CRISPR-Cas9-Mediated Genomic Deletions Protocol in Zebrafish

Since its first application for site-directed mutagenesis, the CRISPR-Cas9 system has revolutionized genome engineering. Here, we present a validated workflow for the generation of targeted genomic deletions in zebrafish, including the design, cloning, and synthesis of single-guide RNAs and Cas9 mRNA, followed by microinjection in zebrafish embryos and subsequent genotype screening for the establishment of a mutant line. The versatility and efficiency of this pipeline makes the generation of zebrafish models a widely used approach in functional genetics. For complete details on the use and execution of this protocol, please refer to Amorim et al. (2020).This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement no. ERC_2015_StG _ 680156 _ZPR). J.B. acknowledges Fundação para a Ciência e a Tecnologia (FCT) for an FCT Scientific Stimulus Grant ( CEECIND/03482/2018 ). J.A. is a PhD fellow from FCT ( SFRH/BD/145110/2019 ). R.B.C. was funded by FCT ( ON2201403-CTO-BPD ) and EMBO (Short-Term Fellowship). We thank Joaquin Letelier, Ana Novoa, and Moises Mallo for important advice establishing the current protocol. We also thank Joana Marques for constant assistance in establishing the protocol

Compressibility of titanosilicate melts

The effect of composition on the relaxed adiabatic bulk modulus (K0) of a range of alkali- and alkaline earth-titanosilicate [X 2 n/n+ TiSiO5 (X=Li, Na, K, Rb, Cs, Ca, Sr, Ba)] melts has been investigated. The relaxed bulk moduli of these melts have been measured using ultrasonic interferometric methods at frequencies of 3, 5 and 7 MHz in the temperature range of 950 to 1600°C (0.02 Pa s < s < 5 Pa s). The bulk moduli of these melts decrease with increasing cation size from Li to Cs and Ca to Ba, and with increasing temperature. The bulk moduli of the Li-, Na-, Ca- and Ba-bearing metasilicate melts decrease with the addition of both TiO2 and SiO2 whereas those of the K-, Rb- and Cs-bearing melts increase. Linear fits to the bulk modulus versus volume fraction of TiO2 do not converge to a common compressibility of the TiO2 component, indicating that the structural role of TiO2 in these melts is dependent on the identity of the cation. This proposition is supported by a number of other property data for these and related melt compositions including heat capacity and density, as well as structural inferences from X-ray absorption spectroscopy (XANES). The compositional dependence of the compressibility of the TiO2 component in these melts explains the difficulty incurred in previous attempts to incorporate TiO2 in calculation schemes for melt compressibility. The empirical relationship KV-4/3 for isostructural materials has been used to evaluate the compressibility-related structural changes occurring in these melts. The alkali metasilicate and disilicate melts are isostructural, independent of the cation. The addition of Ti to the metasilicate composition (i.e. X2TiSiO5), however, results in a series of melts which are not isostructural. The alkaline-earth metasilicate and disilicate compositions are not isostructural, but the addition of Ti to the metasilicate compositions (i.e. XTiSiO5) would appear, on the basis of modulus-volume systematics, to result in the melts becoming isostructural with respect to compressibility

Long-Term Follow-Up of Patients after Acute Kidney Injury: Patterns of Renal Functional Recovery

Background and Objectives: Patients who survive acute kidney injury (AKI), especially those with partial renal recovery, present a higher long-term mortality risk. However, there is no consensus on the best time to assess renal function after an episode of acute kidney injury or agreement on the definition of renal recovery. In addition, only limited data regarding predictors of recovery are available. Design, Setting, Participants, &amp; Measurements: From 1984 to 2009, 84 adult survivors of acute kidney injury were followed by the same nephrologist (RCRMA) for a median time of 4.1 years. Patients were seen at least once each year after discharge until end stage renal disease (ESRD) or death. In each consultation serum creatinine was measured and glomerular filtration rate estimated. Renal recovery was defined as a glomerular filtration rate value $60 mL/min/1.73 m2. A multiple logistic regression was performed to evaluate factors independently associated with renal recovery. Results: The median length of follow-up was 50 months (30–90 months). All patients had stabilized their glomerular filtration rates by 18 months and 83 % of them stabilized earlier: up to 12 months. Renal recovery occurred in 16 patients (19%) at discharge and in 54 (64%) by 18 months. Six patients died and four patients progressed to ESRD during the follow up period. Age (OR 1.09, p,0.0001) and serum creatinine at hospital discharge (OR 2.48, p = 0.007) were independent factors associated with non renal recovery. The acute kidney injury severity, evaluated by peak serum creatinine and nee

Equivalence of plasma p-tau217 with cerebrospinal fluid in the diagnosis of Alzheimer's disease

INTRODUCTION: Plasma biomarkers are promising tools for Alzheimer's disease (AD) diagnosis, but comparisons with more established biomarkers are needed. METHODS: We assessed the diagnostic performance of p-tau181, p-tau217, and p-tau231 in plasma and CSF in 174 individuals evaluated by dementia specialists and assessed with amyloid-PET and tau-PET. Receiver operating characteristic (ROC) analyses assessed the performance of plasma and CSF biomarkers to identify amyloid-PET and tau-PET positivity. RESULTS: Plasma p-tau biomarkers had lower dynamic ranges and effect sizes compared to CSF p-tau. Plasma p-tau181 (AUC = 76%) and p-tau231 (AUC = 82%) assessments performed inferior to CSF p-tau181 (AUC = 87%) and p-tau231 (AUC = 95%) for amyloid-PET positivity. However, plasma p-tau217 (AUC = 91%) had diagnostic performance indistinguishable from CSF (AUC = 94%) for amyloid-PET positivity. DISCUSSION: Plasma and CSF p-tau217 had equivalent diagnostic performance for biomarker-defined AD. Our results suggest that plasma p-tau217 may help reduce the need for invasive lumbar punctures without compromising accuracy in the identification of AD. Highlights: p-tau217 in plasma performed equivalent to p-tau217 in CSF for the diagnosis of AD, suggesting the increased accessibility of plasma p-tau217 is not offset by lower accuracy. p-tau biomarkers in plasma had lower mean fold-changes between amyloid-PET negative and positive groups than p-tau biomarkers in CSF. CSF p-tau biomarkers had greater effect sizes than plasma p-tau biomarkers when differentiating between amyloid-PET positive and negative groups. Plasma p-tau181 and plasma p-tau231 performed worse than p-tau181 and p-tau231 in CSF for AD diagnosis

Cancer Survivor Study (CASUS) on colorectal patients: longitudinal study on physical activity, fitness, nutrition, and its influences on quality of life, disease recurrence, and survival. Rationale and design

Purpose: Evidence suggests that being physically active in combination with a healthy diet contributes to diminish colorectal cancer risk. However, if this is true for colorectal cancer primary prevention, the same is not clear for its recurrence after colorectal cancer treatments. Data on cancer survival are scarce, and there is a need for greater attention on these survivors’ lifestyle behavior. This manuscript describes rationale and design of the Cancer Survival Study (CASUS) on colorectal patients, a longitudinal observational study with the aim of investigating how physical activity, physical fitness, and dietary intake are related with their quality of life, disease recurrence, and survival. Methods: The CASUS on colorectal patients is a longitudinal cohort study on colorectal survivors, aged 18 years or older, recruited 6, 12, and 24 months after surgery. Upon recruitment, patients fill in a battery of questionnaires about physical activity, dietary intake, and quality of life, donate blood samples, do physical fitness tests, and use an accelerometer during 7 days. Repeated analyses will be performed to assess changes over time in physical activity, physical fitness, dietary intake, and other factors in relation to recurrence and survival. Conclusions: Results will contribute to highlight the role of physical activity, physical fitness, and nutrition in the quality of life of colorectal cancer survivors, recurrence, and survival. This study will provide important information for policymakers on the potential benefits of future physical activity and nutritional interventions, which are inexpensive, as a way to improve general health of colorectal cancer survivors.Luisa Soares-Miranda is supported by the grant SFRH/BPD/76947/2011 funded by FCT (QREN-POPH-Type 4.1-Advanced Training, subsidized by the European Social Fund and national funds of MEC), PTDC/DES/099018/2008-FCT/FCOMP-01-0124-FEDER-009573, and the Research Centre in Physical Activity Health and Leisure is supported by FCT: UID/DTP/00617/2013. Carla Costa is supported by the grant SFRH/BPD/96196/2013, funded by FCT (QREN-POPH-Type 4.1-Advanced Training, subsidized by the European Social Fund and national funds of MEC)