Fungal pathogens associated with grapevine trunk diseases in young vineyards in Sicily

After the first report of grapevine decline caused by Botryosphaeriaceae in Sicily in 2007, epidemiological studies carried out in mature vineyards until 2011 confirmed the widespread occurrence of “Botryosphaeria dieback” and the “Esca complex” disease. Dieback symptoms were also recently observed in two young vineyards in Partanna and Castellammare del Golfo in western Sicily (Trapani province). Declining vines were inspected for grapevine trunk disease (GTD) symptoms, and were uprooted and submitted for analyses. Fungal isolates were collected and identified using culturing and molecular analyses. One isolate per identified species was inoculated to three grapevine shoots to evaluate pathogenicity and fulfil Koch’s postulates. Several GTD Botryosphaeriaceae pathogens in the genera Cadophora, Ilyonectria, Neonectria, Phaeoacremonium and Phaeomoniella were isolated from the symptomatic young vines. Artificial inoculation confirmed the pathogenicity of these fungi. In addition, virulence variability was observed among the isolates, with P. chlamydospora causing the largest lesions. The different species were associated with specific symptoms and/or host vine parts, especially in the roots and around the grafting areas. Several fungi associated with Petri disease and black foot were shown to be responsible of young vine decline

Evidence for a quadruplex structure in the polymorphic hs1.2 enhancer of the immunoglobulin heavy chain 3’ regulatory regions and its conservation in mammals

Regulatory regions in the genome can act through a variety of mechanisms that range from the occurrence of histone modifications to the presence of protein-binding loci for self-annealing sequences. The final result is often the induction of a conformational change of the DNA double helix, which alters the accessibility of a region to transcription factors and consequently gene expression. A similar to 300 kb regulatory region on chromosome 14 at the 3' end (3'RR) of immunoglobulin (Ig) heavy-chain genes shows very peculiar features, conserved in mammals, including enhancers and transcription factor binding sites. In primates, the 3'RR is present in two copies, both having a central enhancer named hs1.2. We previously demonstrated the association between different hs1.2 alleles and Ig plasma levels in immunopathology. Here, we present the analysis of a putative G-quadruplex structure (tetraplex) consensus site embedded in a variable number tandem repeat (one to four copies) of hs1.2 that is a distinctive element among the enhancer alleles, and an investigation of its three-dimensional structure using bioinformatics and spectroscopic approaches. We suggest that both the role of the enhancer and the alternative effect of the hs1.2 alleles may be achieved through their peculiar three-dimensional-conformational rearrangement

Extracellular vesicle microRNAs contribute to Notch signaling pathway in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive T-cell malignancy characterized by genotypically-defined and phenotypically divergent cell populations, governed by adaptive landscapes. Clonal expansions are associated to genetic and epigenetic events, and modulation of external stimuli that affect the hierarchical structure of subclones and support the dynamics of leukemic subsets. Recently, small extracellular vesicles (sEV) such as exosomes were also shown to play a role in leukemia. Here, by coupling miRNome, bulk and single cell transcriptome profiling, we found that T-ALL-secreted sEV contain NOTCH1-dependent microRNAs (EV-miRs), which control oncogenic pathways acting as autocrine stimuli and ultimately promoting the expansion/survival of highly proliferative cell subsets of human T-cell leukemias. Of interest, we found that NOTCH1-dependent EV-miRs mostly comprised members of miR-17-92a cluster and paralogues, which rescued in vitro the proliferation of T-ALL cells blocked by γ-secretase inhibitors (GSI) an regulated a network of genes characterizing patients with relapsed/refractory early T-cell progenitor (ETP) ALLs. All these findings suggest that NOTCH1 dependent EV-miRs may sustain the growth/survival of immunophenotypically defined cell populations, altering the cell heterogeneity and the dynamics of T-cell leukemias in response to conventional therapies

Position and sequence conservation in Amniota of polymorphic enhancer HS1.2 within the palindrome of IgH 3'Regulatory Region

<p>Abstract</p> <p>Background</p> <p>The Immunoglobulin heavy chain (IgH) 3' Regulatory Region (3'RR), located at the 3' of the constant alpha gene, plays a crucial role in immunoglobulin production. In humans, there are 2 copies of the 3'RR, each composed of 4 main elements: 3 enhancers and a 20 bp tandem repeat. The single mouse 3'RR differs from the two human ones for the presence of 4 more regulative elements with the double copy of one enhancer at the border of a palindromic region.</p> <p>Results</p> <p>We compared the 3'RR organization in genomes of vertebrates to depict the evolutionary history of the region and highlight its shared features. We found that in the 8 species in which the whole region was included in a fully assembled contig (mouse, rat, dog, rabbit, panda, orangutan, chimpanzee, and human), the shared elements showed synteny and a highly conserved sequence, thus suggesting a strong evolutionary constraint. In these species, the wide 3'RR (~30 kb in human) bears a large palindromic sequence, consisting in two ~3 kb complementary branches spaced by a ~3 kb sequence always including the HS1.2 enhancer. In mouse and rat, HS3 is involved by the palindrome so that one copy of the enhancer is present on each side. A second relevant feature of our present work concerns human polymorphism of the HS1.2 enhancer, associated to immune diseases in our species. We detected a similar polymorphism in all the studied Catarrhini (a primate parvorder). The polymorphism consists of multiple copies of a 40 bp element up to 12 in chimpanzees, 8 in baboons, 6 in macaque, 5 in gibbons, 4 in humans and orangutan, separated by stretches of Cytosine. We show specific binding of this element to nuclear factors.</p> <p>Conclusions</p> <p>The nucleotide sequence of the palindrome is not conserved among evolutionary distant species, suggesting pressures for the maintenance of two self-matching regions driving a three-dimensional structure despite of the inter-specific divergence at sequence level. The information about the conservation of the palindromic structure and the settling in primates of the polymorphic feature of HS1.2 show the relevance of these structures in the control and modulation of the Ig production through the formation of possible three-dimensional structures.</p

Epigenetic Restoration of Fetal-like IGF1 Signaling Inhibits Leukemia Stem Cell Activity

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PKCθ Regulates T-Cell Leukemia-Initiating Activity via Reactive Oxygen Species

Reactive oxygen species (ROS), a by-product of cellular metabolism, damage intracellular macromolecules and, in excess, can promote normal hematopoietic stem cell differentiation and exhaustion1–3. However, mechanisms that regulate ROS levels in leukemia-initiating cells (LICs) and the biological role of ROS in these cells remain largely unknown. We show here the ROSlow subset of CD44+ cells in T-cell acute lymphoblastic leukemia (T-ALL), a malignancy of immature T-cell progenitors, to be highly enriched in the most aggressive LICs, and that ROS are maintained at low levels by downregulation of protein kinase C theta (PKCθ). Strikingly, primary mouse T-ALLs lacking PKCθ show improved LIC activity whereas enforced PKCθ expression in both mouse and human primary T-ALLs compromised LIC activity. We also demonstrate that PKCθ is positively regulated by RUNX1, and that NOTCH1, which is frequently activated by mutation in T-ALL4–6 and required for LIC activity in both mouse and human models7,8, downregulates PKCθ and ROS via a novel pathway involving induction of RUNX3 and subsequent repression of RUNX1. These results reveal key functional roles for PKCθ and ROS in T-ALL and suggest that aggressive biological behavior in vivo could be limited by therapeutic strategies that promote PKCθ expression/activity or ROS accumulation

HS1,2 Ig Enhancer Alleles Association to AIDS Progression in a Pediatric Cohort Infected with a Monophyletic HIV-Strain

Alteration in the humoral immune response has been observed during HIV infection. The polymorphisms of enhancer HS1,2, member of the 3 regulatory region of the Ig heavy chain cluster, may play a role in the variation of the humoral response leading to pathological conditions. To assess the role of the HS1,2 polymorphic variants in the progression of AIDS, the HS1,2-A allelic frequencies were investigated in a cohort of HIV infected pediatric subjects from a nosocomial outbreak with a monophyletic strain of HIV. From a total group of 418 HIV infected children in the outbreak cohort, 42 nonprogressors and 31 progressors without bias due to antiretroviral therapy were evaluated. HS1,2 allele * 1 has been associated with nonprogressors (allelic frequency: 51.19% versus 33.87% in progressors, OR 0.5, and = 0.0437), while allele * 2 has been associated with progression (allelic frequency: 48.39% versus 30.95% in nonprogressors, OR 2.1, and = 0.0393). Further, only subjects carrying allele * 2 in absence of allele * 1, either in homozygous condition for allele * 2 [nonprogressors 2/42 (4.76%), Progressors 7/31 (22.58%), OR 5.8, and = 0.0315] or in combination with other allelic variants [nonprogressors 7/42 (16.67%), Progressors 13/31 (41.93%), OR 3.61, and = 0.0321], have been associated with HIV progression to AIDS. In conclusion, while the HS1,2 allele * 1 has a protective effect on HIV progression when present, allele * 2 is associated with progression toward AIDS when allele * 1 is absent

Polymorphisms and DNA methylation: two ways for functional differences in the 3' regulatory region of the IgH locus

The IgH locus in mouse and human has a 3' regulatory region (3'RR) with multiple DNaseI hypersensitive sites. In the human, but not in the mouse, the sites (HS3, HS1.2 and HS4) are duplicated. One unit is downstream of the Cα-1 gene and a second unit is downstream of the Cα-2 gene. Human HS1,2 enhancers show polymorphic features. In the mouse, HS3A, HS1.2, HS3B and HS4 are enhancers involved in the expression and class switching of immunoglobulin heavy chain genes. A recently identified downstream region, which contains hypersensitive sites HS5, HS6 and HS7, has been hypothesized to serve as an insulator of the Igh locus. This downstream region is associated with marks of active chromatin throughout B cell development and contains binding sites for CTCF, a protein associated with mammalian insulators. CTCF binding to many of its cognate DNA sites is prevented by DNA methylation. Previous studies using genomic Southern analysis have shown changes in DNA methylation in the upstream region of the murine 3' RR during B cell development. In the first part of this work I identified the polymorphic structure of human HS1,2, and its distribution in some populations and in some immunological diseases. The data suggest that the HS1,2 enhancer that lies downstream of the Cα-1 gene has four alleles, one of which, allele *2, is more frequent in some immunological disorders and less frequent in the sub-Saharan region. I have also observed using EMSA that protein binding is different in the four alleles. Furthermore I have studied changes in DNA methylation in the murine 3'RR during B cell development by digesting genomic DNA with methylation-sensitive restriction enzymes, such as HpaII and MaeII, followed by PCR. The data revealed that the 3’RR is methylated in embryonic stem cells. ES cells derived from histone H1 depleted mice showed a reduction in methylation as compared to their respective wildtype counterparts. I have detected a progressive loss of DNA methylation during B cell development. DNase I HS sites HS4, HS5 and HS7 are the earliest regulated and unmethylated sites in cell lines reflecting early stages of B development, while the HS1.2 and HS3B enhancers are unmethylated 3 only in plasma cell lines. DNA methylation is also reduced in spleni

Polymorphisms and DNA methylation: two ways for functional differences in the 3' regulatory region of the IgH locus

The IgH locus in mouse and human has a 3' regulatory region (3'RR) with multiple DNaseI hypersensitive sites. In the human, but not in the mouse, the sites (HS3, HS1.2 and HS4) are duplicated. One unit is downstream of the Cα-1 gene and a second unit is downstream of the Cα-2 gene. Human HS1,2 enhancers show polymorphic features. In the mouse, HS3A, HS1.2, HS3B and HS4 are enhancers involved in the expression and class switching of immunoglobulin heavy chain genes. A recently identified downstream region, which contains hypersensitive sites HS5, HS6 and HS7, has been hypothesized to serve as an insulator of the Igh locus. This downstream region is associated with marks of active chromatin throughout B cell development and contains binding sites for CTCF, a protein associated with mammalian insulators. CTCF binding to many of its cognate DNA sites is prevented by DNA methylation. Previous studies using genomic Southern analysis have shown changes in DNA methylation in the upstream region of the murine 3' RR during B cell development. In the first part of this work I identified the polymorphic structure of human HS1,2, and its distribution in some populations and in some immunological diseases. The data suggest that the HS1,2 enhancer that lies downstream of the Cα-1 gene has four alleles, one of which, allele *2, is more frequent in some immunological disorders and less frequent in the sub-Saharan region. I have also observed using EMSA that protein binding is different in the four alleles. Furthermore I have studied changes in DNA methylation in the murine 3'RR during B cell development by digesting genomic DNA with methylation-sensitive restriction enzymes, such as HpaII and MaeII, followed by PCR. The data revealed that the 3’RR is methylated in embryonic stem cells. ES cells derived from histone H1 depleted mice showed a reduction in methylation as compared to their respective wildtype counterparts. I have detected a progressive loss of DNA methylation during B cell development. DNase I HS sites HS4, HS5 and HS7 are the earliest regulated and unmethylated sites in cell lines reflecting early stages of B development, while the HS1.2 and HS3B enhancers are unmethylated 3 only in plasma cell lines. DNA methylation is also reduced in spleni