Cyanosis in a patient after Fontan palliation due to unrecognised hepatic vein to coronary sinus communication.

A 6-year-old male with heterotaxia, abnormal systemic and pulmonary venous drainage, and a history of Fontan completion presented with desaturations and was found by cardiac catheterisation to have a hepatic vein to coronary sinus connection. This was successfully occluded using an Amplatzer Muscular Ventricular Septal Defect Occluder.

Non-Elective Pediatric Cardiac Catheterization During COVID-19 Pandemic: A New York Center Experience.

BACKGROUND: COVID-19 has led to major changes in hospital systems across the world. In an effort to reduce viral transmission, conserve resources, and in accordance with institutional and state mandates, all elective procedures and surgeries were postponed during the initial outbreak. Guidelines for case selection are limited and management for pediatric catheterization laboratories during this crisis is unprecedented. OBJECTIVES: To report the protocols and case selection of a high-volume pediatric cardiac catheterization laboratory in the epicenter of the novel coronavirus (COVID-19) pandemic. METHODS: All pediatric cardiac catheterization procedures from March 16, 2020 through May 10, 2020 were reviewed. Changes to case selection and periprocedural workflow are described. Data were collected on COVID-19 testing status and primary procedure type, and all procedures were classified by urgency. RESULTS: There were 52 catheterizations performed on 50 patients. Endomyocardial biopsies were the most common procedure (n = 27; 52%). Interventional and diagnostic procedures represented 27% (n = 14) and 21% (n = 11) of cases, respectively. Two emergent procedures (3.8%) were performed on patients with positive COVID-19 testing. Most cases were performed on patients with negative COVID-19 testing (n = 33; 94%). CONCLUSIONS: Adjusting to the COVID-19 pandemic in a high-volume pediatric cardiac catheterization laboratory can be safely and effectively managed by prioritizing emergent and urgent cases and modifying workflow operations. The experience of this center may assist other pediatric cardiac catheterization laboratories in adapting to similar practice changes as the pandemic continues to evolve.

Differential secretion pathways of proteins fused to the Escherichia coli maltose binding protein (MBP) in Pichia pastoris.

The Escherichia coli maltose binding protein (MBP) is an N-terminal fusion partner that was shown to enhance the secretion of some heterologous proteins from the yeast Pichia pastoris, a popular host for recombinant protein expression. The amount of increase in secretion was dependent on the identity of the cargo protein, and the fusions were proteolyzed prior to secretion, limiting its use as a purification tag. In order to overcome these obstacles, we used the MBP as C-terminal partner for several cargo peptides. While the Cargo-MBP proteins were no longer proteolyzed in between these two moieties when the MBP was in this relative position, the secretion efficiency of several fusions was lower than when MBP was located at the opposite end of the cargo protein (MBP-Cargo). Furthermore, fluorescence analysis suggested that the MBP-EGFP and EGFP-MBP proteins followed different routes within the cell. The effect of several Pichia pastoris beta-galactosidase supersecretion (bgs) strains, mutants showing enhanced secretion of select reporters, was also investigated on both MBP-EGFP and EGFP-MBP. While the secretion efficiency, proteolysis and localization of the MBP-EGFP was influenced by the modified function of Bgs13, EGFP-MBP behavior was not affected in the bgs strain. Taken together, these results indicate that the location of the MBP in a fusion affects the pathway and trans-acting factors regulating secretion in P. pastoris.

The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins is initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro-region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57-70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future.

Analysis of the 5' untranslated region (5'UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris.

Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express over one thousand heterologous proteins valued for industrial, pharmaceutical and basic research purposes. In most cases, the 5' untranslated region (UTR) of the alcohol oxidase 1 (AOX1) gene is fused to the coding sequence of the recombinant gene for protein expression in this yeast. Because the effect of the AOX1 5'UTR on protein expression is not known, site-directed mutagenesis was performed in order to decrease or increase the length of this region. Both of these types of changes were shown to affect translational efficiency, not transcript stability. While increasing the length of the 5'UTR clearly decreased expression of a ß-galactosidase reporter in a proportional manner, a deletion analysis demonstrated that the AOX1 5'UTR contains a complex mixture of both positive and negative cis-acting elements, suggesting that the construction of a synthetic 5'UTR optimized for a higher level of expression may be challenging.