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Nicholas Hess

and 5 more

Background: This study investigated the impact of transplanting center donor acceptance patterns on usage of extended-criteria donors (ECDs) and posttransplant outcomes following orthotopic heart transplantation (OHT). Methods: The Scientific Registry of Transplant Recipients was queried to identify heart donor offers and adult, isolated OHT recipients in the United States from 1/1/2013-10/17/2018. Centers were stratified into 3 equal-size terciles based on donor heart acceptance rates (<13.7%, 13.7%-20.2%, >20.2%). Overall survival was compared between recipients of ECDs (≥40 years, left ventricular ejection fraction <60%, distance ≥500 miles, hepatitis B, hepatitis C or human immunodeficiency virus, or ≥50 offers) and recipients of traditional-criteria donors, and among transplanting terciles. Results: A total of 85,505 donor heart offers were made to 133 centers with 15,264 (17.9%) accepted for OHT. High-acceptance programs (>20.2%) more frequently accepted donors with LVEF <60%, HIV, HCV, and/or HBV, ≥50 offers, or distance >500 miles from the transplanting center (each p<0.001). Posttransplant survival was comparable across all three terciles (p=0.11). One- and five-year survival were also similar across terciles when examining recipients of all five ECD factors. Acceptance tier and increasing acceptance rate were not found to have any impact on mortality in multivariable modeling. Of ECD factors, only age ≥40 years was found to have increased hazards for mortality (HR 1.33, 95% CI 1.22-1.46, p<0.001). Conclusions: Of recipients of ECD hearts, outcomes are similar across center-acceptance terciles. Educating less aggressive programs to increase donor acceptance and ECD utilization may yield higher national rates of OHT without major impact on outcomes.

Giulia Zacchello

and 4 more

The timing of germination is a key life-history trait in plants, which is strongly affected by the strength of seed dormancy. Continental-wide variation in seed dormancy has been related to differences in climate and the timing of conditions suitable for seedling establishment. However, for predictions of adaptive potential and consequences of climatic change, information is needed regarding the extent to which seed dormancy varies within climatic regions and the factors driving such variation We planted 17 Italian and 28 Fennoscandian populations of Arabidopsis thaliana in the greenhouse and at two field sites in Italy and Sweden. To identify possible drivers of among-population variation in seed dormancy, we examined the relationship between seed dormancy and climate at the sites where populations were originally sampled. Seed dormancy was on average stronger in the Italian compared to the Fennoscandian populations, but also varied widely within both regions. Estimates of seed dormancy in the three maternal environments were positively correlated, but seeds had on average stronger dormancy when produced in the greenhouse than at the two field sites. Among Fennoscandian populations, seed dormancy tended to increase with increasing summer temperature and decreasing precipitation at the site of origin. In the smaller sample of Italian populations, no significant association was detected between mean seed dormancy and climate at the site of origin. The correlation between seed dormancy and climatic factors in Fennoscandia suggests that at least some of the among-population variation is adaptive and that climate change will affect selection on this trait.

Babu Ram Lamichhane

and 9 more

Conservation of large carnivores like leopards requires large and interconnected habitats. Despite the wide geographic range of the leopard globally, only 17% of their habitat is within protected areas. In Nepal, leopards are distributed widely across the country but their status is not adequately studied which compromised the necessary conservation attention for the species. This study carried out sign-based occupancy survey across the Chure (the Himalayan foothills) range (~19,000 km2) to understand the habitat occupancy of leopards along with the covariates affecting their presence. The model-averaged leopard occupancy in the Chure range was 0.5732 (0.0082 SD) with a detection probability of 0.2554 (0.1142 SE). The top model included wild boar, ruggedness, presence of livestock and human population density as covariates. The β coefficient estimate from the model indicated the wild boar was the primary covariate contributing positively to the leopard occupancy followed by the presence of livestock, ruggedness and human population density. The detection probability of leopard was higher outside the protected areas, less in the densely vegetated areas, and higher in the area where there is a presence of livestock. Enhanced law enforcement and mass awareness activities are necessary to reduce poaching/killing of wild ungulates and leopard in the Chure range and to increase leopard occupancy. In addition, maintaining a sufficient natural prey base can contribute to minimize the livestock depredation and hence, decrease the human-leopard conflict in the Chure range.

Kathryn Luly

and 8 more

Poly(beta-amino ester)s as high-yield transfection reagents for recombinant protein productionKathryn M. Luly1,2, Stephen J. Lee2,3, Huilin Yang2,3, Wentao Wang1,2, Seth D. Ludwig2,3, Haley E. Tarbox4, David R. Wilson1,2,5, Jordan J. Green1,2,3,5,6,7,8,9,10*, Jamie B. Spangler1,2,3,6,7,8*1Department of Biomedical Engineering, Johns Hopkins University School of Medicine, 2Translational Tissue Engineering Center, Johns Hopkins University School of Medicine,3Department of Chemical & Biomolecular Engineering, Johns Hopkins University, 4Department of Chemistry, Johns Hopkins University, 5Institute for Nanobiotechnology, Johns Hopkins University,6Department of Oncology, Johns Hopkins University School of Medicine, 7Bloomberg~Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, 8Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine,9Departments of Neurosurgery and Ophthalmology, Johns Hopkins University School of Medicine, 10Department of Materials Science & Engineering, Johns Hopkins University*Correspondence should be addressed to:Jamie B. Spangler400 N Broadway, Smith 5011, Baltimore, MD 21231(443) 287-1708jamie.spangler@jhu.eduJordan J. Green400 N Broadway, Smith 5017, Baltimore, MD 21231(410) 614-9113green@jhu.eduGrant numbers: R01EY031097, R01EB029455, R01CA228133, P41EB028239, R01CA240339Transient transfection is an essential tool for recombinant protein production, as rapid screening for expression is conducted without stable integration of genetic material into a target cell genome. Poly(ethylenimine) (PEI) is the current gold standard for transient gene transfer, but transfection efficiency and resulting protein yield are limited by the polymer’s toxicity. This study investigated the use of an alternative class of cationic polymers, poly(beta-amino ester)s (PBAEs), for transient transfection of human embryonic kidney 293F (HEK) and Chinese hamster ovary-S (CHO) cell suspensions. In both HEK and CHO cells, several PBAEs demonstrated superior transfection efficiency and production of a cytosolic reporter compared to PEI. This result extended to secreted proteins, as a model PBAE increased the production of three secreted antibodies compared to PEI at scales ranging from 20-2,000 mL. In particular, non-viral gene transfer using the lead PBAE/plasmid DNA nanoparticles led to robust transfection of mammalian cells across different constructs, doses, volumes, and cell types. These results show that PBAEs enhance transfection efficiency and increase protein yield compared to a widespread commercially available reagent, making them attractive candidates as reagents for use in recombinant protein production.Keywords: protein production, transient transfection, poly(beta-amino ester)s, transfection reagentsCurrent research into chemical-based transfection methods focuses largely on optimizing agents for use in the development and production of recombinant proteins. Transient transfection, in which introduced genetic material is not incorporated into the host genome, is especially useful during the high-throughput design and screening of proteins (e.g., candidate biologics) wherein stable expression is not needed. While culture conditions and plasmid design have been popular targets for optimization in transient transfection workflows (Backliwal et al., 2008; Galbraith, Tait, Racher, Birch, & James, 2006), further research into improved transfection reagents has even greater potential for boosting protein yields. Chemical-based transient transfection relies on condensation and encapsulation of plasmid DNA by a biocompatible material into particles which are taken up by target cells; differences in particle size can affect the method of cellular uptake, leading to differences in transfection efficiency (Kim, Sunshine, & Green, 2014). Particles must then escape the endosome and the encapsulating material must degrade to allow for DNA release, nuclear translocation, transcription and subsequent export, and finally translation and processing into fully formed protein (Karlsson, Rhodes, Green, & Tzeng, 2020).Transfection reagent structure and buffering capacity have been demonstrated to influence DNA uptake and escape, making these properties particularly consequential in reagents for transient transfection workflows (Sunshine, Peng, & Green, 2012). Maximizing protonability, for example, facilitates endosomal swelling and consequent rupture via the “proton sponge” effect (Boussif et al., 1995; Bus, Traeger, & Schubert, 2018). Cationic polymers have typically been among the most promising transfection reagents; their charge-based association with DNA into particles offers protection from degradation and offers sufficient buffering capacity to facilitate endosomal escape following cellular uptake (Sunshine et al., 2012).Poly(ethyleneimine) (PEI) is a commercially available cationic polymer used extensively as a transfection reagent that has a high density of protonatable amines, giving rise to high buffering capacity and efficient endosomal escape (Boussif et al., 1995). PEI of average molecular weight 25 kDa is most frequently used in transfection workflows, but its toxicity limits transfection efficiency and, consequently, protein yield (Breunig, Lungwitz, Liebl, & Goepferich, 2007; Yang, Li, Goh, & Li, 2007). Previously, PEI has been conjugated to polyethylene glycol (Petersen et al., 2002) and arginine modified oligo(-alkylaminosiloxane) [P(SiDAAr)n] (Morris & Sharma, 2010) to mitigate cytotoxicity.A promising alternative to PEI, poly(beta-amino ester)s (PBAEs) are a class of cationic polymers used to facilitate efficient gene transferin vitro (Bishop, Kozielski, & Green, 2015). PBAEs are composed of an acrylate base monomer, an amine sidechain, and a terminal end-capping group, each of which can be varied to create a vast library of materials (Akinc, Lynn, Anderson, & Langer, 2003). Hydrolyzable ester linkages allow for degradation of the PBAEs in transfection conditions which allows for use of the polymers at high weight ratios relative to other non-biodegradable materials, maximizing density of buffering amines to facilitate endosomal escape (Sunshine et al., 2012). Their biodegradability also obviates the need for medium replacements or additions, themselves contributors to cell death, which are common where PEI is utilized (Galbraith et al., 2006). These linear polymers are synthesized from inexpensive, commercially available reagents using a two-step polymerization method (Fig. S1A) and are stable long term when stored dry at -20°C (Wilson et al., 2019).Given the high transfection efficacy observed with PBAEs in variousin vitro contexts, we sought to investigate the use of PBAE nanoparticles for transient transfection of suspension cultures in intracellular and secreted protein production workflows (Fig. 1A). We selected four PBAEs with varying base (B), sidechain (S), and end-cap (E) structures to evaluate in comparison with linear 25 kDa PEI: B4-S4-E6 (4-4-6); B4-S5-E7 (4-5-7); B4-S5-E39 (4-5-39); and B5-S3-E6 (5-3-6) (Fig. 1B-C, S1B). Physiochemical characterization of PBAE and PEI nanoparticles in serum-free transfection media indicated that PBAE nanoparticles maintained a smaller size in transfection conditions (approximately 200-350 nm) whereas PEI nanoparticles were prone to aggregation, resulting in sizes over 1 µm (Fig. 1D, S1C). Previous studies indicated that PEI nanoparticles were prone to aggregation in serum-free media due to interactions with salts and a lack of adsorbed proteins that can help stabilize discrete particles and prevent clustering (Ogris et al., 1998; Pezzoli, Giupponi, Mantovani, & Candiani, 2017). Analysis of surface charge revealed that PBAE nanoparticles maintained a positive zeta potential in transfection conditions, whereas PEI nanoparticles exhibited a near neutral surface charge (Fig. 1E, S1C). Shielded surface charge of PEI particles may limit interactions with a charged cell membrane, thus hindering cellular uptake.To determine the optimal DNA dose for production of cytosolic mCherry using various polymer-based transfection agents, we selected a representative PBAE, 2-(3-aminopropylamino)ethanol end-capped poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) (referred to here as 4-4-6), and compared this PBAE to 25 kDa PEI at doses ranging from 0.5 to 4 µg/mL DNA. The polymers were compared in two mammalian cell lines frequently employed for protein expression: human embryonic kidney 293F (HEK) cells and Chinese hamster ovary-S (CHO) cells. Evaluation of mCherry fluorescence over a span of 5 days indicated that peak mCherry expression occurred using the 4-4-6 polymer at 2 µg/mL and 4 µg/mL DNA doses in HEK and CHO cells, respectively (Fig. S1A). Notably, peak mCherry expression in PEI-based transfections was not comparable to that attained by 4-4-6 at any dose. Subsequent time course studies using the optimized DNA dose that compared additional PBAE structures demonstrated significantly increased mCherry expression using 4-4-6, 4-5-7, and 4-5-39 in HEK cells, and using 4-4-6 and 4-5-7 in CHO cells, compared to PEI-mediated transfection (Fig. 2A, 2C). Transfection efficiency, as measured by mCherry-positive cells on day 5, was significantly increased with all PBAEs tested in HEK cells and with 4-4-6, 4-5-7, and 4-5-39 in CHO cells (Fig. 2B, 2D, S2C). Fluorescence microscopy confirmed the increase in mCherry expression (Fig. 2E, S2D). Cell viability, assessed via MTS assay 24 h following transfection, indicated that PBAEs (especially 4-5-39 and 5-3-6) showed greater toxicity than PEI, though notably at a 20- to 30-fold higher weight ratio (Fig. S2B). Importantly, this reduced viability did not result in inferior mCherry expression relative to PEI, with 4-4-6 and 4-5-7 demonstrating superior expression in both HEK and CHO cells (Fig. 2A, C).To demonstrate that the results of these fluorescent protein expression experiments were replicable at scales relevant to the development and production of secreted proteins, we transfected HEK cell cultures of varying volumes with DNA encoding the recombinant antibody 10H2 (Chuntharapai, Lee, Hébert, & Kim, 1994; Patent No. WO/2020/243489, 2020) using either 4-4-6- or PEI-based particles. Based on SDS-PAGE image analysis of small-scale dose titrations (Fig. S3A-D), DNA was dosed at 1 µg/mL for secreted proteins in HEK cells, with polymer weight adjusted accordingly. At volumes ranging from 20-200 mL, transfection with 4-4-6 yielded between 4.5-fold and 8.2-fold more protein than did transfection with PEI (Fig. 3A). To further demonstrate the scalability of enhanced protein expression using PBAEs, 2 L cultures of HEK cells were transfected with DNA encoding either 10H2 or the bispecific antibody BS2 (Patent No. WO/2020/243489, 2020) using either 4-4-6 or PEI. Both the 10H2 and BS2 antibodies were recovered in significantly higher quantities (4.9-fold and 5.6-fold higher, respectively) when 4-4-6 was utilized compared to PEI (Fig. 3B-C). Superiority of PBAE nanoparticles was reproducible across cell lines; transfection of CHO cells with DNA encoding the recombinant antibody 602 (Krieg, Letourneau, Pantaleo, & Boyman, 2010; Létourneau et al., 2010; Patent No. WO/2020/264321, 2020) at an optimized dose of 4 µg/mL resulted in 3.4-fold more protein recovered when 4-4-6 was used compared to PEI (Fig. S3E-G).Taken together, experiments with both cytosolic and secreted proteins demonstrated that PBAEs lead to significantly enhanced protein yields compared to leading commercial reagent PEI in two cell lines that are widely used for protein production. Storage stability and straightforward synthesis from inexpensive chemical monomers further strengthen their attractiveness for use in recombinant protein production across batch scales. Overall, the favorable properties of PBAEs combined with the results herein suggest that these polymers hold promise as superior reagents for transient transfection that can significantly improve protein production workflows.

Bruno Valdigem

and 9 more

BACKGROUND Electrophysiologic Study (EPS) is a diagnostic tool that can further evaluate patients prone to arrhythmic death.OBJECTIVE To shed light on the causes of death in Chagas disease, specially on regards of importance of the VT induction and chances of death, as evaluated by Rassi clinical score. METHODS We evaluated data from 153 patients with Chagas disease from the electrophysiology laboratory from a tertiary center between January of 2011 and January 2013. All patients were evaluated with 1, 2, 3 or 4 ventricular extra-stimuli followed by runs of 10 beats fast ventricular stimulation. If VT or VF is inducible the patients are referred to ICD implant or VT ablation. RESULTS Of a total of 153 patients, 48% were male, mean age 58 ±12 years (24 - 84 years). As for risk of death according to the clinical score, 32% were evaluated as low risk, 35,4% intermediate, 32,6% high risk. NSVT on 24 hour Holter (p=0,009), stimulation on EPS (p<0,001), lower VEFE (p<0,01), cardiomegaly (p<0,001) and high risk on Rassi score (p<0,001) where the more associated variables to ventricular tachyarrithmias. CONCLUSIONS Clinical score is an interesting tool to further stratify patients in higher risk for VT induction during EPS, which is also a marker of higher risk of sudden cardiac death. Some characteristics as Non sustained VT, Pulmonary congestion, cardiomegaly and low voltage QRS were the most relevant determinants for inducible VT on EPS. The duration of NSVT is also important for VT induction in EPS.

Jon Brommer

and 3 more

Adult sex ratio and fecundity are key population parameters in sustainable wildlife management, but inferring these requires estimates of the density of at least three age/sex classes of the population (male and female adults and juveniles). We used an array of 36 wildlife camera traps during 2–3 weeks in autumn prior to harvest during two consecutive years, and recorded white-tailed deer adult males, adult females and fawns from the pictures. Simultaneously, we collected fecal DNA (fDNA) from 92 20mx20m plots placed in 23 clusters of four plots between the camera traps. We identified individuals from fDNA samples with microsatellite markers and estimated the total sex ratio and population density using Spatial Capture Recapture (SCR). The fDNA-SCR analysis concluded equal sex ratio in the first year and female bias in the second year, and no difference in space use between sexes (fawns and adults combined). Camera information was analyzed in a Spatial Capture (SC) framework assuming an informative prior for animals’ space use, either (1) as estimated by fDNA-SCR (same for all age/sex classes), (2) as assumed from the literature (space use of adult males larger than adult females and fawns), (3) by inferring adult male space use from individually-identified males from the camera pictures. These various SC approaches produced plausible inferences on fecundity, but also inferred total density to be lower than the estimate provided by fDNA-SCR in one of the study years. SC approaches where adult male and female were allowed to differ in their space use suggested the population had a female-biased adult sex ratio. In conclusion, SC approaches allowed estimating the pre-harvest population parameters of interest and provided conservative density estimates.

Johanna Melin

and 6 more

Aim: Dapagliflozin improves glycaemic control in patients with type 2 diabetes mellitus (T2DM) and is approved in European and Japanese patients with type 1 diabetes mellitus (T1DM) with inadequate glycaemic control. The objectives of this work were to characterise the dapagliflozin pharmacokinetics (PK) in patients with T1DM, assess the influence of covariates on dapagliflozin PK, and compare dapagliflozin systemic exposure between patients with T1DM and T2DM. Methods: Population PK analysis was performed using a non-linear mixed-effect modelling approach. The analysis included 5,793 dapagliflozin plasma concentrations from 1,150 adult patients with T1DM, collected from one phase 2 (NCT01498185) and two phase 3 studies (DEPICT-1, NCT02268214; DEPICT-2, NCT02460978). Covariate effects were investigated using stepwise covariate modelling. Model-derived area under the concentration-time curve (AUC) was compared with AUC in patients with T2DM. Results: The final two-compartmental model adequately described the dapagliflozin concentrations in patients with T1DM. The estimated apparent clearance was 20.5 L/h. Model-predicted systemic exposure for 5 mg and 10 mg of dapagliflozin indicated dose-proportionality and was comparable between patients with T1DM and T2DM. The identified covariate relationships showed that patients with better renal function (measured as estimated glomerular filtration rate), males, and heavier patients had lower dapagliflozin systemic exposure. Among the covariates studied, no covariates affected dapagliflozin systemic exposure to a clinically relevant extent. Conclusions: Dapagliflozin PK in patients with T1DM was adequately described by the population PK model and no clinically relevant covariates were identified. Dapagliflozin systemic exposure was comparable between patients with T1DM and T2DM. NCT01498185, NCT02268214, NCT02460978

Anna van Aart

and 22 more

Animals like mink, cats and dogs are susceptible to SARS-CoV-2 infection. In the Netherlands, 69 out of 127 mink farms were infected with SARS-CoV-2 between April and November 2020 and all mink on infected farms were culled after SARS-CoV-2 infection to prevent further spread of the virus. On some farms, (feral) cats and dogs were present. This study provides insight into the prevalence of SARS-CoV-2 positive cats and dogs in ten infected mink farms and their possible role in transmission of the virus. Throat and rectal swabs of 101 cats (12 domestic and 89 feral cats) and 13 dogs of ten farms were tested for SARS-CoV-2 using PCR. Serological assays were performed on serum samples from 62 adult cats and all 13 dogs. Whole Genome Sequencing was performed on one cat sample. Cat-to-mink transmission parameters were estimated using data from all ten farms. This study shows evidence of SARS-CoV-2 infection in twelve feral cats and two dogs. Eleven cats (19%) and two dogs (15%) tested serologically positive. Three feral cats (3%) and one dog (8%) tested PCR-positive. The sequence generated from the cat throat swab clustered with mink sequences from the same farm. The calculated rate of mink-to-cat transmission showed that cats on average had a chance of 12% (95%CI 10% to 18%) of becoming infected by mink, assuming no cat-to-cat transmission. As only feral cats were infected it is most likely that infections in cats were initiated by mink, not by humans. Whether both dogs were infected by mink or humans remains inconclusive. This study presents one of the first reports of interspecies transmission of SARS-CoV-2 that does not involve humans, namely mink-to-cat transmission, which should also be considered as a potential risk for spread of SARS-CoV-2.

Moira Giovannoni

and 7 more

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Huseyin Kazan

and 6 more

Objective: To investigate the factors, especially preoperative urinalysis, predicting postoperative early infection after retrograde intrarenal surgery (RIRS) in 1-2 cm renal stones. Methods: Of the 642 patients who underwent RIRS between September 2013 and July 2019, 289 patients with a total stone size of 1-2 cm were included in the study. Patients were divided into two groups as with and without postoperative urinary tract infection. The demographic data and perioperative findings of all patients were retrospectively reviewed. Sterile urine cultures were obtained in all patients during the preoperative 30-day period and urine analysis values were included in the data. Results: Urinary system infection (UTI) was seen in 20 (6.9%) of 289 patients. Patient demographics were similar between groups. There was no statistically significant difference between the two groups in terms of stone diameter and stone localization (median diameter 13.5 vs 15, p=0.285). Patients with postoperative UTI had a higher rate of UTI history (55% vs 20.5%, p=0.000) and longer operative times (62.5 vs 60 min., p=0.008). Rate of pyuria, leukocyte esterase and nitrite positivity were observed more frequently in patients with postoperative UTI. In multivariate analysis, UTI history, prolonged operative time, and nitrite positivity were found to be independent risk factors for postoperative UTI. Conclusion: Nitrite positivity in preoperative urinanalysis, history of UTI and prolonged operation time are the factors that predict the postoperative infection in RIRS for stones between 1-2 cm.

Gunter Sturm

and 38 more

Background: There is controversy whether taking β-blockers or ACE inhibitors (ACEI) is a risk factor for more severe systemic insect sting reactions (SSR) and whether it increases the number or severity of adverse events (AE) during venom immunotherapy (VIT). Methods: In this open, prospective, observational, multicenter trial, we recruited patients with a history of a SSR and indication for VIT. The primary objective of this study was to evaluate whether patients taking β-blockers or ACEI show more systemic AE during VIT compared to patients without such treatment. Results: In total, 1,425 patients were enrolled and VIT was performed in 1,342 patients. Of all patients included, 388 (27.2%) took antihypertensive (AHT) drugs (10.4% took β-blockers, 11.9% ACEI, 5.0% β-blockers and ACEI). Only 5.6% of patients under AHT treatment experienced systemic AE during VIT as compared with 7.4% of patients without these drugs (OR: 0.74, 95% CI: 0.43–1.22, p=0.25). The severity of the initial sting reaction was not affected by the intake of β-blockers or ACEI (OR: 1.14, 95% CI: 0.89–1.46, p=0.29). In total, 210 (17.7%) patients were re-stung during VIT and 191 (91.0%) tolerated the sting without systemic symptoms. Of the 19 patients with VIT treatment failure, 4 took β-blockers, none an ACEI. Conclusions: This trial provides robust evidence that taking β-blockers or ACEI does neither increase the frequency of systemic AE during VIT nor aggravate SSR. Moreover, results suggest that these drugs do not impair effectiveness of VIT. (Funded by Medical University of Graz, Austria; Clinicaltrials.gov number, NCT04269629)

Nazanin Kermani

and 12 more

Background. Patients with severe asthma may have a greater risk of dying from COVID-19 disease. Angiotensin converting enzyme-2 (ACE2) and the enzyme proteases, transmembrane protease serine 2 (TMPRSS2) and FURIN, are needed for viral attachment and invasion into host cells. Methods. We examined microarray mRNA expression of ACE2, TMPRSS2 and FURIN in sputum, bronchial brushing and bronchial biopsies of the European U-BIOPRED cohort. Clinical parameters and molecular phenotypes, including asthma severity, sputum inflammatory cells, lung functions, oral corticosteroid (OCS) use, and transcriptomic-associated clusters, were examined in relation to gene expression levels. Results. ACE2 levels were significantly increased in sputum of severe asthma compared to mild-moderate asthma. In multivariate analyses, sputum ACE2 levels were positively associated with OCS use and male gender. Sputum FURIN levels were significantly related to neutrophils (%) and the presence of severe asthma. In bronchial brushing samples, TMPRSS2 levels were positively associated with male gender and body mass index, whereas FURIN levels with male gender and blood neutrophils. In bronchial biopsies, TMPRSS2 levels were positively related to blood neutrophils. The neutrophilic molecular phenotype characterised by high inflammasome activation expressed significantly higher FURIN levels in sputum than the eosinophilic Type 2-high or the pauci-granulocytic oxidative phosphorylation phenotypes. Conclusion. Levels of ACE2 and FURIN may differ by clinical or molecular phenotypes of asthma. Sputum FURIN expression levels were strongly associated with neutrophilic inflammation and with inflammasome activation. This might indicate the potential for a greater morbidity and mortality outcome from SARS-CoV-2 infection in neutrophilic severe asthma.

Mehmet Pehlivaoğlu

and 5 more

Aim: This study evaluated the effect of intracavitary levobupivacaine infusion diluted with locally applied isotonic solution for pain control in cystoscopy. Methods: Included in this study are 100 patients who had previously undergone transurethral tumor resection for bladder tumor and were followed up by cystoscopy. The patients were randomized into five groups (n = 20). In the first, second, third, and fourth groups, 4, 6, 8, and 10 mL of levobupivacaine HCl (5.0 mg/mL) were mixed with 26, 24, 22, and 20 mL of isotonic solution, respectively. Hence, the total mixture was 30 mL for each group. The fifth group was the control group. In this group, the standard method commonly used in most clinics was utilized. That is, a gel containing Cathejell-2% lidocaine (25 mg lidocaine) was applied. Cystoscopic interventions were performed with a 17.5 Fr rigid cystoscope and 0°, 30°, and 70° lens. During cystoscopy and 30 min later, the pain status of patients was assessed using the Visual Analogue Scale (VAS), and patient satisfaction was questioned. Results: The mean VAS score during and after the cystoscopy procedure was significantly lower in the levobupivacaine groups compared to the lidocaine group. In addition, patient satisfaction in the levobupivacaine groups was significantly higher than in the lidocaine group. No drug-related side-effects were observed in all groups. Conclusion: Thus, levobupivacaine is a more effective drug than lidocaine alone to control pain during cystoscopy. The use of levobupivacaine is recommended to prevent possible complications of general anesthesia by eliminating the need for the aforementioned as well as its cost-saving advantage.

Yanhui Dong

and 4 more

Groundwater age is often used to estimate groundwater recharge through a simplified analytical approach. This estimated recharge is thought to be representative of the mean recharge between the point of entry and the sampling point. However, given the complexity in actual recharge, whether the mean recharge is reasonable is still unclear. This study examined the validity of the method to estimate long-term average groundwater recharge and the possibility of obtaining reasonable spatial recharge pattern. We first validated our model in producing reasonable age distributions using a constant flux boundary condition. We then generated different flow fields and age patterns by using various spatially-varying flux boundary conditions with different magnitudes and wavelengths. Groundwater recharge was estimated and analyzed afterwards using the method at the spatial scale. We illustrated the main findings with a field example in the end. Our results suggest that we can estimate long-term average groundwater recharge with 10% error in many parts of an aquifer. The size of these areas decreases with the increase in both the amplitude and the wavelength. The chance of obtaining a reasonable groundwater recharge is higher if an age sample is collected from the middle of an aquifer and at downstream areas. Our study also indicates that the method can also be used to estimate local groundwater recharge if age samples are collected close to the water table. However, care must be taken to determine groundwater age regardless of conditions.

Xinyi Guan

and 4 more

Adriana Bustamante

and 3 more

Yasser Mubarak

and 1 more

Background: Small Aortic Annulus (AA) is big issue during Aortic Valve Replacement (AVR) necessitating replacement of an undersized prosthetic valve especially with Double Valve Replacement (DVR). Despite that small aortic valve prostheses can lead to Prosthesis-Patient Mismatch (PPM), there remains reluctance to perform aortic root enlargement (ARE) procedures fearing from morbidity and mortality. Objective: To evaluate clinical and echocardiographic outcomes in patients with small aortic annulus undergoing double valve replacement. Methods: The study included 100 consecutive patients underwent DVR for combined rheumatic aortic and mitral valve diseases, between Jan. 2016 and Sept. 2020. Only (50) patients had ARE with DVR. ARE was performed using an autologous or bovine pericardium or Dacron patch by Nicks or Manouguian procedures. The estimated postoperative end-points were mortality, effective orifice areas (EOA), mean aortic pressure gradient and valve-related complications. The least postoperative follow-up period was 6 months. Results: The study included 30 male and 70 female patients with mean age of 35±20 years, body surface area (BSA) of 1.7 ±0.3 m2, aortic annulus diameter was 20±1.4 mm, aortic orifice area was 0.8±0.1 cm2, and mean pressure gradient 85±2.5 mmHg. During follow-up period, there was a mild to moderate paravalvular leak (1%) with, (1%) heart block, and residual gradient on prosthetic aortic valve; that was all in DVR alone. Conclusion: Enlargement of aortic root by Nicks or Manouguian technique is safe and effective in patients with small aortic annulus undergoing double valve replacements.

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