SARS-CoV-2 Proteins Induce Endotoxin Tolerance Hallmarks: A Demonstration in Patients with COVID-19.

According to a large number of reported cohorts, sepsis has been observed in nearly all deceased patients with COVID-19. We and others have described sepsis, among other pathologies, to be an endotoxin tolerance (ET)-related disease. In this study, we demonstrate that the culture of human blood cells from healthy volunteers in the presence of SARS-CoV-2 proteins induced ET hallmarks, including impairment of proinflammatory cytokine production, low MHC class II (HLA-DR) expression, poor T cell proliferation, and enhancing of both phagocytosis and tissue remodeling. Moreover, we report the presence of SARS-CoV-2 blood circulating proteins in patients with COVID-19 and how these levels correlate with an ET status, the viral RNA presence of SARS-CoV-2 in plasma, as well as with an increase in the proportion of patients with secondary infections.

A new metabolic disorder in human cationic amino acid transporter-2 that mimics arginase 1 deficiency in newborn screening.

PURPOSE: We report a patient with a human cationic amino acid transporter 2 (CAT-2) defect discovered due to a suspected arginase 1 deficiency observed in newborn screening (NBS). METHODS: A NBS sample was analyzed using tandem mass spectrometry. Screen results were confirmed by plasma and urine amino acid quantification. Molecular diagnosis was done using clinical exome sequencing. Dimethylated arginines were determined by HPLC and nitrate/nitrite levels by a colorimetric assay. The metabolomic profile was analyzed using 1D nuclear magnetic resonance spectroscopy. RESULTS: A Spanish boy of nonconsanguineous parents had high arginine levels in a NBS blood sample. Plasma and urinary cationic amino acids were high. Arginase enzyme activity in erythrocytes was normal and no pathogenic mutations were identified in the ARG1 gene. Massive parallel sequencing detected two loss-of-function mutations in the SLC7A2 gene. Currently, the child receives a protein-controlled diet of 1.2 g/kg/day with protein-and amino-acid free infant formula, 30 g/day, and is asymptomatic. CONCLUSION: We identified a novel defect in human CAT-2 due to biallelic pathogenic variants in the SLC7A2 gene. The characteristic biochemical profile includes high plasma and urine arginine, ornithine, and lysine levels. NBS centers should know of this disorder since it can be detected in arginase 1 deficiency screening.

Determination of azoxystrobin and chlorothalonil using a methacrylate-based polymer modified with gold nanoparticles as solid-phase extraction sorbent.

This paper describes a novel and sensitive method for extraction, preconcentration, and determination of two important widely used fungicides, azoxystrobin, and chlorothalonil. The developed methodology is based on solid-phase extraction (SPE) using a polymeric material functionalized with gold nanoparticles (AuNPs) as sorbent followed by high-performance liquid chromatography (HPLC) with diode array detector (DAD). Several experimental variables that affect the extraction efficiency such as the eluent volume, sample flow rate, and salt addition were optimized. Under the optimal conditions, the sorbent provided satisfactory enrichment efficiency for both fungicides, high selectivity and excellent reusability (>120 re-uses). The proposed method allowed the detection of 0.05 µg L-1 of the fungicides and gave satisfactory recoveries (75-95 %) when it was applied to drinking and environmental water samples (river, well, tap, irrigation, spring, and sea waters).

Green synthesis of MIL53(Al)-modified paper-based analytical device for efficient extraction of neonicotinoid insecticides from environmental water samples.

BACKGROUND: There is a need to develop low-cost, reliable and portable devices to enhance the efficiency of microextraction techniques in complex samples. Metal-organic frameworks (MOFs) have proven to be promising sorbents due to their well-documented properties. However, their green preparation and combination with paper-based substrates have not been satisfactorily explored to fabricate sustainable sorptive phases. RESULTS: In this work, the hybridization of a paper substrate (as a sustainable support) with MOFs (as a sorptive phase) was carried out by one-pot approach. Concretely, the selected MOF, MIL-53(Al), was in-situ growth onto the paper surface in aqueous solution without the need for high temperature or pressure, thereby aligning with the Green Analytical Chemistry principles. The optimized composite (MIL-53(Al)@cellulose paper) was characterized and evaluated as extraction sorbent for five neonicotinoids (NEOs) (thiamethoxam, clothianidin, imidacloprid, acetamiprid, and thiacloprid). Furthermore, its feasibility was demonstrated by isolating these pollutants from environmental water samples, followed their determination by HPLC coupled to diode array detection. The whole method showed satisfactory analytical performance with recoveries between 86 and 114 %, suitable precision (with RSD lower than 14 %), and limits of detection ranged from 1.0 to 1.6 µg L-1. Besides, the greenness of the method was assessed by application of different existing metrics. The developed extraction device was affordable (<0.08 €/device) and mechanical and chemically stable, being possible its reuse more than 11 cycles, thus demonstrating its suitability for rapid screening of pesticides in environmental samples. SIGNIFICANCE: This report presents, for the first time, the green synthesis of MIL-53(Al)cellulose paper composite and its application as a sorptive phase for the extraction of NEOs from environmental water samples. We believe that the proposed strategy for fabricating these sustainable paper-based sorptive phases paves the way for further hybridizations with other MOFs or materials. Additionally, it opens up large possibilities for their application in extraction of pollutants or other hazardous compounds in aquatic environments.

Changes in CDKN2D , TP53, and miR125a expression: potential role in the evaluation of human amniotic fluid-derived mesenchymal stromal cell fitness.

Human amniotic fluid-derived mesenchymal stromal cells (hAMSC) have become one of the main cell populations used in regenerative medicine and for the study of various clinical disorders. These cells have a great capacity for proliferation and differentiation and do not form teratomas when transplanted into animal models, and their stemness seems to be between embryonic cells and adult mesenchymal cells. Before their use in cell therapy, they must be cultured and expanded in vitro, but the effect this process has on their fitness, a determining factor for the success or failure of cell therapy, is unknown. We undertook a follow-up of gene and microRNAs (miRNAs) expression using microarray of hAMSC for the first 15 passages. Significant changes were noted in the expression of various mRNAs and miRNAs, particularly down-regulation of TP53, increased expression of hsa-miR-125a and up-regulation of CDKN2D . The variations in TP53 and hsa-miR-125a may act as an indicator of the stemness of the hAMSC, whereas CDKN2D may indicate the begging of early senescence process in a p53-independent mechanism. The genes described in this study will help evaluate the fitness of hAMSC, thus guaranteeing their biological quality for use in regenerative medicine.

mRNA-1273 boost after BNT162b2 vaccination generates comparable SARS-CoV-2-specific functional responses in naïve and COVID-19-recovered individuals.

Introduction: COVID-19 vaccines based on mRNA have represented a revolution in the biomedical research field. The initial two-dose vaccination schedule generates potent humoral and cellular responses, with a massive protective effect against severe COVID-19 and death. Months after this vaccination, levels of antibodies against SARS-CoV-2 waned, and this promoted the recommendation of a third vaccination dose. Methods: We have performed an integral and longitudinal study of the immunological responses triggered by the booster mRNA-1273 vaccination, in a cohort of health workers previously vaccinated with two doses of the BNT162b2 vaccine at University Hospital La Paz located in Madrid, Spain. Circulating humoral responses and SARS-CoV-2-specific cellular reactions, after ex vivo restimulation of both T and B cells (cytokines production, proliferation, class switching), have been analyzed. Importantly, all along these studies, the analyses have been performed comparing naïve and subjects recovered from COVID-19, addressing the influence of a previous infection by SARS-CoV-2. Furthermore, as the injection of the third vaccination dose was contemporary to the rise of the Omicron BA.1 variant of concern, T- and B-cell-mediated cellular responses have been comparatively analyzed in response to this variant. Results: All these analyses indicated that differential responses to vaccination due to a previous SARS-CoV-2 infection were balanced following the boost. The increase in circulating humoral responses due to this booster dropped after 6 months, whereas T-cell-mediated responses were more stable along the time. Finally, all the analyzed immunological features were dampened in response to the Omicron variant of concern, particularly late after the booster vaccination. Conclusion: This work represents a follow-up longitudinal study for almost 1.5 years, analyzing in an integral manner the immunological responses triggered by the prime-boost mRNA-based vaccination schedule against COVID-19.

Association between use of enhanced recovery after surgery protocols and postoperative complications after gastric surgery for cancer (POWER 4): a nationwide, prospective multicentre study.

INTRODUCTION: The effectiveness of the Enhanced Recovery After Surgery (ERAS) protocols in gastric cancer surgery remains controversial. METHODS: Multicentre prospective cohort study of adult patients undergoing surgery for gastric cancer. Adherence with 22 individual components of ERAS pathways were assessed in all patients, regardless of whether they were treated in a self-designed ERAS centre. Each centre had a three-month recruitment period between October 2019 and September 2020. The primary outcome was moderate-to-severe postoperative complications within 30 days after surgery. Secondary outcomes were overall postoperative complications, adherence to the ERAS pathway, 30 day-mortality and hospital length of stay (LOS). RESULTS: A total of 743 patients in 72 Spanish hospitals were included, 211 of them (28.4 %) from self-declared ERAS centres. A total of 245 patients (33 %) experienced postoperative complications, graded as moderate-to-severe complications in 172 patients (23.1 %). There were no differences in the incidence of moderate-to-severe complications (22.3% vs. 23.5%; OR, 0.92 (95% CI, 0.59 to 1.41); P = 0.068), or overall postoperative complications between the self-declared ERAS and non-ERAS groups (33.6% vs. 32.7%; OR, 1.05 (95 % CI, 0.70 to 1.56); P = 0.825). The overall rate of adherence to the ERAS pathway was 52% [IQR 45 to 60]. There were no differences in postoperative outcomes between higher (Q1, > 60 %) and lower (Q4, ≤ 45 %) ERAS adherence quartiles. CONCLUSIONS: Neither the partial application of perioperative ERAS measures nor treatment in self-designated ERAS centres improved postoperative outcomes in patients undergoing gastric surgery for cancer. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT03865810.

Evaluation of a low cost cryopreservation system on the biology of human amniotic fluid-derived mesenchymal stromal cells.

BACKGROUND: Human amniotic-derived mesenchymal stromal cells (hAMSC) are a novel population of multipotent stem cells that have been shown to have great potential for use in regenerative medicine. However, procedures to store and preserve hAMSC for future clinical applications have not been explored extensively. METHODS: In this study, we analyzed the influence of cryopreservation, using a protocol based on freezing rate of 1 °C/min, 10% dimethyl sulfoxide as cryoprotectant and a thawing rate >100 °C/min, on hAMSC morphology, proliferation rates, viability, cell cycle, karyotype, immune phenotype and multilineage differentiation potential. RESULTS: This study found that this cryopreservation protocol does not affect the biological properties of hAMSC. DISCUSSION: This shows that this protocol is a viable system for banking hAMSC, with the associated advantages that has a low cost in terms of expense, time and personnel involved and is easy to implement.

Magnetic molecularly imprinted polymer for the simultaneous selective extraction of phenoxy acid herbicides from environmental water samples.

A selective magnetic molecularly imprinted polymer (MMIP) was synthetized with 4-chloro-2-methylphenoxyacetic acid as template and 4-vinylpiridine as monomer in presence of vinylized magnetite nanoparticles. Scanning electron microscopy, nitrogen adsorption-desorption isotherms, Fourier transform infrared spectrometry and vibrating sample magnetometry were applied to characterize the resulting material. The synthesized MMIP was applied as sorbent in magnetic molecularly imprinted solid-phase extraction (MMISPE) for selective extraction of a mixture of the five herbicides 4-chloro-2-methylphenoxyacetic acid (MCPA), 4-(4-chloro-2-methylphenoxy)butyric acid (MCPB), mecoprop (MCPP), fenoxaprop (FEN) and haloxyfop (HAL). Several parameters affecting the extraction conditions were optimized to achieve the best extraction performance. The best MMISPE combined with HPLC-DAD gave detection and quantification limits between 0.33 and 0.71 µg L-1 and 1.1-2.4 µg L-1, respectively, were obtained. The precision of the whole method provided RSD values below 7.3%, and the accuracy was demonstrated by the analysis of several water samples of different origins, with recoveries ranged from 77 to 98%. Moreover, a remarkable re-usability of the MMIP sorbent, more than 65 uses without losses in extraction capacity, was obtained.

Differential Immune Checkpoint and Ig-like V-Type Receptor Profiles in COVID-19: Associations with Severity and Treatment.

Identifying patients' immune system status has become critical to managing SARS-CoV-2 infection and avoiding the appearance of secondary infections during a hospital stay. Despite the high volume of research, robust severity and outcome markers are still lacking in COVID-19. We recruited 87 COVID-19 patients and analyzed, by unbiased automated software, 356 parameters at baseline emergency department admission including: high depth immune phenotyping and immune checkpoint expression by spectral flow cytometry, cytokines and other soluble molecules in plasma as well as routine clinical variables. We identified 69 baseline alterations in the expression of immune checkpoints, Ig-like V type receptors and other immune population markers associated with severity (O2 requirement). Thirty-four changes in these markers/populations were associated with secondary infection appearance. In addition, through a longitudinal sample collection, we described the changes which take place in the immune system of COVID-19 patients during secondary infections and in response to corticosteroid treatment. Our study provides information about immune checkpoint molecules and other less-studied receptors with Ig-like V-type domains such as CD108, CD226, HVEM (CD270), B7H3 (CD276), B7H5 (VISTA) and GITR (CD357), defining these as novel interesting molecules in severe and corticosteroids-treated acute infections.

Cellular and humoral functional responses after BNT162b2 mRNA vaccination differ longitudinally between naive and subjects recovered from COVID-19.

We have analyzed BNT162b2 vaccine-induced immune responses in naive subjects and individuals recovered from coronavirus disease 2019 (COVID-19), both soon after (14 days) and later after (almost 8 months) vaccination. Plasma spike (S)-specific immunoglobulins peak after one vaccine shot in individuals recovered from COVID-19, while a second dose is needed in naive subjects, although the latter group shows reduced levels all along the analyzed period. Despite how the neutralization capacity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mirrors this behavior early after vaccination, both groups show comparable neutralizing antibodies and S-specific B cell levels late post-vaccination. When studying cellular responses, naive individuals exhibit higher SARS-CoV-2-specific cytokine production, CD4+ T cell activation, and proliferation than do individuals recovered from COVID-19, with patent inverse correlations between humoral and cellular variables early post-vaccination. However, almost 8 months post-vaccination, SARS-CoV-2-specific responses are comparable between both groups. Our data indicate that a previous history of COVID-19 differentially determines the functional T and B cell-mediated responses to BNT162b2 vaccination over time.

Lifespan of human amniotic fluid-derived multipotent mesenchymal stromal cells.

BACKGROUND AIMS: Human multipotent mesenchymal stromal cells (hMSC) have become one of the main interests in regenerative medicine because of their ability to differentiate into different lineages. Human amniotic fluid is reported to contain MSC (hAMSC) and therefore may be a useful source of cells for clinical applications. However, our understanding of the behavior of these cells in indefinite in vitro culture conditions is very limited. METHODS: We systematically evaluated and characterized, throughout their whole lifespan, the expansion potential, chromosomal stability, surface and intracellular phenotype and differentiation potential of fibroblastoid hAMSC (F-type hAMSC). RESULTS: Nine F-type hAMSC cultures could be expanded in in vitro culture conditions for 223.25 ± 24.44 days (mean ± SD), during which time 28.96 ± 1.5 passages were made giving rise to 54.95 ± 3.17 population doublings (PD) and an estimated number of accumulated cells of between 1.0 × 10(22) and 9.7 × 10(23), with no visible alterations in the chromosome during their lifespan. All the cultures showed unchanged percentages of strongly positive expressions of the surface markers CD29, CD44, CD73, CD90, CD95, CD105 and HLA-ABC, as well as the embryonic intracellular markers Nanog and Sox2, during their lifespan, whereas the expression of the embryonic surface markers SSEA3, SSEA4, TRA-1-60 and TRA-1-81 fell until it disappeared with progression of the culture. These cells retained their differentiation capacities to adipogenic, chondrogenic and osteogenic lineages throughout their lifespan. CONCLUSIONS: F-type hAMSC exhibit reproducible biologic characteristics, confirming that these cells are ideal candidates for use in regenerative medicine.

Preparation of monolithic polymer-magnetite nanoparticle composites into poly(ethylene-co-tetrafluoroethylene) tubes for uses in micro-bore HPLC separation and extraction of phosphorylated compounds.

This paper describes the fabrication of a novel microbore monolithic column modified with magnetite nanoparticles (MNPs) prepared in a poly(ethylene-co-tetrafluoroethylene) (EFTE) tubing, and its application as stationary phase for the chromatographic separation of phosphorylated compounds. In order to obtain the composite column, a two-step procedure was performed. The formation of a glycidyl methacrylate-based monolith inside the activated ETFE tube was firstly carried out. Then, two incorporation approaches of MNPs in monoliths were investigated. The generic polymer was modified with 3-aminopropyltrimethoxysilane (APTMS) to be subsequently attached to MNP surfaces. Alternatively, APTMS-coated MNPs were firstly prepared and subsequently used for attachment onto the monolith surface through reaction of epoxy groups present in the generic monolith. This last strategy gave a reproducible layer of MNPs coated onto the polymer monolith as well as robust and permeable chromatographic columns. The retention behaviour of this MNP-based composite monolithic column was studied by using small phosphorylated compounds (adenosine phosphates). It was found that the retention of model analytes was ruled by partitioning and adsorption HILIC mechanisms. The columns also exhibited satisfactory performance in the separation of these target compounds, showing good chromatographic behaviour after two months of continued use. These composite monolithic columns were also successfully applied to the extraction of a tryptic digest of ß-casein.

Mutational spectrum of the SPG4 (SPAST) and SPG3A (ATL1) genes in Spanish patients with hereditary spastic paraplegia.

BACKGROUND: Hereditary Spastic Paraplegias (HSP) are characterized by progressive spasticity and weakness of the lower limbs. At least 45 loci have been identified in families with autosomal dominant (AD), autosomal recessive (AR), or X-linked hereditary patterns. Mutations in the SPAST (SPG4) and ATL1 (SPG3A) genes would account for about 50% of the ADHSP cases. METHODS: We defined the SPAST and ATL1 mutational spectrum in a total of 370 unrelated HSP index cases from Spain (83% with a pure phenotype). RESULTS: We found 50 SPAST mutations (including two large deletions) in 54 patients and 7 ATL1 mutations in 11 patients. A total of 33 of the SPAST and 3 of the ATL1 were new mutations. A total of 141 (31%) were familial cases, and we found a higher frequency of mutation carriers among these compared to apparently sporadic cases (38% vs. 5%). Five of the SPAST mutations were predicted to affect the pre-mRNA splicing, and in 4 of them we demonstrated this effect at the cDNA level. In addition to large deletions, splicing, frameshifting, and missense mutations, we also found a nucleotide change in the stop codon that would result in a larger ORF. CONCLUSIONS: In a large cohort of Spanish patients with spastic paraplegia, SPAST and ATL1 mutations were found in 15% of the cases. These mutations were more frequent in familial cases (compared to sporadic), and were associated with heterogeneous clinical manifestations.

Metabolic Serendipities of Expanded Newborn Screening.

Incidental findings on newborn screening (NBS) are results that are not the target of screening within a given NBS program, but rather are found as a result of the screening and resulting diagnostic workup for that target. These findings may not have an immediate clinical impact on the newborn, but are sometimes an additional benefit of NBS programs and may be considered secondary targets of NBS programs. This work describes four case reports that had incidental findings on the NBS, which eventually led to the diagnosis of another metabolic disease instead of the one that was initially suspected. The first case was a new defect in the cationic amino acid transporter-2 (CAT-2), which was oriented as an arginase-1 deficiency in the newborn. The second case was a maternal glutaric aciduria type 1 (GA-1) that mimicked a carnitine transporter deficiency in the newborn. The third report was a case of lysinuric protein intolerance (LPI), which appeared as high levels of citrulline on the NBS. The fourth case was a mother with homocystinuria that was diagnosed during the biochemical study of vitamin B12 status. All cases provide new or interesting data that will help guide differential diagnosis in the future.

Infantile sialidosis: natural history in a preterm infant with two new pathogenic mutations and new ocular findings.

Sialidosis is a rare lysosomal storage disease caused by an α-N-acetyl neuraminidase-1 deficiency due to mutations of the NEU1 gene (6p21). Disease severity varies among patients and is linked to the level of residual neuraminidase activity in vivo. At least 40 disease-causing mutations in the NEU1 gene have been reported. Sialidosis occurs in two main clinical variants: type I, the milder form of the disease, and type II, which is subdivided into congenital, infantile, and juvenile forms. We report the clinical, biochemical, and molecular characterization of a patient with infantile sialidosis type II. The abnormal urinary oligosaccharide profile is described for the first time. The genetic characterization of the patient showed two previously unreported missense mutations in the NEU1 gene: p.R78C (c.232C>T) and p.R290Q (c.869G>A).

Initial Evaluation of Prospective and Parallel Assessments of Cystic Fibrosis Newborn Screening Protocols in Eastern Andalusia: IRT/IRT versus IRT/PAP/IRT.

Identifying newborns at risk for cystic fibrosis (CF) by newborn screening (NBS) using dried blood spot (DBS) specimens provides an opportunity for presymptomatic detection. All NBS strategies for CF begin with measuring immunoreactive trypsinogen (IRT). Pancreatitis-associated protein (PAP) has been suggested as second-tier testing. The main objective of this study was to evaluate the analytical performance of an IRT/PAP/IRT strategy versus the current IRT/IRT strategy over a two-year pilot study including 68,502 newborns. The design of the study, carried out in a prospective and parallel manner, allowed us to compare four different CF-NBS protocols after performing a post hoc analysis. The best PAP cutoff point and the potential sources of PAP false positive results in our non-CF newborn population were also studied. 14 CF newborns were detected, resulting in an overall CF prevalence of 1/4, 893 newborns. The IRT/IRT algorithm detected all CF cases, but the IRT/PAP/IRT algorithm failed to detect one case of CF. The IRT/PAP/IRT with an IRT-dependent safety net protocol was a good alternative to improve sensitivity to 100%. The IRT × PAP/IRT strategy clearly performed better, with a sensitivity of 100% and a positive predictive value (PPV) of 39%. Our calculated optimal cutoffs were 2.31 µg/L for PAP and 167.4 µg2/L2 for IRT × PAP. PAP levels were higher in females and newborns with low birth weight. PAP false positive results were found mainly in newborns with conditions such as prematurity, sepsis, and hypoxic-ischemic encephalopathy.

Effect of intraoperative paravertebral or intravenous lidocaine versus control during lung resection surgery on postoperative complications: A randomized controlled trial.

BACKGROUND: Use of minimally invasive surgical techniques for lung resection surgery (LRS), such as video-assisted thoracoscopy (VATS), has increased in recent years. However, there is little information about the best anesthetic technique in this context. This surgical approach is associated with a lower intensity of postoperative pain, and its use has been proposed in programs for enhanced recovery after surgery (ERAS). This study compares the severity of postoperative complications in patients undergoing LRS who have received lidocaine intraoperatively either intravenously or via paravertebral administration versus saline. METHODS/DESIGN: We will conduct a single-center randomized controlled trial involving 153 patients undergoing LRS through a thoracoscopic approach. The patients will be randomly assigned to one of the following study groups: intravenous lidocaine with more paravertebral thoracic (PVT) saline, PVT lidocaine with more intravenous saline, or intravenous remifentanil with more PVT saline. The primary outcome will be the comparison of the postoperative course through Clavien-Dindo classification. Furthermore, we will compare the perioperative pulmonary and systemic inflammatory response by monitoring biomarkers in the bronchoalveolar lavage fluid and blood, as well as postoperative analgesic consumption between the three groups of patients. We will use an ANOVA to compare quantitative variables and a chi-squared test to compare qualitative variables. DISCUSSION: The development of less invasive surgical techniques means that anesthesiologists must adapt their perioperative management protocols and look for anesthetic techniques that provide good analgesic quality and allow rapid rehabilitation of the patient, as proposed in the ERAS protocols. The administration of a continuous infusion of intravenous lidocaine has proven to be useful and safe for the management of other types of surgery, as demonstrated in colorectal cancer. We want to know whether the continuous administration of lidocaine by a paravertebral route can be substituted with the intravenous administration of this local anesthetic in a safe and effective way while avoiding the risks inherent in the use of regional anesthetic techniques. In this way, this technique could be used in a safe and effective way in ERAS programs for pulmonary resection. TRIAL REGISTRATION: EudraCT, 2016-004271-52; ClinicalTrials.gov, NCT03905837 . Protocol number IGGFGG-2016 version 4.0, 27th April 2017.