Enzymatic dynamic kinetic resolution of racemic N-formyl- and N-carbamoyl-amino acids using immobilized L-N-carbamoylase and N-succinyl-amino acid racemase.

Taking advantage of the catalytic promiscuity of L-carbamoylase from Geobacillus stearothermophilus CECT43 (BsLcar) and N-succinyl-amino acid racemase from Geobacillus kaustophilus CECT4264 (GkNSAAR), we have evaluated the production of different optically pure L-α-amino acids starting from different racemic N-formyl- and N-carbamoyl-amino acids using a dynamic kinetic resolution approach. The enzymes were immobilized on two different solid supports, resulting in improved stability of the enzymes in terms of thermostability and storage when compared to the enzymes in solution. The bienzymatic system retained up to 80 % conversion efficiency after 20 weeks at 4 °C and up to 90 % after 1 week at 45 °C. The immobilization process also resulted in a great enhancement of the activity of BsLcar toward N-formyl-tryptophan, showing for the first time that substrate specificity of L-carbamoylases can be influenced by this approach. The system was effective for the biosynthesis of natural and unnatural L-amino acids (enantiomeric excess (e.e.) >99.5 %), such as L-methionine, L-alanine, L-tryptophan, L-homophenylalanine, L-aminobutyric acid, and L-norleucine, with a higher performance toward N-formyl-α-amino acid substrates. Biocatalyst reuse was studied, and after 10 reaction cycles, over 75 % activity remained.post-print1047 K

Effects of cellulase and xylanase enzymes mixed with increasing doses of Salix babylonica extract on in vitro rumen gas production kinetics of a mixture of corn silage with concentrate

Therefore, the objective of this study was to assess the effects of different doses of plant extracts when combined with exogenous fibrolytic enzymes on in vitro ruminal fermentation of a mixture of corn silage with concentrate.An in vitro gas production (GP) technique was used to investigate the effects of combining different doses of Salix babylonica extract (SB) with exogenous fibrolytic enzymes (EZ) based on xylanase (X) and cellulase (C), or their mixture (XC; 1:1 v/v) on in vitro fermentation characteristics of a total mixed ration of corn silage and concentrate mixture (50:50, w/w) as substrate. Four levels of SB (0, 0.6, 1.2 and 1.8 mL g–1 dry matter (DM)) and four supplemental styles of EZ (1 μL g–1 DM; control (no enzymes), X, C and XC (1:1, v/v) were used in a 4×4 factorial arrangement. In vitro GP (mL g–1 DM) were recorded at 2, 4, 6, 8, 10, 12, 24, 36, 48 and 72 h of incubation. After 72 h, the incubation process was stopped and supernatant pH was determined, and then filtered to determine dry matter degradability (DMD). Fermentation parameters, such as the 24 h gas yield (GY24), in vitro organic matter digestibility (OMD), metabolizable energy (ME), short chain fatty acid concentrations (SCFA), and microbial crude protein production (MCP) were also estimated. Results indicated that there was a SB´EZ interaction (P<0.0001) for the asymptotic gas production (b), the rate of gas production (c), GP from 6 to 72 h, GP2 (P=0.0095), and GP4 (P=0.02). The SB and different combination of enzymes supplementation influenced (P<0.001) in vitro GP parameters after 12 h of incubation; the highest doses of SB (i.e., 1.8 mL g–1 DM), in the absence of any EZ, quadratically increased (P<0.05) the initial delay before GP begins (L) and GP at different incubation times, with lowering b (quadratic effect, P<0.0001) and c (quadratic effect, P<0.0001; linear effect, P=0.0018). The GP was the lowest (P<0.05) when the highest SB level was combined with cellulose. There were SB´EZ interactions (P<0.001) for OMD, ME, the partitioning factor at 72 h of incubation (PF72), GY24, SCFA, MCP (P=0.0143), and pH (P=0.0008). The OMD, ME, GY24 and SCFA with supplementation of SB extract at 1.8 mL g–1 DM were higher (P<0.001) than the other treatments, however

Activation of Neurogenesis in Multipotent Stem Cells Cultured In Vitro and in the Spinal Cord Tissue After Severe Injury by Inhibition of Glycogen Synthase Kinase-3

The inhibition of glycogen synthase kinase-3 (GSK-3) can induce neurogenesis, and the associated activation of Wnt/β-catenin signaling via GSK-3 inhibition may represent a means to promote motor function recovery following spinal cord injury (SCI) via increased astrocyte migration, reduced astrocyte apoptosis, and enhanced axonal growth. Herein, we assessed the effects of GSK-3 inhibition in vitro on the neurogenesis of ependymal stem/progenitor cells (epSPCs) resident in the mouse spinal cord and of human embryonic stem cell-derived neural progenitors (hESC-NPs) and human-induced pluripotent stem cell-derived neural progenitors (hiPSC-NPs) and in vivo on spinal cord tissue regeneration and motor activity after SCI. We report that the treatment of epSPCs and human pluripotent stem cell-derived neural progenitors (hPSC-NPs) with the GSK-3 inhibitor Ro3303544 activates β-catenin signaling and increases the expression of the bIII-tubulin neuronal marker; furthermore, the differentiation of Ro3303544-treated cells prompted an increase in the number of terminally differentiated neurons. Administration of a water-soluble, bioavailable form of this GSK-3 inhibitor (Ro3303544-Cl) in a severe SCI mouse model revealed the increased expression of bIII-tubulin in the injury epicenter. Treatment with Ro3303544-Cl increased survival of mature neuron types from the propriospinal tract (vGlut1, Parv) and raphe tract (5-HT), protein kinase C gamma-positive neurons, and GABAergic interneurons (GAD65/67) above the injury epicenter. Moreover, we observed higher numbers of newly born BrdU/DCX-positive neurons in Ro3303544-Cl-treated animal tissues, a reduced area delimited by astrocyte scar borders, and improved motor function. Based on this study, we believe that treating animals with epSPCs or hPSC-NPs in combination with Ro3303544-Cl deserves further investigation towards the development of a possible therapeutic strategy for SCI

Differential presentation of hypersensitivity reactions to carboplatin and oxaliplatin: Phenotypes, endotypes, and management with desensitization

Background: Drug hypersensitivity reactions (DHRs) to platinum-based drugs are heterogenous and restrict their access, and drug desensitization (DD) has provided a ground-breaking procedure for their re-introduction, although the response is heterogeneous. We aimed to identify the phenotypes, endotypes, and biomarkers of reactions to carboplatin and oxaliplatin and their response to DD. Methods: Seventy-nine patients presenting with DHRs to oxaliplatin (N = 46) and carboplatin (N = 33) were evaluated at the Allergy Departments of two tertiary care hospitals in Spain. Patient symptoms, skin testing, biomarkers, and outcomes of 267 DDs were retrospectively analyzed. Results: Oxaliplatin-reactive patients presented with type I (74%), cytokine release reaction (CRR) (11%), and mixed (Mx) (15%) phenotypes. In contrast, carboplatin reactive patients presented with predominantly type I (85%) and Mx (15%) but no CRRs. Out of 267 DDs, breakthrough reactions (BTRs) to oxaliplatin occurred twice as frequently as carboplatin (32% vs. 15%; p < .05). Phenotype switching from type I to another phenotype was observed in 46% of oxaliplatin DDs compared to 21% of carboplatin DDs. Tryptase was elevated in type I and Mx reactions, and IL-6 in CRR and Mx, indicating different mechanisms and endotypes. Conclusion: Carboplatin and oxaliplatin induced three different types of reactions with defined phenotypes and endotypes amendable to DD. Although most of the initial reactions for both were type I, oxaliplatin presented with unique CRR reactions. During DD, carboplatin reactive patients presented mostly type I BTR, while oxaliplatin-reactive patients frequently switched from type I to CRR, providing a critical difference and the need for personalized DD protocols

Gene Correction Recovers Phagocytosis in Retinal Pigment Epithelium Derived from Retinitis Pigmentosa-Human-Induced Pluripotent Stem Cells.

Hereditary retinal dystrophies (HRD) represent a significant cause of blindness, affecting mostly retinal pigment epithelium (RPE) and photoreceptors (PRs), and currently suffer from a lack of effective treatments. Highly specialized RPE and PR cells interact mutually in the functional retina, therefore primary HRD affecting one cell type leading to a secondary HRD in the other cells. Phagocytosis is one of the primary functions of the RPE and studies have discovered that mutations in the phagocytosis-associated gene Mer tyrosine kinase receptor (MERTK) lead to primary RPE dystrophy. Treatment strategies for this rare disease include the replacement of diseased RPE with healthy autologous RPE to prevent PR degeneration. The generation and directed differentiation of patient-derived human-induced pluripotent stem cells (hiPSCs) may provide a means to generate autologous therapeutically-relevant adult cells, including RPE and PR. However, the continued presence of the MERTK gene mutation in patient-derived hiPSCs represents a significant drawback. Recently, we reported the generation of a hiPSC model of MERTK-associated Retinitis Pigmentosa (RP) that recapitulates disease phenotype and the subsequent creation of gene-corrected RP-hiPSCs using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9. In this study, we differentiated gene-corrected RP-hiPSCs into RPE and found that these cells had recovered both wild-type MERTK protein expression and the lost phagocytosis of fluorescently-labeled photoreceptor outer segments observed in uncorrected RP-hiPSC-RPE. These findings provide proof-of-principle for the utility of gene-corrected hiPSCs as an unlimited cell source for personalized cell therapy of rare vision disorders

Emery-Dreifuss muscular dystrophy Type 1 is associated with a high risk of malignant ventricular arrhythmias and end-stage heart failure

BACKGROUND AND AIMS: Emery-Dreifuss muscular dystrophy (EDMD) is caused by variants in EMD (EDMD1) and LMNA (EDMD2). Cardiac conduction defects and atrial arrhythmia are common to both, but LMNA variants also cause end-stage heart failure (ESHF) and malignant ventricular arrhythmia (MVA). This study aimed to better characterise the cardiac complications of EMD variants. METHODS: Consecutively referred EMD variant-carriers were retrospectively recruited from 12 international cardiomyopathy units. MVA and ESHF incidence in male and female variant-carriers was determined. Male EMD variant-carriers with a cardiac phenotype at baseline (EMDCARDIAC) were compared to consecutively recruited male LMNA variant-carriers with a cardiac phenotype at baseline (LMNACARDIAC). RESULTS: Longitudinal follow-up data were available for 38 male and 21 female EMD variant-carriers (mean [SD] ages 33.4 [13.3] and 43.3 [16.8] years, respectively). Nine (23.6%) males developed MVA and five (13.2%) developed ESHF during a median [IQR] follow-up of 65.0 [24.3, 109.5] months. No female EMD variant-carrier had MVA or ESHF, but nine (42.8%) developed a cardiac phenotype at a median [IQR] age of 58.6 [53.2, 60.4] years. Incidence rates for MVA were similar for EMDCARDIAC and LMNACARDIAC (4.8 and 6.6 per 100 person-years, respectively; log-rank p = 0.49). Incidence rates for ESHF were 2.4 and 5.9 per 100 person-years for EMDCARDIAC and LMNACARDIAC, respectively (log-rank p = 0.09). CONCLUSIONS: Male EMD variant-carriers have a risk of progressive heart failure and ventricular arrhythmias similar to that of male LMNA variant-carriers. Early implantable cardioverter defibrillator implantation and heart failure drug therapy should be considered in male EMD variant-carriers with cardiac disease

CD69 expression on regulatory T cells protects from immune damage after myocardial infarction.

Increasing evidences advocate for an important function of T cells in controlling immune homeostasis and pathogenesis after myocardial infarction (MI), although the underlying molecular mechanisms remain elusive. In this study, a broad analysis of immune markers in 283 patients revealed a significant CD69 overexpression on Treg cells after MI. Our results in mice showed that CD69 expression on Treg cells increased survival after left-anterior-descending coronary artery (LAD)-ligation. Cd69-/- mice developed strong IL-17+ γδT cell responses after ischemia that increased myocardial inflammation and, consequently, worsened cardiac function. CD69+ Treg cells, by induction of AhR-dependent CD39 ectonucleotidase activity, induced apoptosis and decreased IL-17A production in γδT cells. Adoptive transfer of CD69+ Treg cells to Cd69-/- mice after LAD-ligation reduced IL-17+ γδT cell recruitment, thus increasing survival. Consistently, clinical data from two independent cohorts of patients indicated that increased CD69 expression in peripheral blood cells after acute MI was associated with a lower risk of re-hospitalization for heart failure (HF) after 2.5 years of follow-up. This result remained significant after adjustment for age, sex and traditional cardiac damage biomarkers. Our data highlight CD69 expression on Treg cells as a potential prognostic factor and a therapeutic option to prevent HF after MI.This study was supported by competitive grants from the Ministerio de Ciencia e Innovación (MCIN), through the Carlos III Institute of Health (ISCIII)-Fondo de Investigación Sanitaria (PI22/01759) to P.M.; RTI2018-094727-B-100 to J. M-G; Comunidad de Madrid grants S2017/BMD-3671-INFLAMUNE-CM to P.M. and FSM.; Fundació La Marató TV3 (20152330 31) to J.M-G and F.S-M.; Ministerio de Ciencia e Innovación (MCIN) RTI2018-099357-B-I00, and CIBERFES (CB16/10/00282), Human Frontier Science Program (grant RGP0016/2018), and Leducq Transatlantic Networks (17CVD04) to JAE. AC is supported by Marie Skłodowska- Curie grant (agreement No. 713673). R.B-D. is supported by Formación de Profesorado Universitario (FPU16/02780) program from the Spanish Ministry of Education, Culture and Sports. The CNIC is supported by the ISCIII, the MCIN and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Early Tracheostomy for Managing ICU Capacity During the COVID-19 Outbreak: A Propensity-Matched Cohort Study

10 p.Background: During the first wave of the COVID-19 pandemic, shortages of ventilators and ICU beds overwhelmed health care systems. Whether early tracheostomy reduces the duration of mechanical ventilation and ICU stay is controversial. Research question: Can failure-free day outcomes focused on ICU resources help to decide the optimal timing of tracheostomy in overburdened health care systems during viral epidemics? Study design and methods: This retrospective cohort study included consecutive patients with COVID-19 pneumonia who had undergone tracheostomy in 15 Spanish ICUs during the surge, when ICU occupancy modified clinician criteria to perform tracheostomy in Patients with COVID-19. We compared ventilator-free days at 28 and 60 days and ICU- and hospital bed-free days at 28 and 60 days in propensity score-matched cohorts who underwent tracheostomy at different timings (≤ 7 days, 8-10 days, and 11-14 days after intubation). Results: Of 1,939 patients admitted with COVID-19 pneumonia, 682 (35.2%) underwent tracheostomy, 382 (56%) within 14 days. Earlier tracheostomy was associated with more ventilator-free days at 28 days (≤ 7 days vs > 7 days [116 patients included in the analysis]: median, 9 days [interquartile range (IQR), 0-15 days] vs 3 days [IQR, 0-7 days]; difference between groups, 4.5 days; 95% CI, 2.3-6.7 days; 8-10 days vs > 10 days [222 patients analyzed]: 6 days [IQR, 0-10 days] vs 0 days [IQR, 0-6 days]; difference, 3.1 days; 95% CI, 1.7-4.5 days; 11-14 days vs > 14 days [318 patients analyzed]: 4 days [IQR, 0-9 days] vs 0 days [IQR, 0-2 days]; difference, 3 days; 95% CI, 2.1-3.9 days). Except hospital bed-free days at 28 days, all other end points were better with early tracheostomy. Interpretation: Optimal timing of tracheostomy may improve patient outcomes and may alleviate ICU capacity strain during the COVID-19 pandemic without increasing mortality. Tracheostomy within the first work on a ventilator in particular may improve ICU availability

Grupo español de cirugía torácica asistida por videoimagen: método, auditoría y resultados iniciales de una cohorte nacional prospectiva de pacientes tratados con resecciones anatómicas del pulmón

Introduction: our study sought to know the current implementation of video-assisted thoracoscopic surgery (VATS) for anatomical lung resections in Spain. We present our initial results and describe the auditing systems developed by the Spanish VATS Group (GEVATS). Methods: we conducted a prospective multicentre cohort study that included patients receiving anatomical lung resections between 12/20/2016 and 03/20/2018. The main quality controls consisted of determining the recruitment rate of each centre and the accuracy of the perioperative data collected based on six key variables. The implications of a low recruitment rate were analysed for '90-day mortality' and 'Grade IIIb-V complications'. Results: the series was composed of 3533 cases (1917 VATS; 54.3%) across 33 departments. The centres' median recruitment rate was 99% (25-75th:76-100%), with an overall recruitment rate of 83% and a data accuracy of 98%. We were unable to demonstrate a significant association between the recruitment rate and the risk of morbidity/mortality, but a trend was found in the unadjusted analysis for those centres with recruitment rates lower than 80% (centres with 95-100% rates as reference): grade IIIb-V OR=0.61 (p=0.081), 90-day mortality OR=0.46 (p=0.051). Conclusions: more than half of the anatomical lung resections in Spain are performed via VATS. According to our results, the centre's recruitment rate and its potential implications due to selection bias, should deserve further attention by the main voluntary multicentre studies of our speciality. The high representativeness as well as the reliability of the GEVATS data constitute a fundamental point of departure for this nationwide cohort