Common carotid artery diameter, blood flow velocity and wave intensity responses at rest and during exercise in young healthy humans: a reproducibility study

The aim of this study was to assess the reproducibility of non-invasive, ultrasound-derived wave intensity (WI) in humans at the common carotid artery. Common carotid artery diameter and blood velocity of 12 healthy young participants were recorded at rest and during mild cycling, to assess peak diameter, change in diameter, peak velocity, change in velocity, time derivatives, non-invasive wave speed and WI. Diameter, velocity and WI parameters were fairly reproducible. Diameter variables exhibited higher reproducibility than corresponding velocity variables (intra-class correlation coefficient [ICC] = 0.79 vs. 0.73) and lower dispersion (coefficient of variation [CV] = 5% vs. 9%). Wave speed had fair reproducibility (ICC = 0.6, CV = 16%). WI energy variables exhibited higher reproducibility than corresponding peaks (ICC = 0.78 vs. 0.74) and lower dispersion (CV = 16% vs. 18%). The majority of variables had higher ICCs and lower CVs during exercise. We conclude that non-invasive WI analysis is reliable both at rest and during exercise

Non-invasive assessment of the common carotid artery hemodynamics with Increasing exercise workrate using wave intensity analysis

Non-invasively determined local wave speed (c) and wave intensity (WI) parameters provide insight into arterial stiffness and cardiac-vascular interactions in response to physiological perturbations. However, the effects of incremental exercise and subsequent recovery on c and WI are not fully established. We examined the changes in c and WI parameters in the common carotid artery (CCA) during exercise and recovery in 8 young healthy male athletes. Ultrasound measurements of CCA diameter (D) and blood flow velocity (U) were acquired at rest, during 5 stages of incremental exercise (up to 70% maximum workrate) and throughout 1 h of recovery and non-invasive WI analysis (DU approach) was performed. During exercise, c increased (+136%), showing increased stiffness with workrate. All peak and area of forward compression (FCW), backward compression (BCW) and forward expansion waves (FEW) increased during exercise (+452%, +700%, +900%, respectively). However, WI reflection indices and CCA resistance did not significantly change from rest to exercise. Further, wave speed and magnitude of all waves returned to baseline within 5 min of recovery, suggesting the effects of exercise in the investigated parameters of young healthy individuals were transient. In conclusion, incremental exercise was associated with an increase in local CCA stiffness and increases in all wave parameters, indicative of enhanced ventricular contractility and improved late-systolic blood flow deceleration

Deletion of LBR N-terminal domains recapitulates Pelger-Huet anomaly phenotypes in mouse without disrupting X chromosome inactivation

Mutations in the gene encoding Lamin B receptor (LBR), a nuclear-membrane protein with sterol reductase activity, have been linked to rare human disorders. Phenotypes range from a benign blood disorder, such as Pelger-Huet anomaly (PHA), affecting the morphology and chromatin organization of white blood cells, to embryonic lethality as for Greenberg dysplasia (GRBGD). Existing PHA mouse models do not fully recapitulate the human phenotypes, hindering efforts to understand the molecular etiology of this disorder. Here we show, using CRISPR/Cas-9 gene editing technology, that a 236bp N-terminal deletion in the mouse Lbr gene, generating a protein missing the N-terminal domains of LBR, presents a superior model of human PHA. Further, we address recent reports of a link between Lbr and defects in X chromosome inactivation (XCI) and show that our mouse mutant displays minor X chromosome inactivation defects that do not lead to any overt phenotypes in vivo. We suggest that our N-terminal deletion model provides a valuable pre-clinical tool to the research community and will aid in further understanding the etiology of PHA and the diverse functions of LBR

Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery

Gene expression can be activated or suppressed using CRISPR--Cas9 systems. However, tools that enable dose-dependent activation of gene expression without the use of exogenous transcription regulatory proteins are lacking. Here we describe chemical epigenetic modifiers (CEMs) designed to activate the expression of target genes by recruiting components of the endogenous chromatin-activating machinery, eliminating the need for exogenous transcriptional activators. The system has two parts: catalytically inactive Cas9 (dCas9) in complex with FK506-binding protein (FKBP) and a CEM consisting of FK506 linked to a molecule that interacts with cellular epigenetic machinery. We show that CEMs upregulate gene expression at target endogenous loci up to 20-fold or more depending on the gene. We also demonstrate dose-dependent control of transcriptional activation, function across multiple diverse genes, reversibility of CEM activity and specificity of our best-in-class CEM across the genome.Activation of gene expression with chemical epigenetic modifier

Polycomb-mediated repression of EphrinA5 promotes growth and invasion of glioblastoma

Glioblastoma (GBM) is the most common and most aggressive intrinsic brain tumour in adults. Integrated transcriptomic and epigenomic analyses of glioblastoma initiating cells (GIC) in a mouse model uncovered a novel epigenetic regulation of EfnA5. In this model, Bmi1 enhances H3K27me3 at the EfnA5 locus and reinforces repression of selected target genes in a cellular context-dependent fashion. EfnA5 mediates Bmi1-dependent proliferation and invasion in vitro and tumour formation in an allograft model. Importantly, we show that this novel Polycomb feed-forward loop is also active in human GIC and we provide pre-clinical evidence of druggability of the EFNA5 signalling pathway in GBM xenografts overexpressing Bmi1

Chemical genomics reveals histone deacetylases are required for core regulatory transcription

Identity determining transcription factors (TFs), or core regulatory (CR) TFs, are governed by cell-type specific super enhancers (SEs). Drugs to selectively inhibit CR circuitry are of high interest for cancer treatment. In alveolar rhabdomyosarcoma, PAX3-FOXO1 activates SEs to induce the expression of other CR TFs, providing a model system for studying cancer cell addiction to CR transcription. Using chemical genetics, the systematic screening of chemical matter for a biological outcome, here we report on a screen for epigenetic chemical probes able to distinguish between SE-driven transcription and constitutive transcription. We find that chemical probes along the acetylation-axis, and not the methylation-axis, selectively disrupt CR transcription. Additionally, we find that histone deacetylases (HDACs) are essential for CR TF transcription. We further dissect the contribution of HDAC isoforms using selective inhibitors, including the newly developed selective HDAC3 inhibitor LW3. We show HDAC1/2/3 are the co-essential isoforms that when co-inhibited halt CR transcription, making CR TF sites hyper-accessible and disrupting chromatin looping

Seleção de isolados de trichoderma spp. para o controle de sclerotinia sclerotiorum, agente causal do mofo-branco do feijoeiro.

O mofo-branco (Sclerotinia sclerotiorum) é uma das doenças mais destrutiva do feijoeiro nos plantios de outono-inverno, quando os dias são mais curtos e as temperaturas amenas (15-25oC). O controle químico é caro e, como medida isolada, pode ter eficiência baixa. Várias espécies de Trichoderma são antagonistas ou parasitas de escleródios do patógeno no solo. Porém, os isolados mais utilizados como agentes de biocontrole são favorecido por temperaturas acima de 25oC. O uso desses agentes em áreas e/ou épocas de temperaturas amenas pode ser pouco eficiente. Objetivou-se selecionar isolados de Trichoderma spp. eficientes em inibir a germinação e parasitar escleródios do patógeno em temperaturas amenas. Vinte isolados do antagonista foram avaliados. Escleródios foram enterrados no solo em vasos e foram aplicados os tratamentos: testemunha; isolados de Trichoderma spp. (107 conídios/mL e volume de calda de 300 L/ha) e Cerconil (dose recomendada). Após cinco dias a 22±2ºC, os escleródios foram recuperados e transferidos para discos de cenoura sobre ágar-água. Avaliaram-se o número de escleródios germinados e o número de escleródios parasitados após 10 dias. O ensaio foi conduzido duas vezes em delineamento inteiramente ao acaso com sete repetições. Os isolados ALF111 e ALF409 consistentemente inibiram a germinação e parasitaram mais de 80% dos escleródios. Além destes, os isolados ALF02, ALF57, ALF324 e ALF402 se destacaram. O isolado 172H inibiu a germinação, porém não foi capaz de parasitar os escleródios, o que sugere a ocorrência de outros mecanismos de ação, como antibiose. Concluiu-se que os isolados selecionados são candidatos potenciais para o controle do mofo-branco em cultivos de outono-inverno.Documentos, IAC, Campinas, v. 79, p. 98-101, 2007

Miswired Enhancer Logic Drives a Cancer of the Muscle Lineage

Core regulatory transcription factors (CR TFs) establish enhancers with logical ordering during embryogenesis and development. Here we report that in fusion-positive rhabdomyosarcoma, a cancer of the muscle lineage, the chief oncogene PAX3-FOXO1 is driven by a translocated FOXO1 super enhancer (SE) restricted to a late stage of myogenesis. Using chromatin conformation capture techniques, we demonstrate that the extensive FOXO1 cis-regulatory domain interacts with PAX3. Furthermore, RNA sequencing and chromatin immunoprecipitation sequencing data in tumors bearing rare PAX translocations implicate enhancer miswiring across all fusion-positive tumors. HiChIP of H3K27ac showed connectivity between the FOXO1 SE, additional intra-domain enhancers, and the PAX3 promoter. We show that PAX3-FOXO1 transcription is diminished when this network of enhancers is ablated by CRISPR. Our data reveal a hijacked enhancer network that disrupts the stepwise CR TF logic of normal skeletal muscle development (PAX3 to MYOD to MYOG), replacing it with an "infinite loop" enhancer logic that locks rhabdomyosarcoma in an undifferentiated stage