Social determinants of gastrointestinal malformation mortality in Brazil: a national study.

Introduction: In Brazil, approximately 5% are born with a congenital disorder, potentially fatal without surgery. This study aims to evaluate the relationship between gastrointestinal congenital malformation (GICM) mortality, health indicators, and socioeconomic factors in Brazil. Methods: GICM admissions (Q39-Q45) between 2012 and 2019 were collected using national databases. Patient demographics, socioeconomic factors, clinical management, outcomes, and the healthcare workforce density were also accounted for. Pediatric Surgical Workforce density and the number of neonatal intensive care units in a region were extracted from national datasets and combined to create a clinical index termed 'NeoSurg'. Socioeconomic variables were combined to create a socioeconomic index termed 'SocEcon'. Simple linear regression was used to investigate if the temporal changes of both indexes were significant. The correlation between mortality and the different indicators in Brazil was evaluated using Pearson's correlation coefficient. Results: Over 8 years, Brazil recorded 12804 GICM admissions. The Southeast led with 6147 cases, followed by the Northeast (2660), South (1727), North (1427), and Midwest (843). The North and Northeast reported the highest mortality, lowest NeoSurg, and SocEcon Index rates. Nevertheless, mortality rates declined across regions from 7.7% (2012) to 3.9% (2019), a 51.7% drop. The North and Midwest experienced the most substantial reductions, at 63% and 75%, respectively. Mortality significantly correlated with the indexes in nearly all regions (p<0.05). Conclusion: Our study highlights the correlation between social determinants of health and GICM mortality in Brazil, using two novel indexes in the pediatric population. These findings provide an opportunity to rethink and discuss new indicators that could enhance our understanding of our country and could lead to the development of necessary solutions to tackle existing challenges in Brazil and globally.

Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference.

The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents, and better "evolvability" of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in Thermus thermophilus through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein. To generate a monomeric folding reporter, we have increased the thermostability of the moderately thermostable Hph5 variant of the hygromycin B phosphotransferase from Escherichia coli to meet the method requirements. The final Hph17 variant showed 1.5 °C higher melting temperature (T m) and 3-fold longer half-life at 65 °C compared to parental Hph5, with no changes in the steady-state kinetic parameters. Additionally, we demonstrate the validity of the reporter by stabilizing the 2-keto-3-deoxy-l-rhamnonate aldolase from E. coli (YfaU). The most thermostable multiple-mutated variants thus obtained, YfaU99 and YfaU103, showed increases of 2 and 2.9 °C in T m compared to the wild-type enzyme but severely lower retro-aldol activities (150- and 120-fold, respectively). After segregation of the mutations, the most thermostable single variant, Q107R, showed a T m 8.9 °C higher, a 16-fold improvement in half-life at 60 °C and higher operational stability than the wild-type, without substantial modification of the kinetic parameters.

Thermostability enhancement of the Pseudomonas fluorescens esterase I by in vivo folding selection in Thermus thermophilus.

Prolonged stability is a desired property for the biotechnological application of enzymes since it allows its reutilization, contributing to making biocatalytic processes more economically competitive with respect to chemical synthesis. In this study, we have applied selection by folding interference at high temperature in Thermus thermophilus to obtain thermostable variants of the esterase I from Pseudomonas fluorescens (PFEI). The most thermostable variant (Q11L/A191S) showed a melting temperature (Tm ) of 77.3 ± 0.1°C (4.6°C higher than the wild-type) and a half-life of over 13 hr at 65°C (7.9-fold better than the wild-type), with unchanged kinetic parameters. Stabilizing mutations Q11L and A191S were incorporated into PFEI variant L30P, previously described to be enantioselective in the hydrolysis of the (-)-enantiomer of the Vince lactam. The final variant Q11L/L30P/A191S showed a significant improvement in thermal stability (Tm of 80.8 ± 0.1°C and a half-life of 65 min at 75°C), while retaining enantioselectivity (E > 100). Structural studies revealed that A191S establishes a hydrogen bond network between a V-shaped hairpin and the α/ß hydrolase domain that leads to higher rigidity and thus would contribute to explaining the increase in stability.