The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 ...protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.
Display omitted
•CRISPRi enables robust gene repression and activation in human cells•CRISPRi knockdown is specific with minimal off-target effects in human cells•CRISPRi can effectively repress endogenous genes in human and yeast•dCas9 enables modular and programmable RNA-guided genome regulation in eukaryotes
Catalytically inactive CRISPR can be targeted to specific loci in human and yeast cells to specifically repress and activate transcription. The study demonstrates the potential for adapting CRISPRi for multiple modes of transcriptional control, chromatin modification, and regulatory element mapping in a broad range of eukaryotes.
In this study, the authors examined the correlates of psychological aggression victimization and perpetration among a community sample of 145 heterosexual couples. For both women and men, ...psychological aggression victimization was associated with greater psychological distress, anxiety, and physical health symptoms beyond the effects of physical aggression. Psychological aggression victimization was also uniquely associated with higher levels of depression for women. Trait anger and poor relationship adjustment were the strongest correlates of psychological aggression perpetration across genders. Childhood father-to-child and father-to-mother aggressions were associated with psychological aggression perpetration for men only, suggesting possible distinct etiologies across genders. These data highlight the importance of the further development of models for psychological aggression in both women and men.
Sexual reproduction is restricted to eukaryotic species and involves the fusion of haploid gametes to form a diploid cell that subsequently undergoes meiosis to generate recombinant haploid forms. ...This process has been extensively studied in the unicellular yeast Saccharomyces cerevisiae, which exhibits separate regulatory control over mating and meiosis. Here we address the mechanism of sexual reproduction in the related hemiascomycete species Candida lusitaniae. We demonstrate that, in contrast to S. cerevisiae, C. lusitaniae exhibits a highly integrated sexual program in which the programs regulating mating and meiosis have fused. Profiling of the C. lusitaniae sexual cycle revealed that gene expression patterns during mating and meiosis were overlapping, indicative of co-regulation. This was particularly evident for genes involved in pheromone MAPK signalling, which were highly induced throughout the sexual cycle of C. lusitaniae. Furthermore, genetic analysis showed that the orthologue of IME2, a 'diploid-specific' factor in S. cerevisiae, and STE12, the master regulator of S. cerevisiae mating, were each required for progression through both mating and meiosis in C. lusitaniae. Together, our results establish that sexual reproduction has undergone significant rewiring between S. cerevisiae and C. lusitaniae, and that a concerted sexual cycle operates in C. lusitaniae that is more reminiscent of the distantly related ascomycete, Schizosaccharomyces pombe. We discuss these results in light of the evolution of sexual reproduction in yeast, and propose that regulatory coupling of mating and meiosis has evolved multiple times as an adaptation to promote the haploid lifestyle.
Macroautophagy (hereafter referred to as autophagy) is a conserved process that promotes cellular homeostasis through the degradation of cytosolic components, also known as cargo. During autophagy, ...cargo is sequestered into double-membrane vesicles called autophagosomes, which are predominantly transported in the retrograde direction to the perinuclear region to fuse with lysosomes, thus ensuring cargo degradation.1 The mechanisms regulating directional autophagosomal transport remain unclear. The ATG8 family of proteins associates with autophagosome membranes2 and plays key roles in autophagy, including the movement of autophagosomes. This is achieved via the association of ATG8 with adaptor proteins like FYCO1, involved in the anterograde transport of autophagosomes toward the cell periphery.1,3–5 We previously reported that phosphorylation of LC3B/ATG8 on threonine 50 (LC3B-T50) by the Hippo kinase STK4/MST1 is required for autophagy through unknown mechanisms.6 Here, we show that STK4-mediated phosphorylation of LC3B-T50 reduces the binding of FYCO1 to LC3B. In turn, impairment of LC3B-T50 phosphorylation decreases starvation-induced perinuclear positioning of autophagosomes as well as their colocalization with lysosomes. Moreover, a significantly higher number of LC3B-T50A-positive autophagosomes undergo aberrant anterograde movement to axonal tips in mammalian neurons and toward the periphery of mammalian cells. Our data support a role of a nutrient-sensitive STK4-LC3B-FYCO1 axis in the regulation of the directional transport of autophagosomes, a key step of the autophagy process, via the post-translational modification of LC3B.
Display omitted
•STK4-mediated LC3B phosphorylation lowers LC3B binding to the transport protein FYCO1•LC3B phosphorylation inhibition decreases retrograde transport of autophagosomes•Block of LC3B phosphorylation also compromises autophagosome-lysosome association•The STK4-LC3B-FYCO1 axis is a nutrient-sensitive, autophagy-regulatory pathway
Nieto-Torres et al. describe that phosphorylation of the autophagy protein LC3B is critical for the retrograde transport of autophagosomes within cells. Block of LC3B phosphorylation leads to aberrant autophagosome transport and association with lysosomes. This pathway may be relevant for autophagy-related pathologies, such as neurodegeneration.
Contaminants of emerging concern (CECs), such as pharmaceuticals, personal care products, pesticides, synthetic and natural hormones and industrial chemicals, are frequently released into the ...environment because of the inability of conventional processes in municipal wastewater treatment plants to remove them. Some examples of alternative options to remove such pollutants are photo-Fenton and ozone-based processes, which are two techniques widely studied in Ibero-American countries. In fact, this region has been responsible for delivering frequently publications and conferences on advanced oxidation processes. This work is a critical review of recent developments in the intensification of the two aforementioned advanced oxidation techniques for CECs elimination in the Ibero-American region. Specifically for the photo-Fenton process (pF), this study analyses strategies such as iron-complexation with artificial substances (e.g., oxalic acid and ethylenediamine-N,N′-disuccinic acid) and natural compounds (such as humic-like substances, orange juice or polyphenols) and hybrid processes with ultrasound. Meanwhile, for ozonation, the enhancement of CECs degradation by adding hydrogen peroxide (i.e., peroxone), ultraviolet or solar light, and combining (i.e., photolytic ozonation) with catalysts (i.e., catalytic ozonation) was reviewed. Special attention was paid to how efficient these techniques are for removing contaminants from water matrices, and any potentialities and weak points of the intensified processes.
Display omitted
•A critical review on intensification of two widely investigated processes in Ibero-America is shown.•Strategies for contaminants of emerging concern removal by photo-Fenton and O3 are analyzed.•After application of intensified process ecotoxicity and pollutants transformations should be tested.•Understanding the role of secondary radicals and metals-leaching from catalysts is needed.•Life cycle analyses and economic assessments should be carried out in future researches.
Macroautophagy/autophagy is a multi-step process that leads to cargo degradation via the fusion of hydrolases-containing lysosomes with cargo-loaded autophagosomes. For this process to occur, ...autophagosomes are directionally transported by molecular motors toward the nucleus, where they fuse with lysosomes for cargo degradation. The molecular basis for this regulation, including the cell machinery required for this directional transport, has not been fully identified. Using a combination of proteomic and live-imaging approaches in mammalian cells, including primary neurons, we describe that the phosphorylation of the autophagosome protein Atg8/LC3B by the Hippo kinase STK4/MST1, an event we previously reported to be required for autophagy completion, reduces the binding of the transport-related protein FYCO1 to MAP1LC3B/LC3B. This event in turn allows the proficient microtubule-based transport of autophagosomes toward the perinuclear area, thus facilitating the contact of autophagosomes with lysosomes. In the absence of LC3B phosphorylation, autophagosomes undergo aberrant transport including increased movement toward the cell periphery resulting in reduced autophagosome-lysosome colocalization. Thus, LC3B phosphorylation modulates the directional transport of autophagosomes to meet with lysosomes in the perinuclear area, a crucial event in ensuring autophagic degradation of cargo.
The ability of the ultrasound (US) combined with peroxymonosulfate (PMS), and a carbonaceous material (BC) was evaluated in the degradation of a model pollutant (acetaminophen, ACE). The US/BC/PMS ...system was compared with other possible systems (US, oxidation by PMS, BC adsorption, BC/PMS, US/PMS, and US/BC. The effect of the ultrasonic frequency (40, 375, and 1135 kHz) on the kinetics and synergy of the ACE removal was evaluated. In the US system, kinetics was favored at 375 kHz due to the increased production of hydroxyl radicals (HO•), but this did not improve in the US/PMS and US/BC systems. However, synergistic and antagonistic effects were observed at the low and high frequencies where the production of radicals is less efficient but there is an activation of PMS through mechanical effects. US/BC/PMS at 40 kHz was the most efficient system obtaining ∼95% ACE removal (40 μM) in the first 10 min of treatment, and high synergy (S = 10.30). This was promoted by disaggregation of the carbonaceous material, increasing the availability of catalytic sites where PMS is activated. The coexistence of free-radical and non-radical pathways was analyzed. Singlet oxygen (1O2) played the dominant role in degradation, while HO• and sulfate radicals (SO4•–), scarcely generated at low frequency, play a minimum role. Performance in hospital wastewater (HWW), urine, and seawater (SW) evidenced the competition of organic matter by BC active sites and reactive species and the removal enhancement when Cl− is present. Besides, toxicity decreased by ∼20% after treatment, being the system effective after three cycles of reuse.
Display omitted
•Frequency has kinetics and synergistic effects on the ultrasound/biochar/PMS system.•The hybrid system at 40 kHz was the most efficient system for acetaminophen removal.•Biochar disaggregation at 40 kHz improves adsorption, catalysis, and mass transfer.•1O2 had the dominant role, while the radical pathway (HO•, SO4•–) was secondary•Hospital wastewater and urine inhibited removal, but Cl− in seawater intensified it.
Abstract
The enhancement of the ultrasound system by adding diverse oxidants to remove a model contaminant (acetaminophen, ACE) in water was investigated. Different parameters were evaluated to study ...their effect on both the degradation kinetics and the synergy of the combination. The variables studied were the ultrasonic frequency (575, 858, and 1135 kHz), type of oxidant (hydrogen peroxide, sodium peroxydisulfate (or persulfate, PDS), and potassium peroxymonosulfate (PMS)), ACE concentration (4, 8, and 40 µM), and oxidant concentration (0.01, 0.1, 1, and 5 mM). Particular interest was placed on synergistic effects, implying that one process (or both) is activated by the other to lead to greater efficiency. Interestingly, the parameters that led to the higher synergistic effects did not always lead to the most favorable degradation kinetics. An increase in ACE removal of 20% was obtained using the highest frequency studied (1135 kHz), PMS 0.1 mM, and the highest concentration of ACE (40 µM). The intensification of degradation was mainly due to the ability of ultrasound to activate oxidants and produce extra hydroxyl radicals (HO
•
) or sulfate radicals (SO
4
•–
). Under these conditions, treatment of ACE spiked into seawater, hospital wastewater, and urine was performed. The hospital wastewater matrix inhibited ACE degradation slightly, while the urine components inhibited the pollutant degradation completely. The inhibition was mainly attributed to the competing organic matter in the effluents for the sono-generated radical species. On the contrary, the removal of ACE in seawater was significantly intensified due to “salting out” effects and the production of the strong oxidant HOCl from the reaction of chloride ions with PMS.
Prévalence et incidence de la démence : revue systématique et méta-analyse.
Contexte:
La démence est une maladie neurologique fréquente touchant de nombreuses personnes âgées. Elle est la cause de la ...perte de l’indépendance, d’une qualité de vie altérée, d’une mortalité prématurée et constitue un fardeau important pour les soignants. Elle entraîne une utilisation et un coût élevé de soins de santé par ces patients. Nous avons effectué une revue systématique et une méta-analyse à jour de la prévalence et de l’incidence de la démence à travers le monde.
Méthodologie:
Nous avons identifié les études pertinentes publiées entre 2000 (1985 pour les publications canadiennes) et juillet 2012 dans les bases de données MEDLINE et EMBASE. Les articles choisis pour un examen du texte intégral ont été inclus dans l’examen systématique s’ils fournissaient une estimation originale à l’échelle populationnelle de l’incidence et/ou de la prévalence de la démence. Nous avons également recherché des études additionnelles dans la liste de références incluse dans ces articles. Deux évaluateurs ont revu indépendamment les résumés et le texte intégral des publications ainsi que l’extraction des données et ils en ont évalué la qualité. Nous avons utilisé des modèles à effets aléatoires et/ou de méta-régression pour générer des estimations regroupées par âge, sexe, milieu (communauté, institution ou les deux), critères diagnostiques utilisés, lieu (continent), et année de collecte des données.
Résultats:
Parmi les 16 066 résumés revus, 707 articles ont été choisis pour une revue du texte intégral. En tout, 160 articles rencontraient les critères d’inclusion. Chez les individus de 60 ans et plus demeurant dans la communauté, les estimés regroupés de prévalence ponctuelle et annuelle de démence étaient de 48,62 (IC à 95%: 41,98 à 56,32) et 69,07 (IC à 95%: 52,36 à 91,11) par 1 000 personnes respectivement. Le taux d’incidence regroupée (même âge et contexte) était de 17,18 (IC à 95%: 13,90 à 21,23) par 1 000 personnes-années alors que la proportion d’incidence annuelle était de 52,85 (IC à 95%: 33,08 à 84,42) par 1 000. Un âge croissant des participants était associé à une prévalence et à une incidence plus élevées de démence. La prévalence annuelle pour une période donnée était plus élevée en Amérique du Nord qu’en Amérique du Sud, en Europe ou en Asie (en ordre décroissant de prévalence par période) et plus élevée dans un contexte institutionnel par rapport à la communauté et aux deux contextes combinés. Le sexe, les critères diagnostiques (sauf pour la proportion d’incidence) et l’année de la collecte des données n’étaient pas associés à des estimations de prévalence ou d’incidence significativement différentes au point de vue statistique, bien que les estimations étaient constamment plus élevées chez les femmes que chez les hommes.
Conclusions:
La démence est une maladie neurologique fréquente chez les individus plus âgés. Nous avons identifié d’importantes lacunes dans les connaissances sur l’épidémiologie, particulièrement en ce qui concerne l’incidence de la démence dans les pays à revenu faible et intermédiaire. Des estimations exactes de la prévalence et de l’incidence de la démence sont nécessaires pour la planification des services de santé et des services sociaux qui seront requis par une population vieillissante.
Soft tissue sarcomas (STS) are low-incidence tumors whose clinical and histopathological factors are associated with adverse oncological outcomes. This study evaluated prognostic factors (PF) ...associated with tumor recurrence and overall survival (OS) in patients diagnosed with STS of the extremities, treated at the Instituto Nacional de Cancerología (INC), Bogotá, Colombia.
An analytical observational study of a historical cohort was carried out, including patients diagnosed with STS and managed surgically in the Functional Unit for Breast and Soft Tissue Tumors of the INC from January 2008 to December 2018.
A total of 227 patients were included; 74.5% had tumors greater than 5 cm. Most patients (29.1%) were in stage IIIB at diagnosis. Age was associated with higher mortality (HR = 1.01; CI95%: 1-1.02;
= 0.048). Tumor persistence at admission to the INC (HR = 2.34; CI95%: 1.25-4.35;
= 0.007) and histologic grade III (HR = 5.36; CI95%: 2.29-12.56;
= <0.001) showed statistical significance in the multivariate analysis for recurrence of any type, as did the PFs associated with a higher risk of local recurrence (HR = 2.85; CI95%: 1.23-6.57;
= 0.014 and HR = 6.09; CI95%: 2.03-18.2;
= 0.001), respectively. Tumor size (HR = 1.03; CI95%: 1-1.06;
= 0.015) and histologic grade III (HR = 4.53; CI95%: 1.42-14.49;
= 0.011) were associated with a higher risk of distant recurrence.
This cohort showed that in addition to histologic grade and tumor size, tumor persistence at the time of admission has an impact on disease recurrence, so STS should be managed by a multidisciplinary team with experience in this pathology in high-volume reference centers.
PDF
