In vitro genome editing for testing potential gRNAs in CRISPR/Cas9 strategy

Motivation: CRISPR/Cas9 is a promising strategy that improves the efficacy of homology recombination, opening a wide number of possibilites for genome editing. The use of this technology on the reparation of single nucleotide mutations is being under investigation to recover the phenotype of a number of diseases, as the Crigler-Najjar syndrome. Methods: The genomic target sequence of the Ugt1a1 mouse gene was cloned into vectors that were designed to assess efficiency in the generation of double strand brakes (DSB) by engineered nucleases. We have designed different sgRNAs targeting this genomic region. The vectors were transfected into Hek293 together with a plasmid expressing the Cas9 nucelase and the sgRNA, generating a DSB in the target sequence. After recombination, the inactive luciferase gene recovers activity, which is proportional to the cutting efficiency of the nuclease. Then, luciferase and T7 assays were used to determine the activity of gRNAs to target the specific genome locus. Results: Depending on the DNA sequence where is located the gRNA and, specially, the PAM sequences, we have obtained different results on the activity of our gRNAs. However, different factors, as the type of cells transfected or the vectors used, can affect the final activity of the gRNAs and thus, the whole CRISPR/Cas9 activity.Conclusions: With this project, it has been demonstrated the importance of a good design and optimization of the protocols to choose the most efficient gRNAs for CRISPR/Cas9 machinery, as well as in the vectors used to synthetize all the components needed

In vivo cranial bone strain and bite force in the agamid lizard Uromastyx geyri

In vivo bone strain data are the most direct evidence of deformation and strain regimes in the vertebrate cranium during feeding and can provide important insights into skull morphology. Strain data have been collected during feeding across a wide range of mammals; in contrast, in vivo cranial bone strain data have been collected from few sauropsid taxa. Here we present bone strain data recorded from the jugal of the herbivorous agamid lizard Uromastyx geyri along with simultaneously recorded bite force. Principal and shear strain magnitudes in Uromastyx geyri were lower than cranial bone strains recorded in Alligator mississippiensis, but higher than those reported from herbivorous mammals. Our results suggest that variations in principal strain orientations in the facial skeleton are largely due to differences in feeding behavior and bite location, whereas food type has little impact on strain orientations. Furthermore, mean principal strain orientations differ between male and female Uromastyx during feeding, potentially because of sexual dimorphism in skull morphology

Validation of the Predictive Model of the European Society of Cardiology for Early Mortality in Acute Pulmonary Embolism

Background \u2003Acute pulmonary embolism (PE) is burdened by high mortality, especially within 30 days from the diagnosis. The development and the validation of predictive models for the risk of early mortality allow to differentiate patients who can undergo home treatment from those who need admission into intensive care units. Methods \u2003To validate the prognostic model for early mortality after PE diagnosis proposed by the European Society of Cardiology (ESC) in 2014, we analyzed data of a cohort of 272 consecutive patients with acute PE, observed in our hospital during a 10-year period. Moreover, we evaluated the additional contribution of D-dimer, measured at PE diagnosis, in improving the prognostic ability of the model. All cases of PE were objectively diagnosed by angiography chest CT scan or perfusion lung scan. Results \u2003The overall mortality rate within 30 days from PE diagnosis was 10% (95% confidence interval [CI]: 6.4-13.5%). According to the ESC prognostic model, the risk of death increased 3.23 times in the intermediate-low-risk category, 5.55 times in the intermediate-high-risk category, and 23.78 times in the high-risk category, as compared with the low-risk category. The receiver operating characteristic analysis showed a good discriminatory power of the model (area under the curve [AUC]\u2009=\u20090.77 [95% CI: 0.67-0.87]), which further increased when D-dimer was added (AUC\u2009=\u20090.85 [95% CI: 0.73-0.96]). Conclusion \u2003This study represents a good validation of the ESC predictive model whose performance can be further improved by adding D-dimer plasma levels measured at PE diagnosis

Comparative study of three low-tech soilless systems for the cultivation of geranium (Pelargonium zonale): A commercial quality assessment

The study evaluated the feasibility of simplified hydroponics for the growth of rooted cuttings of geranium (Pelargonium zonale) for commercial purposes in local farms in Northern Italy. Tested systems included a control where soilless system on substrate (peat) (T-1), usually adopted by local farmers, was compared against an open-cycle drip system on substrate (peat) (T-2), and a Nutrient Film Technique system (T-3). For commercial features, assessed parameters included flowering degree (flowering timing, numbers of inflorescences plant−1, and number of flowers inflorescence−1), numbers of leaves plant−1, number of branches plant−1, final height of plant, and the aesthetic-commercial assessment index. Assessed parameters also included fresh and dry weight, SPAD Index, the water consumption, and the water use efficiency (WUE). The soilless systems typology significantly affected rooted cuttings growth, commercial features, and WUE. The adoption of an open-cycle drip system (T-2) resulted in a significant improvement of all the crop commercial characteristics as compared with other treatments, making plants more attractive for the market. The water consumption was higher in T-2 as compared with T-1 and T-3, but it allowed for the highest fresh weight, and therefore also the highest WUE. The results indicate that the typology of soilless system significantly enhances the commercial characteristics of geranium

Gravitational Tunneling in Lorentz Violating Gravity

Black holes in Lorentz violating gravity, such as Einstein--Aether or Horava--Lifshitz Gravity, are drastically different from their general relativistic siblings. Although they allow for superluminal motion in their vicinity, they still exhibit an absolute causal boundary in the form of a universal horizon. By working in the tunneling picture for a gravitating scalar field, we show that universal horizons emit Hawking radiation in a manner akin to standard results in General Relativity, with a temperature controlled by the high-energy behavior of the dispersion relation of the gravitating field, and in agreement with alternative derivations in the literature. Our results substantiate the link between the universal horizon and thermodynamics in Lorentz violating theories.Comment: 16 pages, 2 figures, references updated, typos fixe

Winegrape berry skin thickness determination: comparison between histological observation and texture analysis determination.

We analyzed the relation between the assessment of grape berry skin thickness by means of histology sections and instrumental mechanical properties measurements. Berry skin of Vitis vinifera L. cultivar Corvina vineyards from Valpolicella Valpantena zone (Verona, Italy) were tested, evidencing a strong correlation between the two thickness determination methods. The middle or equatorial berry skin portion was found to be the less variable in instrumental skin thickness determination. In addition, unlike other studies, no correlation between the skin thickness and cell layers number was found

A Major Fraction of Fibronectin Present in the Extracellular Matrix of Tissues Is Plasma-derived

The origin of the fibronectin (FN) found in the extracellular matrix of tissues has not been defined experimentally. Previous studies suggest that there is contribution from both local tissue production and transfer from plasma, but the extent of this phenomenon has not been addressed. We have shown before that engineered mice constitutively expressing extra domain A-containing FN (EDA+FN) have a significant decrease of FN levels in plasma and most tissues. We showed that hepatocytes modified to produce EDA+FN have normal extracellular matrix-FN levels but secrete less soluble FN. When we performed a liver-specific EDA-exon deletion in these animals, FN levels were restored both in plasma and tissues. Therefore, an important fraction of tissue FN, approximately an equal amount of that produced by the tissue itself, is actually plasma-derived, suggesting that plasma is an important source of tissue FN. The present results have potential significance for understanding the contributions of plasma FN, and perhaps other plasma proteins, in the modulation of cellular activities and in the formation of the extracellular matrix of tissues

TEM Nanostructural Investigation of Ag-Conductive Filaments in Polycrystalline ZnO-Based Resistive Switching Devices

Memristive devices based on a resistive switching mechanism are considered very promising for nonvolatile memory and unconventional computing applications, even though many details of the switching mechanisms are not yet fully understood. Here, we report a nanostructural study by means of high-resolution transmission electron microscopy and spectroscopy techniques of a Ag/ZnO/Pt memristive device. To ease the localization of the filament position for its characterization, we propose to use the guiding effect of regular perturbation arrays obtained by FIB technology to assist the filament formation. HRTEM and EDX were used to identify the composition and crystalline structure of the so-obtained conductive filaments and surrounding regions. It was determined that the conducting paths are composed mainly of monocrystalline Ag, which remains polycrystalline in some circumstances, including the zone where the switching occurs and at secondary filaments created at the grain boundaries of the polycrystalline ZnO matrix. We also observed that the ZnO matrix shows a degraded quality in the switching zone, while it remains unaltered in the rest of the memristive device