Cytotoxic polyfunctionality maturation of cytomegalovirus-pp65-specific CD4 + and CD8 + T-cell responses in older adults positively correlates with response size

Cytomegalovirus (CMV) infection is one of the most common persistent viral infections in humans worldwide and is epidemiologically associated with many adverse health consequences during aging. Previous studies yielded conflicting results regarding whether large, CMV-specific T-cell expansions maintain their function during human aging. In the current study, we examined the in vitro CMV-pp65-reactive T-cell response by comprehensively studying five effector functions (i.e., interleukin-2, tumor necrosis factor-α, interferon-γ, perforin, and CD107a expression) in 76 seropositive individuals aged 70 years or older. Two data-driven, polyfunctionality panels (IL-2-associated and cytotoxicity-associated) derived from effector function co-expression patterns were used to analyze the results. We found that, CMV-pp65-reactive CD8 + and CD4 + T cells contained similar polyfunctional subsets, and the level of polyfunctionality was related to the size of antigen-specific response. In both CD8 + and CD4 + cells, polyfunctional cells with high cytotoxic potential accounted for a larger proportion of the total response as the total response size increased. Notably, a higher serum CMV-IgG level was positively associated with a larger T-cell response size and a higher level of cytotoxic polyfunctionality. These findings indicate that CMV-pp65-specific CD4 + and CD8 + T cell undergo simultaneous cytotoxic polyfunctionality maturation during aging

Transgene-Induced Gene Silencing Is Not Affected by a Change in Ploidy Level

BACKGROUND: Whole genome duplication, which results in polyploidy, is a common feature of plant populations and a recurring event in the evolution of flowering plants. Polyploidy can result in changes to gene expression and epigenetic instability. Several epigenetic phenomena, occurring at the transcriptional or post-transcriptional level, have been documented in allopolyploids (polyploids derived from species hybrids) of Arabidopsis thaliana, yet findings in autopolyploids (polyploids derived from the duplication of the genome of a single species) are limited. Here, we tested the hypothesis that an increase in ploidy enhances transgene-induced post-transcriptional gene silencing using autopolyploids of A. thaliana. METHODOLOGY/PRINCIPAL FINDINGS: Diploid and tetraploid individuals of four independent homozygous transgenic lines of A. thaliana transformed with chalcone synthase (CHS) inverted repeat (hairpin) constructs were generated. For each line diploids and tetraploids were compared for efficiency in post-transcriptional silencing of the endogenous CHS gene. The four lines differed substantially in their silencing efficiency. Yet, diploid and tetraploid plants derived from these plants and containing therefore identical transgene insertions showed no difference in the efficiency silencing CHS as assayed by visual scoring, anthocyanin assays and quantification of CHS mRNA. CONCLUSIONS/SIGNIFICANCE: Our results in A. thaliana indicated that there is no effect of ploidy level on transgene-induced post-transcriptional gene silencing. Our findings that post-transcriptional mechanisms were equally effective in diploids and tetraploids supports the use of transgene-driven post-transcriptional gene silencing as a useful mechanism to modify gene expression in polyploid species

A Pathogenic Mechanism in Huntington's Disease Involves Small CAG-Repeated RNAs with Neurotoxic Activity

Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approaches

AUX1-mediated root hair auxin influx governs SCFTIR1/AFB-type Ca2+ signaling

Auxin is a key regulator of plant growth and development, but the causal relationship between hormone transport and root responses remains unresolved. Here we describe auxin uptake, together with early steps in signaling, in Arabidopsis root hairs. Using intracellular microelectrodes we show membrane depolarization, in response to IAA in a concentration- and pH-dependent manner. This depolarization is strongly impaired in aux1 mutants, indicating that AUX1 is the major transporter for auxin uptake in root hairs. Local intracellular auxin application triggers Ca2+ signals that propagate as long-distance waves between root cells and modulate their auxin responses. AUX1-mediated IAA transport, as well as IAA- triggered calcium signals, are blocked by treatment with the SCFTIR1/AFB - inhibitor auxinole. Further, they are strongly reduced in the tir1afb2afb3 and the cngc14 mutant. Our study reveals that the AUX1 transporter, the SCFTIR1/AFB receptor and the CNGC14 Ca2+ channel, mediate fast auxin signaling in roots

Action potentials in abscisic acid-deficient tomato mutant generated spontaneously and evoked by electrical stimulation

Action potentials generated spontaneously (SAPs) and evoked by electrical stimulation (APs) in tomato plants (Solanum lycopersicum L.) cv. Micro-Tom ABA-deficient mutants (sitiens—MTsit) and its wild type (MTwt) were characterized by continuous monitoring of electrical activity for 66 h and by application of an electrical stimulation supplied extracellularly. MTsit generated SAPs which spread along the stem, including petioles and roots with an amplitude of 44.6 ± 4.4 mV, half-time (t½) of 33.1 ± 2.9 s and velocity of 5.4 ± 1.0 cm min−1. Amplitude and velocity were 43 and 108 % higher in MTsit than in MTwt, respectively. The largest number of SAPs was registered in the early morning in both genotypes. MTsit was less responsive to electrical stimuli. The excitation threshold and the refractory period were greater in MTsit than in MTwt. After current application, APs were generated in the MTwt with 21.2 ± 2.4 mV amplitude and propagated with 5.6 ± 0.5 cm min−1 velocity. Lower intensity stimuli did not trigger APs in these plants. In MTsit APs were measured with amplitude of 26.8 ± 4.8 mV and propagated with velocity of 8.5 ± 0.1 cm min−1

Deregulated MicroRNAs in Myotonic Dystrophy Type 2

Myotonic Dystrophy Type-2 (DM2) is an autosomal dominant disease caused by the expansion of a CCTG tetraplet repeat. It is a multisystemic disorder, affecting skeletal muscles, the heart, the eye, the central nervous system and the endocrine system. Since microRNA (miRNA) expression is disrupted in Myotonic Dystrophy Type-1 and many other myopathies, miRNAs deregulation was studied in skeletal muscle biopsies of 13 DM2 patients and 13 controls. Eleven miRNAs were deregulated: 9 displayed higher levels compared to controls (miR-34a-5p, miR-34b-3p, miR-34c-5p, miR-146b-5p, miR-208a, miR-221-3p and miR-381), while 4 were decreased (miR-125b-5p, miR-193a-3p, miR-193b-3p and miR-378a-3p). To explore the relevance of DM2 miRNA deregulation, the predicted interactions between miRNA and mRNA were investigated. Global gene expression was analyzed in DM2 and controls and bioinformatic analysis identified more than 1,000 miRNA/mRNA interactions. Pathway and function analysis highlighted the involvement of the miRNA-deregulated mRNAs in multiple aspects of DM2 pathophysiology. In conclusion, the observed miRNA dysregulations may contribute to DM2 pathogenetic mechanisms

Evolution of MicroRNA Genes in Oryza sativa and Arabidopsis thaliana: An Update of the Inverted Duplication Model

The origin and evolution of microRNA (miRNA) genes, which are of significance in tuning and buffering gene expressions in a number of critical cellular processes, have long attracted evolutionary biologists. However, genome-wide perspectives on their origins, potential mechanisms of their de novo generation and subsequent evolution remain largely unsolved in flowering plants. Here, genome-wide analyses of Oryza sativa and Arabidopsis thaliana revealed apparently divergent patterns of miRNA gene origins. A large proportion of miRNA genes in O. sativa were TE-related and MITE-related miRNAs in particular, whereas the fraction of these miRNA genes much decreased in A. thaliana. Our results show that the majority of TE-related and pseudogene-related miRNA genes have originated through inverted duplication instead of segmental or tandem duplication events. Based on the presented findings, we hypothesize and illustrate the four likely molecular mechanisms to de novo generate novel miRNA genes from TEs and pseudogenes. Our rice genome analysis demonstrates that non-MITEs and MITEs mediated inverted duplications have played different roles in de novo generating miRNA genes. It is confirmed that the previously proposed inverted duplication model may give explanations for non-MITEs mediated duplication events. However, many other miRNA genes, known from the earlier proposed model, were rather arisen from MITE transpositions into target genes to yield binding sites. We further investigated evolutionary processes spawned from de novo generated to maturely-formed miRNA genes and their regulatory systems. We found that miRNAs increase the tunability of some gene regulatory systems with low gene copy numbers. The results also suggest that gene balance effects may have largely contributed to the evolution of miRNA regulatory systems

Transgene Silencing and Transgene-Derived siRNA Production in Tobacco Plants Homozygous for an Introduced AtMYB90 Construct

Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2–R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3′ half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production

Identification and Pathway Analysis of microRNAs with No Previous Involvement in Breast Cancer

microRNA expression signatures can differentiate normal and breast cancer tissues and can define specific clinico-pathological phenotypes in breast tumors. In order to further evaluate the microRNA expression profile in breast cancer, we analyzed the expression of 667 microRNAs in 29 tumors and 21 adjacent normal tissues using TaqMan Low-density arrays. 130 miRNAs showed significant differential expression (adjusted P value = 0.05, Fold Change = 2) in breast tumors compared to the normal adjacent tissue. Importantly, the role of 43 of these microRNAs has not been previously reported in breast cancer, including several evolutionary conserved microRNA*, showing similar expression rates to that of their corresponding leading strand. The expression of 14 microRNAs was replicated in an independent set of 55 tumors. Bioinformatic analysis of mRNA targets of the altered miRNAs, identified oncogenes like ERBB2, YY1, several MAP kinases, and known tumor-suppressors like FOXA1 and SMAD4. Pathway analysis identified that some biological process which are important in breast carcinogenesis are affected by the altered microRNA expression, including signaling through MAP kinases and TP53 pathways, as well as biological processes like cell death and communication, focal adhesion and ERBB2-ERBB3 signaling. Our data identified the altered expression of several microRNAs whose aberrant expression might have an important impact on cancer-related cellular pathways and whose role in breast cancer has not been previously described

Ubiquitous Expression of CUG or CAG Trinucleotide Repeat RNA Causes Common Morphological Defects in a Drosophila Model of RNA-Mediated Pathology

Expanded DNA repeat sequences are known to cause over 20 diseases, including Huntington’s disease, several types of spinocerebellar ataxia and myotonic dystrophy type 1 and 2. A shared genetic basis, and overlapping clinical features for some of these diseases, indicate that common pathways may contribute to pathology. Multiple mechanisms, mediated by both expanded homopolymeric proteins and expanded repeat RNA, have been identified by the use of model systems, that may account for shared pathology. The use of such animal models enables identification of distinct pathways and their ‘molecular hallmarks’ that can be used to determine the contribution of each pathway in human pathology. Here we characterise a tergite disruption phenotype in adult flies, caused by ubiquitous expression of either untranslated CUG or CAG expanded repeat RNA. Using the tergite phenotype as a quantitative trait we define a new genetic system in which to examine ‘hairpin’ repeat RNA-mediated cellular perturbation. Further experiments use this system to examine whether pathways involving Muscleblind sequestration or Dicer processing, which have been shown to mediate repeat RNA-mediated pathology in other model systems, contribute to cellular perturbation in this model