPENDENT RNA POLYMERASE 2 (RDR2), CLASSY1 (CLSY1), and RNA–DIRECTED DNA METHYLATION 4 (RDM4), suggesting that the upstream siRNA generating portion of the RdDM pathway may be more physically coupled than previously envisioned. A homeodomain protein, SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1), was also found to co-purify with NRPD1; and we demonstrate that SHH1 is required for de novo and maintenance DNA methylation, as well as for the accumulation of siRNAs at specific loci, confirming it is a bonafide component of the RdDM pathway
NOF1 Encodes an Arabidopsis Protein Involved in the Control of rRNA Expression
The control of ribosomal RNA biogenesis is essential for the regulation of protein synthesis in eukaryotic cells. Here, we report the characterization of NOF1 that encodes a putative nucleolar protein involved in the control of rRNA expression in Arabidopsis. The gene has been isolated by T-DNA tagging and its function verified by the characterization of a second allele and genetic complementation of the mutants. The nof1 mutants are affected in female gametogenesis and embryo development. This result is consistent with the detection of NOF1 mRNA in all tissues throughout plant life's cycle, and preferentially in differentiating cells. Interestingly, the closely related proteins from zebra fish and yeast are also necessary for cell division and differentiation. We showed that the nof1-1 mutant displays higher rRNA expression and hypomethylation of rRNA promoter. Taken together, the results presented here demonstrated that NOF1 is an Arabidopsis gene involved in the control of rRNA expression, and suggested that it encodes a putative nucleolar protein, the function of which may be conserved in eukaryotes
The De Novo Cytosine Methyltransferase DRM2 Requires Intact UBA Domains and a Catalytically Mutated Paralog DRM3 during RNA–Directed DNA Methylation in Arabidopsis thaliana
Eukaryotic DNA cytosine methylation can be used to transcriptionally silence repetitive sequences, including transposons and retroviruses. This silencing is stable between cell generations as cytosine methylation is maintained epigenetically through DNA replication. The Arabidopsis thaliana Dnmt3 cytosine methyltransferase ortholog DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) is required for establishment of small interfering RNA (siRNA) directed DNA methylation. In mammals PIWI proteins and piRNA act in a convergently evolved RNA–directed DNA methylation system that is required to repress transposon expression in the germ line. De novo methylation may also be independent of RNA interference and small RNAs, as in Neurospora crassa. Here we identify a clade of catalytically mutated DRM2 paralogs in flowering plant genomes, which in A.thaliana we term DOMAINS REARRANGED METHYLTRANSFERASE3 (DRM3). Despite being catalytically mutated, DRM3 is required for normal maintenance of non-CG DNA methylation, establishment of RNA–directed DNA methylation triggered by repeat sequences and accumulation of repeat-associated small RNAs. Although the mammalian catalytically inactive Dnmt3L paralogs act in an analogous manner, phylogenetic analysis indicates that the DRM and Dnmt3 protein families diverged independently in plants and animals. We also show by site-directed mutagenesis that both the DRM2 N-terminal UBA domains and C-terminal methyltransferase domain are required for normal RNA–directed DNA methylation, supporting an essential targeting function for the UBA domains. These results suggest that plant and mammalian RNA–directed DNA methylation systems consist of a combination of ancestral and convergent features
Plant ARGONAUTEs: Features, Functions and Unknowns
ARGONAUTEs (AGOs) are the effector proteins in eukaryotic small RNA(sRNA)–
based gene silencing pathways controlling gene expression and transposon activity. In
plants, AGOs regulate key biological processes such as development, response to
stress, genome structure and integrity, and pathogen defense. Canonical functions of
plant AGO–sRNA complexes include the endonucleolytic cleavage or translational
inhibition of target RNAs, and the methylation of target DNAs. Here, I provide a brief
update on the major features, molecular functions and biological roles of plant AGOs.
A special focus is given to the more recent discoveries related to emerging molecular
or biological functions of plant AGOs, as well as to the major unknowns in the plant
AGO field.This work
was supported by an Individual Fellowship from the European
Union’s Horizon 2020 research and innovation program under
the Marie Skłodowska-Curie grant agreement No. 655841 to A.C.Carbonell Olivares, A. (2017). Plant ARGONAUTEs: Features, Functions and Unknowns. En Plant Argonaute Proteins: Methods and Protocols. Springer Link. 1-21. https://doi.org/10.1007/978-1-4939-7165-7_1121Meister G (2013) Argonaute proteins: functional insights and emerging roles. Nat Rev Genet 14(7):447–459. doi: 10.1038/nrg3462Huntzinger E, Izaurralde E (2011) Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat Rev Genet 12(2):99–110. doi: 10.1038/nrg2936Cerutti H, Casas-Mollano JA (2006) On the origin and functions of RNA-mediated silencing: from protists to man. Curr Genet 50(2):81–99. doi: 10.1007/s00294-006-0078-xFang X, Qi Y (2016) RNAi in plants: an argonaute-centered view. 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Effects of SR 49059, a new orally active and specific vasopressin V1 receptor antagonist, on vasopressin-induced vasoconstriction in humans
We have evaluated the efficacy of SR 49059, a new orally active and specific vasopressin V1 receptor antagonist (arginine-vasopressin [AVP]), in the blockade of the vascular effects of exogenous AVP in healthy subjects. In preliminary experiments, two procedures to measure the V1 vascular effects of AVP were assessed. First, the AVP-induced changes in skin blood flow were investigated by the injection of increasing doses of AVP intradermally, with or without a previous local vasodilation with calcitonin gene-related peptide (CGRP). In a second protocol, AVP was infused intra-arterially, and the changes in radial artery diameter and blood flow were measured. The intradermal injection of AVP caused significant decreases in skin blood flow, and the use of CGRP increased the sensitivity of the method by a factor of 10(2) to 10(3). AVP infused intra-arterially caused dose-dependent decreases in the radial artery diameter and blood flow. In the main study, the potency and efficacy of SR 49059 to block the AVP-induced changes in skin blood flow were assessed in 12 healthy men with a double-blind, triple crossover study design. The subjects were randomized to receive a placebo orally and 30 mg and 300 mg of the antagonist at a 1-week interval. The subjects were then further randomized to evaluate the efficacy of the same doses of the antagonist to block the vasoconstriction of the radial artery induced by an intra-arterial infusion of AVP. SR 49059 inhibits, dose-dependently and significantly, the AVP-induced changes in skin blood flow, with a peak effect occurring between 2 and 6 hours after injection. In addition, the 300-mg dose of SR 49059 completely blocked the vasoconstriction of the radial artery induced by AVP. In conclusion, skin blood-flow measurement, after intradermal injection of AVP on a skin area vasodilated with CGRP, is an effective method to investigate the V1 vascular effect of AVP in humans. SR 49059 is a potent and specific antagonist of V1 receptors, which blocks the AVP-induced vasoconstriction
From combinations to multitarget-directed ligands: A continuum in Alzheimer's disease polypharmacology
The continued drug discovery failures in complex neurodegenerative diseases, including Alzheimer's disease (AD), has raised questions about the classical paradigm \u201cone-drug, one-target, one-disease.\u201d In parallel, the ever-increasing awareness of the multiplicity of the underlying pathways has led to the affirmation of polypharmacological approaches. Polypharmacology, which broadly embodies the use of pharmaceutical agents acting on multiple targets, seems to be the best way to restore the complex diseased network and to provide disease-modifying effects in AD. In this review, our aim is to provide a roadmap into a world that is still only partly explored and that should be seen as a continuum of pharmacological opportunities, from drug combinations to multitarget-directed ligands (both codrugs and hybrids). Each modality has unique features that can be effectively exploited by medicinal chemists. We argue that understanding their advantages and drawbacks is very helpful in choosing a proper approach and developing successful AD multitarget drug-discovery endeavors. We also briefly dwell on (co)target validation, an aspect that is quite often neglected, but critical for an efficient clinical translation. We substantiate our discussion with instructive examples taken from the recent literature. Our wish is that, in spite of the specter of the high attrition rates, best researchers preferring to enter, stay, and progress in the field would help grow the sector and develop AD polypharmacology to full potential
From combinations to multitarget‐directed ligands: A continuum in Alzheimer's disease polypharmacology
Sigma-1 Receptor Agonists Induce Oxidative Stress in Mitochondria and Enhance Complex I Activity in Physiological Condition but Protect Against Pathological Oxidative Stress
Increased Amyloid-β Peptide-Induced Memory Deficits in Phospholipid Transfer Protein (PLTP) Gene Knockout Mice
Oxidative stress is recognized as one of the earliest and most intense pathological processes in Alzheimer's disease (AD), and the antioxidant vitamin E has been shown to efficiently prevent amyloid plaque formation and neurodegeneration. Plasma phospholipid transfer protein (PLTP) has a major role in vitamin E transfers in vivo, and PLTP deficiency in mice is associated with reduced brain vitamin E levels. To determine the impact of PLTP on amyloid pathology in vivo, we analyzed the vulnerability of PLTP-deficient (PLTP-KO) mice to the toxic effects induced by intracerebroventricular injection of oligomeric amyloid-β(25–35) (Aβ(25–35)) peptide, a non-transgenic model of AD. Under basal conditions, PLTP-KO mice showed increased cerebral oxidative stress, increased brain Aβ(1–42) levels, and a lower expression of the synaptic function marker synaptophysin, as compared with wild-type mice. This PLTP-KO phenotype was associated with increased memory impairment 1 week after Aβ(25–35) peptide injection. Restoration of brain vitamin E levels in PLTP-KO mice through a chronic dietary supplementation prevented Aβ(25–35)-induced memory deficits and reduced cerebral oxidative stress and toxicity. We conclude that PLTP, through its ability to deliver vitamin E to the brain, constitutes an endogenous neuroprotective agent. Increasing PLTP activity may offer a new way to develop neuroprotective therapies