Whole Transcriptome Analysis of Notochord-Derived Cells during Embryonic Formation of the Nucleus Pulposus.
Recapitulation of developmental signals represents a promising strategy for treating intervertebral disc degeneration. During development, embryonic notochord-derived cells (NDCs) are the direct progenitors of cells that populate the adult nucleus pulposus (NP) and are an important source of secreted signaling molecules. The objective of this study was to define global gene expression profiles of NDCs at key stages of embryonic disc formation. NDCs were isolated from Shh-cre;ROSA:YFP mice at embryonic day 12.5 and postnatal day 0, representing opposite ends of the notochord to NP transformation. Differences in global mRNA abundance across this developmental window were established using RNA-Seq. Protein expression of selected molecules was confirmed using immunohistochemistry. Principal component analysis revealed clustering of gene expression at each developmental stage with more than 5000 genes significantly differentially expressed between E12.5 and P0. There was significantly lower mRNA abundance of sonic hedgehog pathway elements at P0 vs E12.5, while abundance of elements of the transforming growth factor-beta and insulin-like growth factors pathways, and extracellular matrix components including collagen 6 and aggrecan, were significantly higher at P0. This study represents the first transcriptome-wide analysis of embryonic NDCs. Results suggest signaling and biosynthesis of NDCs change dramatically as a function of developmental stage.
Proteoliposome-based full-length ZnT8 self-antigen for type 1 diabetes diagnosis on a plasmonic platform.
Identified as a major biomarker for type 1 diabetes (T1D) diagnosis, zinc transporter 8 autoantibody (ZnT8A) has shown promise for staging disease risk and disease diagnosis. However, existing assays for ZnT8 autoantibody (ZnT8A) are limited to detection by soluble domains of ZnT8, owing to difficulties in maintaining proper folding of a full-length ZnT8 protein outside its native membrane environment. Through a combined bioengineering and nanotechnology approach, we have developed a proteoliposome-based full-length ZnT8 self-antigen (full-length ZnT8 proteoliposomes; PLR-ZnT8) for efficient detection of ZnT8A on a plasmonic gold chip (pGOLD). The protective lipid matrix of proteoliposomes improved the proper folding and structural stability of full-length ZnT8, helping PLR-ZnT8 immobilized on pGOLD (PLR-ZnT8/pGOLD) achieve high-affinity capture of ZnT8A from T1D sera. Our PLR-ZnT8/pGOLD exhibited efficient ZnT8A detection for T1D diagnosis with ∼76% sensitivity and ∼97% specificity (n = 307), superior to assays based on detergent-solubilized full-length ZnT8 and the C-terminal domain of ZnT8. Multiplexed assays using pGOLD were also developed for simultaneous detection of ZnT8A, islet antigen 2 autoantibody, and glutamic acid decarboxylase autoantibody for diagnosing T1D.
Methionine Sulfoxide Reductase A (MsrA) and Its Function in Ubiquitin-Like Protein Modification in Archaea.
Methionine sulfoxide reductase A (MsrA) is an antioxidant enzyme found in all domains of life that catalyzes the reduction of methionine-S-sulfoxide (MSO) to methionine in proteins and free amino acids. We demonstrate that archaeal MsrA has a ubiquitin-like (Ubl) protein modification activity that is distinct from its stereospecific reduction of MSO residues. MsrA catalyzes this Ubl modification activity, with the Ubl-activating E1 UbaA, in the presence of the mild oxidant dimethyl sulfoxide (DMSO) and in the absence of reductant. In contrast, the MSO reductase activity of MsrA is inhibited by DMSO and requires reductant. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis reveals that MsrA-dependent Ubl conjugates are associated with DNA replication, protein remodeling, and oxidative stress and include the Ubl-modified MsrA, Orc3 (Orc1/Cdc6), and Cdc48d (Cdc48/p97 AAA+ ATPase). Overall, we found archaeal MsrA to have opposing MSO reductase and Ubl modifying activities that are associated with oxidative stress responses and controlled by exposure to mild oxidant.IMPORTANCE Proteins that are damaged by oxidative stress are often targeted for proteolysis by the ubiquitin-proteasome system (UPS). The mechanisms that control this response are poorly understood, especially under conditions of mild oxidative stress when protein damage is modest. Here, we discovered a novel function of archaeal MsrA in guiding the Ubl modification of target proteins in the presence of mild oxidant. This newly reported activity of MsrA is distinct from its stereospecific reduction of methionine-S-sulfoxide to methionine residues. Our results are significant steps forward, first, in elucidating a protein factor that guides Ubl modification in archaea, and second, in providing an insight into oxidative stress responses that can trigger Ubl modification in a cell.
Central Nervous System Responses of the Oriental migratory, Locusta migratoria manilensis, to Fungal Infection.
Responses of the central nervous system (CNS) to microbial challenge and the interplay between the CNS and the immune system are important for defending against pathogen attack. We have examined the CNS transcriptional response of Locusta migratoria manilensis to infection by the locust-specific fungal pathogen, Metarhizium acridum. CNS responses were examined during spore attachment, fungal germination and pre-penetration of the cuticle, and cuticle penetration/hemocoel ingress and proliferation. Effects were seen at the earliest time points (4 h post-infection) and the number of differentially expressed genes (DEGs) was highest during late mycosis (72 h post-infection). Significantly affected neurological pathways included genes involved in serotonergic, cholinergic, dopaminergic, GABAergic, and glutamergic synapse responses, as well as pathways responsible for synaptic vesicle cycle, long-term potentiation and depression, and neurotrophin and retrograde endocannabinoid signaling. In addition, a significant number of immune related DEGs were identified. These included components of the Toll, Imd and JAK/STAT pathways, consistent with interactions between the CNS and immune systems. The activation of immune response related CNS genes during early stage infection highlights the rapid detection of microbial pathogens and suggests an important role for the CNS in modulating immunity potentially via initiating behavioral adaptations along with innate immune responses.
Heterologous Expression of Pteris vittata Arsenite Antiporter PvACR3;1 Reduces Arsenic Accumulation in Plant Shoots.
Arsenic (As) is a toxic carcinogen so it is crucial to decrease As accumulation in crops to reduce its risk to human health. Arsenite (AsIII) antiporter ACR3 protein is critical for As metabolism in organisms, but it is lost in flowering plants. Here, a novel ACR3 gene from As-hyperaccumulator Pteris vittata, PvACR3;1, was cloned and expressed in Saccharomyces cerevisiae (yeast), Arabidopsis thaliana (model plant), and Nicotiana tabacum (tobacco). Yeast experiments showed that PvACR3;1 functioned as an AsIII-antiporter to mediate AsIII efflux to an external medium. At 5 μM AsIII, PvACR3;1 transgenic Arabidopsis accumulated 14-29% higher As in the roots and 55-61% lower As in the shoots compared to WT control, showing lower As translocation. Besides, transgenic tobacco under 5 μM AsIII or AsV also showed similar results, indicating that expressing PvACR3;1 gene increased As retention in plant roots. Moreover, observation of PvACR3;1-green fluorescent protein fusions in transgenic Arabidopsis showed that PvACR3;1 protein localized to the vacuolar membrane, indicating that PvACR3;1 mediated AsIII sequestration into vacuoles, consistent with increased root As. In addition, soil experiments showed ∼22% lower As in the shoots of transgenic tobacco than control. Thus, our study provides a potential strategy to limit As accumulation in plant shoots, representing the first report to decrease As translocation by sequestrating AsIII into vacuoles, shedding light on engineering low-As crops to improve food safety.
Inhibition of the lytic cycle of Kaposi’s sarcoma-associated herpesvirus by cohesin factors following de novo infection.
Establishment of Kaposi’s sarcoma-associated herpesvirus (KSHV) latency following infection is a multistep process, during which polycomb proteins are recruited onto the KSHV genome, which is crucial for the genome-wide repression of lytic genes during latency. Strikingly, only a subset of lytic genes are expressed transiently in the early phase of infection prior to the binding of polycomb proteins onto the KSHV genome, which raises the question what restricts lytic gene expression in the first hours of infection. Here, we demonstrate that both CTCF and cohesin chromatin organizing factors are rapidly recruited to the viral genome prior to the binding of polycombs during de novo infection, but only cohesin is required for the genome-wide inhibition of lytic genes. We propose that cohesin is required for the establishment of KSHV latency by initiating the repression of lytic genes following infection, which is an essential step in persistent infection of humans.
The dental caries pathogen Streptococcus mutans is continually exposed to several types of stress in the oral biofilm environment. Oxidative stress generated by reactive oxygen species has a major impact on the establishment, persistence and virulence of S. mutans.Here, we combined fluorescent reporter-promoter fusions with single-cell imaging to study the effects of reactive oxygen species on activation of genetic competence in S. mutans Exposure to paraquat, which generates superoxide anion, produced a qualitatively different effect on activation of expression of the gene for the master competence regulator, ComX, than did treatment with hydrogen peroxide (H2O2), which can yield hydroxyl radical. Paraquat suppressed peptide-mediated induction of comX in a progressive and cumulative fashion, whereas the response to H2O2 displayed a strong threshold behavior. Low concentrations of H2O2 had little effect on induction of comX or the bacteriocin gene cipB, but expression of these genes declined sharply if extracellular H2O2 exceeded a threshold concentration. These effects were not due to decreased reporter gene fluorescence. Two different threshold concentrations were observed in the response to H2O2, depending on the gene promoter that was analyzed and the pathway by which the competence regulon was stimulated. The results show that paraquat and H2O2 affect the S. mutans competence signaling pathway differently, and that some portions of the competence signaling pathway are more sensitive to oxidative stress than others.ImportanceStreptococcus mutans inhabits the oral biofilm where it plays an important role in the development of dental caries. Environmental stresses such as oxidative stress influence the growth of S. mutans and its important virulence-associated behaviors, such as genetic competence. S. mutans competence development is a complex behavior that involves two different signaling peptides and can exhibit cell-to-cell heterogeneity. Although oxidative stress is known to influence S. mutans competence, it is not understood how oxidative stress interacts with the peptide signaling or affects heterogeneity. In this study we used fluorescent reporters to probe the effect of reactive oxygen species on competence signaling at the single cell level. Our data show that different reactive oxygen species have different effects on S. mutans competence, and that some portions of the signaling pathway are more acutely sensitive to oxidative stress than others.
Sepsis is a major cause of morbidity and mortality, especially at the extremes of age. To understand the human age-specific transcriptomic response to sepsis, a multi-cohort, pooled analysis was conducted on adults, children, infants, and neonates with and without sepsis. Nine public whole-blood gene expression datasets (636 patients) were employed. Age impacted the transcriptomic host response to sepsis. Gene expression from septic neonates and adults was more dissimilar whereas infants and children were more similar. Neonates showed reductions in inflammatory recognition and signaling pathways compared to all other age groups. Likewise, adults demonstrated decreased pathogen sensing, inflammation, and myeloid cell function, as compared to children. This may help to explain the increased incidence of sepsis-related organ failure and death in adults. The number of dysregulated genes in septic patients was proportional to age and significantly differed among septic adults, children, infants, and neonates. Overall, children manifested a greater transcriptomic intensity to sepsis as compared to the other age groups. The transcriptomic magnitude for adults and neonates was dramatically reduced as compared to children and infants. These findings suggest that the transcriptomic response to sepsis is age-dependent, and diagnostic and therapeutic efforts to identify and treat sepsis will have to consider age as an important variable.
T lymphocytes constitute a major effector cell population in autoimmune type 1 diabetes. Despite essential functions of mitochondria in regulating activation, proliferation, and apoptosis of T cells, little is known regarding T cell metabolism in the progression of human type 1 diabetes. In this study, we report, using two independent cohorts, that T cells from patients with type 1 diabetes exhibited mitochondrial inner-membrane hyperpolarization (MHP). Increased MHP was a general phenotype observed in T cell subsets irrespective of prior antigen exposure, and was not correlated with HbA1C levels, subject age, or duration of diabetes. Elevated T cell MHP was not detected in subjects with type 2 diabetes. T cell MHP was associated with increased activation-induced IFNγ production, and activation-induced IFNγ was linked to mitochondria-specific ROS production. T cells from subjects with type 1 diabetes also exhibited lower intracellular ATP levels. In conclusion, intrinsic mitochondrial dysfunction observed in type 1 diabetes alters mitochondrial ATP and IFNγ production; the latter is correlated with ROS generation. These changes impact T cell bioenergetics and function.
PaxA, but not PaxC, is required for cnidocyte development in the sea anemone Nematostella vectensis.
Pax genes are a family of conserved transcription factors that regulate many aspects of developmental morphogenesis, notably the development of ectodermal sensory structures including eyes. Nematostella vectensis, the starlet sea anemone, has numerous Pax orthologs, many of which are expressed early during embryogenesis. The function of Pax genes in this eyeless cnidarian is unknown.
Here, we show that PaxA, but not PaxC, plays a critical role in the development of cnidocytes in N. vectensis. Knockdown of PaxA results in a loss of developing cnidocytes and downregulation of numerous cnidocyte-specific genes, including a variant of the transcription factor Mef2. We also demonstrate that the co-expression of Mef2 in a subset of the PaxA-expressing cells is associated with the development with a second lineage of cnidocytes and show that knockdown of the neural progenitor gene SoxB2 results in downregulation of expression of PaxA, Mef2, and several cnidocyte-specific genes. Because PaxA is not co-expressed with SoxB2 at any time during cnidocyte development, we propose a simple model for cnidogenesis whereby a SoxB2-expressing progenitor cell population undergoes division to give rise to PaxA-expressing cnidocytes, some of which also express Mef2.
The role of PaxA in cnidocyte development among hydrozoans has not been studied, but the conserved role of SoxB2 in regulating the fate of a progenitor cell that gives rise to neurons and cnidocytes in Nematostella and Hydractinia echinata suggests that this SoxB2/PaxA pathway may well be conserved across cnidarians.
Type 1 Interferons Potentiate Human CD8+ T Cell Cytotoxicity Through a STAT4 and Granzyme B Dependent Pathway.
Events defining the progression to human type 1 diabetes (T1D) have remained elusive owing to the complex interaction between genetics, the immune system, and the environment. Type 1 Interferons (T1-IFN) are known to be a constituent of the auto-inflammatory milieu within the pancreas of patients with T1D. However, the capacity of IFNα/β to modulate human activated auto-reactive CD8+ T cell (CTL) responses within the islets of patients with T1D have not been investigated. Here, we engineer human beta cell specific CTLs and demonstrate that T1-IFN augments cytotoxicity by inducing rapid phosphorylation of STAT4, resulting in direct binding at the granzyme B (GZMB) promoter within 2 hours of exposure. The current findings provide novel insights concerning the regulation of effector function by T1-IFN in human antigen-experienced CD8+ T cells and provide a mechanism by which the presence of T1-IFN potentiates diabetogenicity within the autoimmune islet.
A Defective mRNA Cleavage and Polyadenylation Complex Facilitates Expansions of Transcribed (GAA)n Repeats Associated with Friedreich’s Ataxia.
Expansions of microsatellite repeats are responsible for numerous hereditary diseases in humans, including myotonic dystrophy and Friedreich’s ataxia. Whereas the length of an expandable repeat is the main factor determining disease inheritance, recent data point to genomic trans modifiers that can impact the likelihood of expansions and disease progression. Detection of these modifiers may lead to understanding and treating repeat expansion diseases. Here, we describe a method for the rapid, genome-wide identification of trans modifiers for repeat expansion in a yeast experimental system. Using this method, we found that missense mutations in the endoribonuclease subunit (Ysh1) of the mRNA cleavage and polyadenylation complex dramatically increase the rate of (GAA)n repeat expansions but only when they are actively transcribed. These expansions correlate with slower transcription elongation caused by the ysh1 mutation. These results reveal an interplay between RNA processing and repeat-mediated genome instability, confirming the validity of our approach.
Persistence of Pancreatic Insulin mRNA Expression and Proinsulin Protein in Type 1 Diabetes Pancreata.
The canonical notion that type 1 diabetes (T1D) results following a complete destruction of β cells has recently been questioned as small amounts of C-peptide are detectable in patients with long-standing disease. We analyzed protein and gene expression levels for proinsulin, insulin, C-peptide, and islet amyloid polypeptide within pancreatic tissues from T1D, autoantibody positive (Ab+), and control organs. Insulin and C-peptide levels were low to undetectable in extracts from the T1D cohort; however, proinsulin and INS mRNA were detected in the majority of T1D pancreata. Interestingly, heterogeneous nuclear RNA (hnRNA) for insulin and INS-IGF2, both originating from the INS promoter, were essentially undetectable in T1D pancreata, arguing for a silent INS promoter. Expression of PCSK1, a convertase responsible for proinsulin processing, was reduced in T1D pancreata, supportive of persistent proinsulin. These data implicate the existence of β cells enriched for inefficient insulin/C-peptide production in T1D patients, potentially less susceptible to autoimmune destruction.
NOTE: These abstracts were retrieved from the U.S. National Library of Medicine website managed in collaboration with the U.S. National Library of Medicine