Infants' and toddlers' digital media use and mothers' mental health: A comparative study before and during the COVID-19 pandemic.

This study compared children's and mothers' digital media use and mothers' mental health in two samples: one accessed before (Group 1; N = 257; M = 33.18 years; SD = 4.79) and the other accessed during (Group 2; N = 256; M = 33.51 years; SD = 4.96) the COVID-19 pandemic in Brazil. Mothers of children up to 3 years old (Group 1: M = 17.95 months, SD = 9.85; Group 2: M = 16.48 months, SD = 10.15) answered an online survey. Bivariate analysis, factorial ANOVA tests, and multiple linear regression were performed. Results suggest that mothers' and children's media use duration was higher during the pandemic only among children over 12 months. Mothers' media use duration (ß = .18) and mothers' intention to offer media (ß = .23) contributed to the explanation of children's media use duration (F(4, 474) = 16.81; p < .001; R2  = .12; R2 adjusted = .117). Higher mothers' common mental disorders symptoms were also positively correlated to mothers' intention to offer media to children both before and during the pandemic. Results suggest that interventions focusing on infants and toddlers screen time reduction should target maternal aspects such as mental health, maternal screen time, and intention to offer media, taking into account the mothers' needs when planning these actions.

Este estudio comparó el uso de los medios digitales por parte de los niños y las madres con la salud mental de las madres en dos grupos muestra: uno al cual se tuvo acceso antes (Grupo 1: N = 257; M = 33.18 años; SD = 4.79) y el otro al cual se tuvo acceso durante (Grupo 2; N = 256; M = 33.51 años; SD = 4.96) la pandemia del COVID-19 en Brasil. Las madres de niños de hasta tres años (Grupo 1: M = 17.95 meses, SD = 9.85; Grupo 2: M = 16.48 meses, SD = 10.15) respondieron una encuesta electrónica. Los análisis bivariados los exámenes factoriales ANOVA, así como múltiples regresiones lineales se llevaron a cabo. Los resultados indican que la duración de uso de los medios por parte de las madres y los niños fue más alta durante la pandemia sólo entre niños de más de 12 meses. La duración de uso de los medios por parte de las madres (ß = 0.18) y la intención de las madres de ofrecer los medios (ß = 0.23) contribuyeron a explicar la duración de uso de los medios por parte de los niños (F(4,474) = 16,81; p < .001; R2 = .12; R2 ajustado = .117). Más altos síntomas de trastornos mentales comunes en las madres se correlacionaron también positivamente con la intención de las madres de ofrecer los medios a los niños tanto antes como durante la pandemia. Los resultados indican que las intervenciones enfocadas en reducir el tiempo frente a la pantalla de infantes y niños pequeñitos deben dirigirse a los aspectos maternos como la salud mental, el tiempo de la madre frente a la pantalla, así como la intención de ofrecer los medios, tomando en cuenta las necesidades de las madres cuando se planeen estas acciones.

Cette étude a comparé l'utilisation des médias numériques des enfants et des mères et la santé mentale des mères chez deux échantillons: l'un accédé avant la pandémie du Covid-19 (Groupe 1; N = 257; M = 33,18 ans; SD = 4,79) et l'autre accédé durant la pandémie du covid-19 (Groupe 2; N = 256; M = 33,51 ans; SD = 4,96) au Brésil. Les mères d'enfants jusqu'à l'âge de trois ans (Groupe 1: M = 17,95 mois, SD = 9,85; Groupe 2: M = 16,48 mois, SD = 10,15) ont répondu à un questionnaire en ligne. Une analyse à deux variables, des tests ANOVA factoriels, et une régression linéaire multiple ont été faits. Les résultats suggèrent que la durée de l'utilisation média des mères et des enfants a été plus élevée durant la pandémie uniquement pour les enfants de plus de 12 mois. La durée de l'utilisation média des mères (ß = 0,18) et l'intention des mères à offrir le média (ß = 0,23) a contribué à l'explication de la durée de l'utilisation média des enfants (F(4, 474) = 16,81; p <,001; R2 = ,12; R2 adjusté = ,117). Plus de symptômes communs de troubles mentaux des mères était aussi lié de manière positive à l'intention des mères d'offrir le média à la fois avant et durant la pandémie. Les résultats suggèrent que les interventions s'attachant à la réduction du temps d'écran des bébés et des petits enfants devraient cibler des aspects maternels comme la santé mentale, le temps d'écran maternel, et l'intention d'offrir le média, prenant en compte les besoins des mères en planifiant ces actions.

The Gene Sculpt Suite: a set of tools for genome editing.

The discovery and development of DNA-editing nucleases (Zinc Finger Nucleases, TALENs, CRISPR/Cas systems) has given scientists the ability to precisely engineer or edit genomes as never before. Several different platforms, protocols and vectors for precision genome editing are now available, leading to the development of supporting web-based software. Here we present the Gene Sculpt Suite (GSS), which comprises three tools: (i) GTagHD, which automatically designs and generates oligonucleotides for use with the GeneWeld knock-in protocol; (ii) MEDJED, a machine learning method, which predicts the extent to which a double-stranded DNA break site will utilize the microhomology-mediated repair pathway; and (iii) MENTHU, a tool for identifying genomic locations likely to give rise to a single predominant microhomology-mediated end joining allele (PreMA) repair outcome. All tools in the GSS are freely available for download under the GPL v3.0 license and can be run locally on Windows, Mac and Linux systems capable of running R and/or Docker. The GSS is also freely available online at www.genesculpt.org.

Advanced glycation endproducts produced by in vitro glycation of type I collagen modulate the functional and secretory behavior of dorsal root ganglion cells cultivated in two-dimensional system.

Advanced glycation end-products (AGEs) are proteins/lipids that are glycated upon sugar exposure and are often increased during inflammatory diseases such as osteoarthritis and neurodegenerative disorders. Here, we developed an extracellular matrix (ECM) using glycated type I collagen (ECM-GC), which produced similar levels of AGEs to those detected in the sera of arthritic mice. In order to determine whether AGEs were sufficient to stimulate sensory neurons, dorsal root ganglia (DRGs) cells were cultured on ECM-GC or ECM-NC-coated plates. ECM-GC or ECM-NC were favorable for DRG cells expansion. However, ECM-GC cultivated neurons displayed thinner F-actin filaments, rounded morphology, and reduced neuron interconnection compared to ECM-NC. In addition, ECM-GC did not affect RAGE expression levels in the neurons, although induced rapid p38, MAPK and ERK activation. Finally, ECM-GC stimulated the secretion of nitrite and TNF-α by DRG cells. Taken together, our in vitro glycated ECM model suitably mimics the in vivo microenvironment of inflammatory disorders and provides new insights into the role of ECM impairment as a nociceptive stimulus.

Hyperglycemia reduces integrin subunits alpha v and alpha 5 on the surface of dermal fibroblasts contributing to deficient migration.

Deficient wound healing is a common multifactorial complication in diabetic patients, but the cellular and molecular mechanisms involved are poorly defined. In the present study, we analyzed the effects of hyperglycemia on integrins expression in rat dermal fibroblasts and addressed its role in cell adhesion and migration. Diabetes Mellitus was induced in rats by streptozotocin injection and maintained for 30 days. Primary cultures of dermal fibroblasts from control and diabetic rats were maintained under low glucose (5 mM D-glucose) or high glucose (30 mM D-glucose) for 7 days. Cell adhesion and migration were studied by kymography, transwell, and time-lapse assays, and the expressions of integrin subunits αv and α5 were studied by immunocytochemistry and western blotting. Fibroblasts derived from diabetic rats confirmed a reduced migration speed and delayed spreading compared to fibroblasts derived from control rats. The membrane fraction of diabetic-derived fibroblasts showed a decrease of integrin subunits α5 and αv, which was confirmed by immunocytochemistry assays. A reduction in the pericellular fibronectin matrix was also observed. The exposure of diabetic-derived cells to a higher concentration of exogenous fibronectin improved migration velocity and the expression of αv but did not completely restore their migration capacity. In conclusion, the mechanisms involved in the deleterious effects of Diabetes Mellitus on wound healing include the ability of fibroblasts to secrete and to adhere to fibronectin.

Fitness Screens Map State-Specific Glioblastoma Stem Cell Vulnerabilities.

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults and is driven by self-renewing glioblastoma stem cells (GSCs) that persist after therapy and seed treatment refractory recurrent tumors. GBM tumors display a high degree of intra- and inter-tumoral heterogeneity that is a prominent barrier to targeted treatment strategies. This heterogeneity extends to GSCs that exist on a gradient between two transcriptional states or subtypes termed developmental and injury-response. Drug targets for each subtype are needed to effectively target GBM. To identify conserved and subtype-specific genetic dependencies across a large and heterogeneous panel of GSCs, we designed the GBM5K targeted gRNA library and performed fitness screens in a total of 30 patient-derived GSC cultures. The focused CRISPR screens identified the most conserved subtype-specific vulnerabilities in GSCs and elucidated the functional dependency gradient existing between the developmental and injury-response states. Developmental-specific fitness genes were enriched for transcriptional regulators of neurodevelopment, whereas injury-response-specific fitness genes were highlighted by several genes implicated in integrin and focal adhesion signaling. These context-specific vulnerabilities conferred differential sensitivity to inhibitors of ß1 integrin, FAK, MEK and OLIG2. Interestingly, the screens revealed that the subtype-specific signaling pathways drive differential cyclin D (CCND1 vs. CCND2) dependencies between subtypes. These data provide biological insight and mechanistic understanding of GBM heterogeneity and point to opportunities for precision targeting of defined GBM and GSC subtypes to tackle heterogeneity.

Correction: Bufalo et al. Human Sensory Neuron-like Cells and Glycated Collagen Matrix as a Model for the Screening of Analgesic Compounds. Cells 2022, 11, 247.

In the original publication [...].

Treatment of long-term stored DNA--comparison between different methods to obtain high-quality material.

Long-term stored DNA can be sometimes the only source of genetic material of an organism that does not exist anymore, but a research interest still persists. However, there is a lack of information about useful methods to improve quality from such type of material. In this study, we compared four different protocols using DNA samples collected in 1998. Fresh DNA was also tested aiming to check the differences between these two material types. Sixteen samples of each DNA type treated with phenol-chloroform with PEG 5.0%, silica-gel membrane spin column, PEG 7.5%, and glass-fiber matrix spin column were submitted to spectrophotometer measurements, electrophoresis, PCR, and RFLP-PCR to assess the best method concerning yield, quality, and purity. Based on the results, purification with PEG 7.5% was considered the best method to treat aged DNA samples. In addition to the efficiency, this protocol has low cost. Analyzing the data, we also conclude that long-term stored DNA may be considered a reliable and potential resource for future molecular studies.

Reducing harm and promoting positive media use strategies: new perspectives in understanding the impact of preschooler media use on health and development.

Most children grow up in homes with easy access to multiple screens. Screen use by children between the ages of 0 to 5 has become a worldwide preoccupation. In the present narrative review, we examine child and parent screen use and its contribution to physical, cognitive, and social developmental outcomes. As research has mostly focused on the adverse consequences of screen media, we aim to depict both the negative and the positive influences of screen usage. To provide a more nuanced portrait of the potential benefits and harms of screen use, we examine how consequences of media use vary according to the content of media (ex., educational, violent), context (ex., using screens during mealtimes), and the nature (ex., passive vs active use) of child screen use. Our review supports existing screen time guidelines and recommendations and suggests that media content, the context of use, and the nature of child use, as well as the parent's own screen use, be considered clinically. Future research should seek to clarify how these dimensions jointly contribute to child screen use profiles and associated consequences. Finally, child sex, behavioral/temperamental difficulties, and family adversity appear to contribute to child screen use and its consequences and should be considered in future research. Suggestions for harm-reduction approaches are discussed.

Prospective associations between child screen time and parenting stress and later inattention symptoms in preschoolers during the COVID-19 pandemic.

Introduction: Child attention skills are critical for supporting self-regulation abilities, especially during the first years of life. On the other hand, inattention symptoms in preschoolers have been associated with poor school readiness, literacy skills and academic achievement. Previous research has linked excessive screen time with increased inattention symptoms in early childhood. However, most research has only focused on TV exposure and did not investigate this association during the COVID-19 pandemic. This atypical context has increased screen time in children worldwide, including preschoolers. We hypothesize that higher levels of child screen media and parenting stress at age 3.5 will be associated with higher child inattention symptoms at age 4.5. Method: This study draws on participants followed longitudinally over the span of 2-years for an investigation of Canadian preschoolers' screen media use during the pandemic (N = 315, 2020). A follow-up with this sample was completed in 2021 (N = 264). Results: Analyses using multiple linear regression, revealed a positive association between child screen time at age 3.5 and inattention symptoms at 4.5 years. Parental stress was also positively associated with child inattention symptoms. Associations were observed above individual (child age, inhibitory control, and sex) and family (parent education and family income) characteristics. Discussion: These results confirmed our hypothesis and highlight that preschooler screen use and parenting stress may undermine attentional skills. Since attention is a crucial component for children development, behavior and academic outcomes, our study reinforces the importance for parents of adopting healthy media habits.

Evaluation of the Amazonian fruit Ambelania acida: Chemical and nutritional studies.

Ambelania acida is native to the Amazon region, with few published studies of its fruits. We examined the proximate composition of its fruits, including minerals, fatty acids, volatile organic compounds (VOCs), as well as its antioxidant capacity. The protein contents (2.61%) of the pulp and seeds (13.6%) were higher than observed in other taxa of the family or in other tropical fruits. Peel and pulp showed high contents of potassium, calcium, and magnesium, and the potassium content in the pulp was 1125 mg/100 g. The peel had higher contents of total phenolics, tannins, and ortho-diphenols than the pulp, as well as better antioxidant activity as evidenced by 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), Ferric Reducing Antioxidant Power (FRAP), and Fe2+ chelating activity assays. GC-MS analyses identified 42 VOCs in the peel and pulp, with more than 90% being classified as terpenes. Eleven types of fatty acids were identified in the lipid fractions of the peel, pulp, and seeds. Linoleic acid, an essential fatty acid for humans, was the principal fatty acid in the edible portion of the fruit, therefore, evidencing its nutritionally significant profile for the fruits when considering the relationship among polyunsaturated, saturated, and monounsaturated fatty acids. The information gathered here indicates that this native fruit is a healthy food source and its cultivation and consumption should be stimulated.

Human Sensory Neuron-like Cells and Glycated Collagen Matrix as a Model for the Screening of Analgesic Compounds.

Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (ß-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions.

Cre/lox regulated conditional rescue and inactivation with zebrafish UFlip alleles generated by CRISPR-Cas9 targeted integration.

The ability to regulate gene activity spatially and temporally is essential to investigate cell-type-specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish. However, simple and efficient methods for isolation of stable, Cre/lox regulated zebrafish alleles are lacking. Here, we applied our GeneWeld CRISPR-Cas9 targeted integration strategy to generate floxed alleles that provide robust conditional inactivation and rescue. A universal targeting vector, UFlip, with sites for cloning short homology arms flanking a floxed 2A-mRFP gene trap, was integrated into an intron in rbbp4 and rb1. rbbp4off and rb1off integration alleles resulted in strong mRFP expression,>99% reduction of endogenous gene expression, and recapitulated known indel loss-of-function phenotypes. Introduction of Cre led to stable inversion of the floxed cassette, loss of mRFP expression, and phenotypic rescue. rbbp4on and rb1on integration alleles did not cause phenotypes in combination with a loss-of-function mutation. Addition of Cre led to conditional inactivation by stable inversion of the cassette, gene trapping and mRFP expression, and the expected mutant phenotype. Neural progenitor Cre drivers were used for conditional inactivation and phenotypic rescue to showcase how this approach can be used in specific cell populations. Together these results validate a simplified approach for efficient isolation of Cre/lox-responsive conditional alleles in zebrafish. Our strategy provides a new toolkit for generating genetic mosaics and represents a significant advance in zebrafish genetics.

Endogenous zebrafish proneural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration.

We previously reported efficient precision targeted integration of reporter DNA in zebrafish and human cells using CRISPR/Cas9 and short regions of homology. Here, we apply this strategy to isolate zebrafish Cre recombinase drivers whose spatial and temporal restricted expression mimics endogenous genes. A 2A-Cre recombinase transgene with 48 bp homology arms was targeted into proneural genes ascl1b, olig2 and neurod1. We observed high rates of germline transmission ranging from 10 to 100% (2/20 olig2; 1/5 neurod1; 3/3 ascl1b). The transgenic lines Tg(ascl1b-2A-Cre)is75, Tg(olig2-2A-Cre)is76, and Tg(neurod1-2A-Cre)is77 expressed functional Cre recombinase in the expected proneural cell populations. Somatic targeting of 2A-CreERT2 into neurod1 resulted in tamoxifen responsive recombination in the nervous system. The results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of the targeted genes. This approach provides a straightforward, efficient, and cost-effective method to generate cell type specific zebrafish Cre and CreERT2 drivers, overcoming challenges associated with promoter-BAC and transposon mediated transgenics.

Crotoxin Inhibits Endothelial Cell Functions in Two- and Three-dimensional Tumor Microenvironment.

Antitumor property of Crotoxin (CTX), the major toxin from Crotalus durissus terrificus snake venom, has been demonstrated in experimental animal models and clinical trials. However, the direct action of this toxin on the significant events involved in neovascularization, which are essential for tumor growth and survival, has not been confirmed. This study investigated the effects of CTX on the key parameters of neovascularization in two- and three-dimensional culture models. Murine endothelial cell lines derived from thymus hemangioma (t.End.1) were treated at different concentrations of CTX (6.25-200 nM). Endothelial cell proliferation, cell adhesion, and actin cytoskeletal dynamics on laminin (10 µg/ml), type I collagen (10 µg/ml), and fibronectin (3 µg/ml) were evaluated along with the endothelial cell migration and formation of capillary-like tubes in 3D Matrigel. CTX concentration of 50 nM inhibited tube formation on 3D Matrigel and impaired cell adhesion, proliferation, and migration under both culture medium and tumor-conditioned medium. These actions were not accountable for the loss of cell viability. Inhibition of cell adhesion to different extracellular matrix components was related to the reduction of αv and α2 integrin distribution and cytoskeletal actin polymerization (F-actin), accompanied by inhibition of focal adhesion kinase (FAK), Rac1 (GTPase) signaling proteins, and actin-related protein 2/3 (Arp 2/3) complex. This study proved that CTX inhibits the major events involved in angiogenesis, particularly against tumor stimuli, highlighting the importance of the anti-angiogenic action of CTX in inhibition of tumor progression.

Application of the Southwell Plot method to determine equilibrium time in phosphate adsorption.

Among the different factors that influence the liquid-solid adsorption technique, equilibrium time is one of the most relevant and requires a large number of experiments over a long period of time for its determination. This work evaluates the Southwell Plot as a further tool that can contribute to determining the equilibrium time in adsorption processes. It can also optimize the operating conditions in a batch system for the removal of phosphate in adsorbents produced from domestic sewage sludge and clam shell residue. Sewage sludge and clam shell residues were ground, sieved and sintered at 700 °C for 1 h. The material was characterized by thermal analyses (TG/DTG), chemical analysis (EDX), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and adsorption studies. The kinetic studies were investigated by varying the initial concentration of the phosphate solution and mass of the adsorbent. The equilibrium time was determined by applying the Southwell Plot method to the kinetic data and the results showed some fluctuations as a function of the adsorbent mass. At 0.30 g of the adsorbent in 30 mL of the phosphate solution, regardless of the initial phosphate concentration, the equilibrium time determined by the Southwell Plot was 4 h. The maximum phosphate adsorption capacity in this condition, determined by the Langmuir equation, was 49.45 mg g-1.

GeneWeld: Efficient Targeted Integration Directed by Short Homology in Zebrafish.

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Multiple design strategies for zebrafish gene targeting have previously been reported with widely varying frequencies for germline recovery of integration alleles. The GeneWeld protocol and pGTag (plasmids for Gene Tagging) vector series provide a set of resources to streamline precision gene targeting in zebrafish. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at a CRISPR/Cas induced DNA double-strand break. The pGTag vectors contain reporters flanked by a universal CRISPR sgRNA sequence to liberate the targeting cassette in vivo and expose homology arms for homology-driven integration. Germline transmission rates for precision-targeted integration alleles range 22-100%. Our system provides a streamlined, straightforward, and cost-effective approach for high-efficiency gene targeting applications in zebrafish. Graphic abstract: GeneWeld method for CRISPR/Cas9 targeted integration.

Symptoms of Common Mental Disorders in Brazilian Parents During the COVID-19 Pandemic: Associated Factors.

COVID-19 pandemic has become a significant international public health problem. In addition to dealing with the pandemic's impact on mental health, parents need to cope with specific changes in their routines caused by social distance measures. This study aimed to investigate common mental disorders (CMD) symptoms in Brazilian parents during the COVID-19 pandemic and its associated factors. A total of 232 Brazilian parents ranging from 20 to 48 years old (M = 33.85; SD = 4.83) with children aged 1-36 months (M = 17.00; SD = 9,87) participated in an online survey. Parents answered a sociodemographic questionnaire, Self-Report Questionnaire (SRQ-20), Perceived Stress Scale (PSS-4), and Parenting Sense of Competence Scale. Chi-square tests, correlations, and multiple linear regression were performed. Results showed that parents' symptoms of CMD were negatively associated to perceived parental competence (ß =- 0.130; p = 0.011) and family income (ß =- 0.190; p = 0.024). Furthermore, perceived stress was the most related variable to parents' symptoms of CMD (ß = 0.618; p < 0.001), showing a positive association. The model explained 49.5% of the variation. Results suggest that lower family income may increase symptoms of CMD in Brazilian parents, which is a concern in a country of high social inequality. Parental sense of competence may be a relevant protective factor. Interventions targeting parental competence and stress reduction should be considered to address the mental health impacts of the pandemic.

Amblyomin-X, a recombinant Kunitz-type inhibitor, regulates cell adhesion and migration of human tumor cells.

In a tumor microenvironment, endothelial cell migration and angiogenesis allow cancer to spread to other organs causing metastasis.  Indeed, a number of molecules that are involved in cytoskeleton re-organization and intracellular signaling have been investigated for their effects on tumor cell growth and metastasis. Alongside that, Amblyomin-X, a recombinant Kunitz-type protein, has been shown to reduce metastasis and tumor growth in in vivo experiments. In the present report, we provide a mechanistic insight to these antitumor effects, this is,  Amblyomin-X modulates Rho-GTPases and uPAR signaling, and reduces the release of MMPs, leading to disruption of the actin cytoskeleton and decreased cell migration of tumor cell lines. Altogether, our data support a role for Amblyomin-X as a novel potential antitumor drug. ABBREVIATIONS: Amb-X: Amblyomin-X; ECGF: endotelial cell growth factor; ECM: extracellular matrix; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HUVEC: human umbilical vein endothelial cell; LRP1: low-density lipoprotein receptor-related protein; MMP: matrix metalloproteinase; HPI-4: hedgehog pathway inhibitor 4; PAI-1: plasminogen activator inhibitor 1; PMA: phorbol 12-myristate-13-acetate; TFPI: tissue factor pathway inhibitor; uPA: urokinase plasminogen activator; uPAR: uPA receptor.

Efficient targeted integration directed by short homology in zebrafish and mammalian cells.

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22-100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.

Determinants of organochlorine levels detectable in the amniotic fluid of women from Tenerife Island (Canary Islands, Spain).

Organochlorines (OCs) tend to accumulate in human tissues and can be measured in amniotic fluid (AF). The detection of OCs in AF samples reflects intrauterine exposure of human beings to these persistent organic pollutants. The present study was performed to evaluate the level of contamination of AF by OCs in 100 pregnant women from Tenerife Island (Canary Islands, Spain). Gas chromatography/mass spectrometry (GC/MS) was used to identify and quantify the analytes, including 7 polychlorobiphenyl (PCB) congeners and 18 OC pesticides and metabolites. The majority of the AF samples (67%) showed some detectable OC-residue, hexachlorobenzene (HCB) being the most frequently detected compound (66% of the samples) and at the highest concentration (median 0.023 ng/ml). Lindane was also detected in 28% of the samples. Inverse associations were found between previous lactation and hexachlorocyclohexane isomers (HCH) and cyclodienes in the group of younger women (p = 0.037 and p = 0.027, respectively). Unexpectedly, serum values of HCB (r = -0.414; p = 0.04), gamma-HCH (r = -0.294; p = 0.035), and SigmaOCs (r = -0.350; p = 0.014) were negatively related to age. Even more, women with detectable levels of HCH isomers were younger (33.9 +/- 4.9 years) than women with undetectable levels of them (36.1 +/- 4.9 years; p = 0.035). We conclude that approximately one in two fetuses in the Canary Islands is exposed to OCs in utero, and that, therefore, the exposure of young women from these Islands to some HCH isomers persists nowadays. Because prenatal exposure to these chemicals may be a causative factor in adverse health trends, further studies are required to enhance preventive measures.