Deglycosylated RBD produced in Pichia pastoris as a low-cost sera COVID-19 diagnosis tool and a vaccine candidate.

During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes.

Concordance among venous thromboembolism risk assessment models. / Concordancia entre las escalas de predicción de riesgo de tromboembolia venosa.

BACKGROUND AND OBJECTIVES: There are a lot of venous thromboembolism risk assessment models with the aim of predicting the individual risk of venous thromboembolism (VTE), although most have not been externally validated. The objective of our study was to analyse the concordance among the Padua, PRETEMED, IMPROVE and MEDENOX scores. PATIENTS AND METHODS: Observational cohort study with 602 patients admitted to Medical Services of the Hospital Universitario Santa Lucía. Concordance was calculated using the kappa index (KI) between the four risk prediction scales and the VTE occurring within 90 days of hospitalisation. RESULTS: Patients considered were those with a high risk of VTE, 78%, 56%, 69% and 59% according to Padua, PRETEMED, IMPROVE and MEDENOX scores respectively. A KI=0.60 was observed among Padua-IMPROVE scores; KI=0.44 among IMPROVE-PRETEMED, KI=0.43 among PRETEMED-MEDENOX, KI=0.33 among Padua-PRETEMED, KI=0.27 among IMPROVE-MEDENOX and a KI=0.24 among Padua-MEDENOX. During follow-up, nine cases of VTE (1.5%) were observed. CONCLUSIONS: The highest degree of agreement was observed among the Padua and IMPROVE scores. There is no good agreement between any of the predictive risk scales for VTE in medical patients. The Padua score was the best predictor of VTE episodes at 90 days among high risk patients.

Trends in hospital admissions for pulmonary embolism in Spain from 2002 to 2011.

The aim of our study was to analyse changes in the incidence, diagnostic procedures, comorbidity, length of hospital stay, costs and in-hospital mortality of patients hospitalised for pulmonary embolism in Spain over a 10-year period. We included all patients who were hospitalised for pulmonary embolism (ICD-9-CM codes 415.11 and 415.19) as the primary diagnosis between 2002 and 2011. Data were collected from the National Hospital Discharge Database, covering the entire Spanish population. 115 671 patients were admitted. The overall crude incidence increased from 20.44 per 100 000 inhabitants in 2002 to 32.69 in 2011 (p<0.05). In 2002, 13.3% of patients had a Charlson comorbidity index>2, and in 2011 the prevalence increased to 20.8% (p<0.05). Mean length of hospital stay was 12.7 days in 2002 and decreased to 9.99 in 2011 (p<0.05). During the study period, mean cost per patient increased from €3915 to €4372 (p<0.05). In-hospital mortality decreased from 12.9% in 2002 to 8.32% in 2011 (p<0.05). The increase in the use of computed tomographic pulmonary angiography over time was associated with increased incidence and lower mortality. Our results revealed an increase in the incidence of hospitalised pulmonary embolism patients from 2002 to 2011 with concomitant increase in comorbidities and cost. However, length of hospital stay and in-hospital mortality decreased.

Factors associated with elevated pulmonary arterial pressure levels on the echocardiographic assessment in patients with prior pulmonary embolism.

BACKGROUND: Factors associated with the detection of raised systolic pulmonary artery pressure (sPAP) levels in patients with a prior episode of pulmonary embolism (PE) are not well known. METHODS: We used the RIETE Registry database to identify factors associated with the finding of sPAP levels ≥50 mm Hg on trans-thoracic echocardiography, in 557 patients with a prior episode of acute, symptomatic PE. RESULTS: Sixty-two patients (11.1%; 95% CI: 8.72-14.1) had sPAP levels ≥50 mm Hg. These patients were more likely women, older, and more likely had chronic lung disease, heart failure, renal insufficiency or leg varicosities than those with PAP levels <50mm Hg. During the index PE event, they more likely had recent immobility, and more likely presented with hypoxemia, increased sPAP levels, atrial fibrillation, or right bundle branch block. On multivariate analysis, women aged ≥70 years (hazard ratio [HR]: 2.0; 95% CI: 1.0-3.7), chronic heart or chronic lung disease (HR: 2.4; 95% CI: 1.3-4.4), atrial fibrillation at PE presentation (HR: 2.8; 95% CI: 1.3-6.1) or varicose veins (HR: 1.8; 95% CI: 1.0-3.3) were all associated with an increased risk to have raised sPAP levels. Chronic heart disease, varicose veins, and atrial fibrillation were independent predictors in women, while chronic heart disease, atrial fibrillation, a right bundle branch block or an S1Q3T3 pattern on the electrocardiogram were independent predictors in men. CONCLUSIONS: Women aged ≥70 years more likely had raised sPAP levels than men after a PE episode. Additional variables influencing this risk seem to differ according to gender.

Chronological assessment of mast cell-mediated gut dysfunction and mucosal inflammation in a rat model of chronic psychosocial stress.

Life stress and mucosal inflammation may influence symptom onset and severity in certain gastrointestinal disorders, particularly irritable bowel syndrome (IBS), in connection with dysregulated intestinal barrier. However, the mechanism responsible remains unknown. Crowding is a validated animal model reproducing naturalistic psychosocial stress, whose consequences on gut physiology remain unexplored. Our aims were to prove that crowding stress induces mucosal inflammation and intestinal dysfunction, to characterize dynamics in time, and to evaluate the implication of stress-induced mast cell activation on intestinal dysfunction. Wistar-Kyoto rats were submitted to 15 days of crowding stress (8 rats/cage) or sham-crowding (2 rats/cage). We measured spontaneous and corticotropin-releasing factor-mediated release of plasma corticosterone. Stress-induced intestinal chrono-pathobiology was determined by measuring intestinal inflammation, epithelial damage, mast cell activation and infiltration, and intestinal barrier function. Corticosterone release was higher in crowded rats throughout day 15. Stress-induced mild inflammation, manifested earlier in the ileum and the colon than in the jejunum. While mast cell counts remained mostly unchanged, piecemeal degranulation increased along time, as the mucosal content and luminal release of rat mast cell protease-II. Stress-induced mitochondrial injury and increased jejunal permeability, both events strongly correlated with mast cell activation at day 15. Taken together, we have provided evidences that long-term exposure to psychosocial stress promotes mucosal inflammation and mast cell-mediated barrier dysfunction in the rat bowel. The notable resemblance of these findings with those in some IBS patients, support the potential interest and translational validity of this experimental model for the research of stress-sensitive intestinal disorders, particularly IBS.

Structural selection of a native fold by peptide recognition. Insights into the thioredoxin folding mechanism.

Thioredoxins (TRXs) are monomeric alpha/beta proteins with a fold characterized by a central twisted beta-sheet surrounded by alpha-helical elements. The interaction of the C-terminal alpha-helix 5 of TRX against the remainder of the protein involves a close packing of hydrophobic surfaces, offering the opportunity of studying a fine-tuned molecular recognition phenomenon with long-range consequences on the acquisition of tertiary structure. In this work, we focus on the significance of interactions involving residues L94, L99, E101, F102, L103 and L107 on the formation of the noncovalent complex between reduced TRX1-93 and TRX94-108. The conformational status of the system was assessed experimentally by circular dichroism, intrinsic fluorescence emission and enzymic activity; and theoretically by molecular dynamics simulations (MDS). Alterations in tertiary structure of the complexes, resulting as a consequence of site specific mutation, were also examined. To distinguish the effect of alanine scanning mutagenesis on secondary structure stability, the intrinsic helix-forming ability of the mutant peptides was monitored experimentally by far-UV CD spectroscopy upon the addition of 2,2,2-trifluoroethanol, and also theoretically by Monte Carlo conformational search and MDS. This evidence suggests a key role of residues L99, F102 and L103 on the stabilization of the secondary structure of alpha-helix 5, and on the acquisition of tertiary structure upon complex formation. We hypothesize that the transition between a partially folded and a native-like conformation of reduced TRX1-93 would fundamentally depend on the consolidation of a cooperative tertiary unit based on the interaction between alpha-helix 3 and alpha-helix 5.

A closer look at mucosal inflammation in irritable bowel syndrome: sex- and gender-related disparities--quantity, quality, or both?

Irritable bowel syndrome remains a bothersome and frustrating disorder that imposes a heavy and growing socio-economic toll on its sufferers, two-thirds of whom are women, and on health care systems. The biomedical community must take a giant step forward into the discipline of women's gastrointestinal health. Efforts and accomplishments, such as the one reported in this month's issue by Cremon et al., are certainly welcome.

[Stress-mast cell axis and regulation of gut mucosal inflammation: from intestinal health to an irritable bowel]. / Eje estrés-mastocito y regulación de la inflamación en la mucosa intestinal: desde la salud intestinal hasta el intestino irritable.

The functional gastrointestinal disorders and the irritable bowel syndrome, in particular, represent one of the commonest causes of medical consultation and the most frequent diagnosis raised by the gastroenterologists. Despite their high prevalence, the aetiology and pathophysiology of these functional digestive disorders remains unclear and specific diagnostic markers and clearly effective therapeutic options are lacking as well. These factors generate an important impairment in the quality of life in these patients and a growing sanitary burden. Recent studies showing the presence of low grade intestinal mucosal inflammation along with mast cell hyperplasia may contribute to the development and perpetuation of visceral hypersensitivity and dismotility patterns and epithelial barrier abnormalities, characteristic of the irritable bowel syndrome. In this article we will review the role of the stress-mast cell axis in the modulation of the gut mucosal inflammation and in the pathophysiology of the irritable bowel syndrome.