CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications
but identifying unwanted off-target mutations is important for clinical translation
. A ...well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.
Purpose
To evaluate the clinical performance and survival rate of posterior monolithic zirconia fixed partial dentures over a 2‐year period.
Material and Methods
A total of 20 patients, requiring 20 ...posterior fixed partial dentures were included in the study. Tooth preparations were scanned, and restorations were milled and cemented with a resin cement. The restorations were assessed for the quality of the surface and the color, anatomical form and marginal integrity. Periodontal status was assessed by determining the plaque index, gingival index, pocket depth, and margin index of the abutment teeth. Data were statistically analyzed using the Friedman and the Wilcoxon signed‐rank tests with the Bonferroni correction.
Results
The survival rate at 2 years was 100%, and no biological or technical complications were observed. All restorations were assessed as satisfactory. The results obtained for gingival index and plaque index were better at 2 years follow‐up, than at baseline. The margin index remained stable throughout the follow‐up period. No differences in periodontal parameters were observed between abutment and control teeth.
Conclusions
The high survival rate after 2 years suggest that monolithic zirconia may be an acceptable alternative to metal‐ceramic and veneered zirconia restorations in the posterior region. Additional long‐term, controlled studies are necessary to confirm the results.
Background Understanding the enduring respiratory consequences of severe COVID-19 is crucial for comprehensive patient care. This study aims to evaluate the impact of post-COVID conditions on ...respiratory sequelae of severe acute respiratory distress syndrome (ARDS). Methods We examined 88 survivors of COVID-19-associated severe ARDS six months post-intensive care unit (ICU) discharge. Assessments included clinical and functional evaluation as well as plasma biomarkers of endothelial dysfunction, inflammation, and viral response. Additionally, an in vitro model using human umbilical vein endothelial cells (HUVECs) explored the direct impact of post-COVID plasma on endothelial function. Results Post-COVID patients with impaired gas exchange demonstrated persistent endothelial inflammation marked by elevated ICAM-1, IL-8, CCL-2, and ET-1 plasma levels. Concurrently, systemic inflammation, evidenced by NLRP3 overexpression and elevated levels of IL-6, sCD40-L, and C-reactive protein, was associated with endothelial dysfunction biomarkers and increased in post-COVID patients with impaired gas exchange. T-cell activation, reflected in CD69 expression, and persistently elevated levels of interferon-beta (IFN-beta) further contributed to sustained inflammation. The in vitro model confirmed that patient plasma, with altered levels of sCD40-L and IFN-beta proteins, has the capacity to alter endothelial function. Conclusions Six months post-ICU discharge, survivors of COVID-19-associated ARDS exhibited sustained elevation in endothelial dysfunction biomarkers, correlating with the severity of impaired gas exchange. NLRP3 inflammasome activity and persistent T-cell activation indicate on going inflammation contributing to persistent endothelial dysfunction, potentially intensified by sustained viral immune response. Keywords: Acute respiratory distress syndrome, Post-COVID-19 syndrome, Endothelial dysfunction, ICAM-1, Inflammation
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Introduction
This study evaluates the effects of 25 mL of three types of oils extra‐virgin olive oil (EVOO), olive oil (OO), and sunflower oil (SO) on postprandial (3 h) satiety markers and variables ...related to metabolic status and inflammation in non‐obese patients (n = 6) and in those with morbid obesity (n = 6), before and 1 year after Roux‐en‐Y gastric by‐pass (RYGB).
Methods and Results
After EVOO intake, serum acylated ghrelin decreases and GLP1 increases more than with OO and SO. EVOO causes a higher increase of insulin and lower postprandial hypertriglyceridemia and free fatty acid levels than with OO and SO. EVOO decreases TNFα and IL6 expression in peripheral blood mononuclear cells, with OO inducing intermediate effects and SO inducing an increase of these proinflammatory markers. These results are observed in non‐obese patients and in those with morbid obesity after RYGB. However, patients with morbid obesity before RYGB show a profound alteration of this response.
Conclusion
EVOO produces more beneficial effects than OO, which has lower amounts of minor components, and SO, which has PUFA as its main component. RYGB produces an improvement in the metabolic response to all three types of oils in patients with morbid obesity.
Extra‐virgin olive oil produces more beneficial effects than olive oil, which has lower amounts of minor components, and sunflower oil, which has polyunsaturated fatty acids as its main component. These results are observed in non‐obese patients and in those with morbid obesity after bariatric surgery. However, patients with morbid obesity before bariatric surgery show a profound alteration of this response.
Although not currently recommended, spirometry during hospitalization due to exacerbation of chronic obstructive pulmonary disease (COPD) is an opportunity to enhance the diagnosis of this disease. ...The aim of the present study was to assess the usefulness and reliability of spirometry before hospital discharge, comparing it to measurements obtained during clinical stability.
This prospective longitudinal observational study compares spirometry results before and 8 weeks after discharge in consecutive patients admitted for COPD exacerbation. Concordance between results was assessed by the Kappa index, intraclass correlation coefficient, and Bland-Altman graphs.
From an initial population of 179 COPD patients, 100 completed the study (mean age 67.8 years, 83% men, 35% active smokers, FEV1 at clinical stability 40.3%). Forty-nine patients could not complete the study because they did not reach clinical stability. In three patients with obstructive spirometry during admission, the results were normal at follow-up. In the remaining patients, the COPD diagnosis was confirmed at stability with acceptable concordance. In 27 cases, spirometry improved more than 200 mL.No variables were found to be associated with this improvement or to explain it.
This study provides information on the role of spirometry prior to hospital discharge in patients admitted for COPD exacerbation, demonstrating that it is a valid and reproducible method, representing an opportunity toimprove COPD diagnosis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
CD39/NTPDase1 has emerged as an important molecule that contributes to maintain inflammatory and coagulatory homeostasis. Various studies have hypothesized the possible role of CD39 in COVID-19 ...pathophysiology since no confirmatory data shed light in this regard. Therefore, we aimed to quantify CD39 expression on COVID-19 patients exploring its association with severity clinical parameters and ICU admission, while unraveling the role of purinergic signaling on thromboinflammation in COVID-19 patients. We selected a prospective cohort of patients hospitalized due to severe COVID-19 pneumonia (n=75), a historical cohort of Influenza A pneumonia patients (n=18) and sex/age-matched healthy controls (n=30). CD39 was overexpressed in COVID-19 patients' plasma and immune cell subsets and related to hypoxemia. Plasma soluble form of CD39 (sCD39) was related to length of hospital stay and independently associated with intensive care unit admission (adjusted odds ratio 1.04, 95%CI 1.0-1.08, p=0.038), with a net reclassification index of 0.229 (0.118-0.287; p=0.036). COVID-19 patients showed extracellular accumulation of adenosine nucleotides (ATP and ADP), resulting in systemic inflammation and pro-coagulant state, as a consequence of purinergic pathway dysregulation. Interestingly, we found that COVID-19 plasma caused platelet activation, which was successfully blocked by the P2Y
receptor inhibitor, ticagrelor. Therefore, sCD39 is suggested as a promising biomarker for COVID-19 severity. As a conclusion, our study indicates that CD39 overexpression in COVID-19 patients could be indicating purinergic signaling dysregulation, which might be at the basis of COVID-19 thromboinflammation disorder.
This study assesses crop residues in the EU from major crops using empirical models to predict crop residues from yield statistics; furthermore it analyses the inter‐annual variability of those ...estimates over the period 1998‐2015, identifying its main drivers across Europe. The models were constructed based on an exhaustive collection of experimental data from scientific papers for the crops: wheat, barley, rye, oats, triticale, rice, maize, sorghum, rapeseed, sunflower, soybean, potato and sugarbeet. We discuss the assumptions on the relationship between yield and the harvest index, adopted by previous studies, to interpret the experimental data, quantify the uncertainties of these models, and establish the premises to implement them at regional scale –i.e., NUTS level 3– within the EU. To cope this, we created a consolidated sub‐national statistical data along with an algorithm able to aggregate (figures are provided at country level) and disaggregate (production at 25 km grid is provided assupplementary material) estimates. The total lignocellulosic biomass production in the EU28 over the review period, according to our models, is 419 Mt, from which wheat is the major contributor (155 Mt). Our results show that maize and rapeseed are the two crops with the highest residue yield, respectively 8.9 and 8.6 t ha‐1. The spatial analysis revealed that these three crops, which, according to our results, are feedstocks highly suitable a priori for second generation biofuels in the EU and are unevenly distributed across Europe. Weather fluctuation was identified as the major driver in residue production from cereals, while, in the case of starch crops and oilseeds – which are predominant in northern Europe – corresponded to the marked production trend likely influenced by the agricultural policies and agro‐management over the review period. Our results, among others, could help to understand and quantify the ecological boundaries of the bioeconomy from agriculture.
Although the production of advanced biofuels have been explicitly supported by the EU since 2015, there are still economic and technological challenges to be addressed. The quantitative assessment of lignocellulosic biomass of residues production is an essential step for that and constitutes our main scope. To that end, we constructed empirical models to predict crop residues estimates from yield statistics. Furthermore, we analysed the inter‐annual variability of those estimates over the period 1998–2015, identifying its main drivers across Europe. Our results, among others, could help to understand and quantify the ecological boundaries of the bioeconomy from the agricultural sector.
COVID-19 has emerged as a devastating disease in the last 2 years. Many authors appointed to the importance of kallikrein-kinin system (KKS) in COVID-19 pathophysiology as it is involved in ...inflammation, vascular homeostasis, and coagulation. We aim to study the bradykinin cascade and its involvement in severity of patients with COVID-19. This is an observational cohort study involving 63 consecutive patients with severe COVID-19 pneumonia and 27 healthy subjects as control group. Clinical laboratory findings and plasma protein concentration of KKS peptides bradykinin (BK), BK1-8, KKS proteins high–molecular weight kininogen (HK), and KKS enzymes carboxypeptidase N subunit 1 (CPN1), kallikrein B1 (KLKB1), angiotensin converting enzyme 2 (ACE2), and C1 esterase inhibitor (C1INH) were analyzed. We detected dysregulated KKS in patients with COVID-19, characterized by an accumulation of BK1-8 in combination with decreased levels of BK. Accumulated BK1-8 was related to severity of patients with COVID-19. A multivariate logistic regression model retained BK1-8, BK, and D-dimer as independent predictor factors to intensive care unit (ICU) admission. A Youden’s optimal cutoff value of −0.352 was found for the multivariate model score with an accuracy of 92.9%. Multivariate model score-high group presented an odds ratio for ICU admission of 260.0. BK1-8 was related to inflammation, coagulation, and lymphopenia. Our data suggest that BK1-8/BK plasma concentration in combination with D-dimer levels might be retained as independent predictors for ICU admission in patients with COVID-19. Moreover, we reported KKS dysregulation in patients with COVID-19, which was related to disease severity by means of inflammation, hypercoagulation, and lymphopenia.
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