The E3 ligase adapter cereblon targets the C-terminal cyclic imide degron.

The ubiquitin E3 ligase substrate adapter cereblon (CRBN) is a target of thalidomide and lenalidomide1, therapeutic agents used in the treatment of haematopoietic malignancies2-4 and as ligands for targeted protein degradation5-7. These agents are proposed to mimic a naturally occurring degron; however, the structural motif recognized by the thalidomide-binding domain of CRBN remains unknown. Here we report that C-terminal cyclic imides, post-translational modifications that arise from intramolecular cyclization of glutamine or asparagine residues, are physiological degrons on substrates for CRBN. Dipeptides bearing the C-terminal cyclic imide degron substitute for thalidomide when embedded within bifunctional chemical degraders. Addition of the degron to the C terminus of proteins induces CRBN-dependent ubiquitination and degradation in vitro and in cells. C-terminal cyclic imides form adventitiously on physiologically relevant timescales throughout the human proteome to afford a degron that is endogenously recognized and removed by CRBN. The discovery of the C-terminal cyclic imide degron defines a regulatory process that may affect the physiological function and therapeutic engagement of CRBN.

Accessing Femoral Arteries Less than 3 mm in Diameter is Associated with Increased Incidence of Loss of Pulse Following Cardiac Catheterization in Infants.

To evaluate whether avoidance of a risk factor associated with loss of pulse (LOP) following femoral artery (FA) catheterization in infants identified from previous study, was associated with decreased incidence of LOP during a prospective evaluation. Since initiation of routine ultrasound guided femoral arterial access (UGFAA) for infants undergoing catheterization in Jan 2003-Dec 2011 (Period-1), our incidence of LOP had stayed steady. Prospective evaluation between Jan 2012-Dec 2014 (Period-2), identified FA-diameter < 3 mm as risk factor for LOP. Between Jan 2015-Dec 2018 (Period-3), an initiative to avoid UGFAA for FA-diameter < 3 mm was implemented to determine whether that led to a decreased incidence of LOP. FA-diameter was measured prior to USGFAA and ratio of outer diameter of arterial sheath to luminal diameter of cannulated artery (OD/AD ratio) was calculated during Periods-2 and 3. The incidence and risk factors for LOP were assessed during the three periods. FA-access rates dropped significantly during Period-3 (56.7% vs. 93.8% and 90.4% during Periods-1 and 2, respectively, p < 0.001). Incidence of LOP in Period-3 decreased to 2.7% compared to 12.5% (Period-1) and 17.4% (Period-2) (p < 0.001). By multivariate analysis, FA size < 3 mm and an OD/AD ratio > 40% were the only significant independent predictors for LOP (OR 6.48, 95% CI 2.3-11.42, p < 0.001 and OR 4.16, 95% CI 1.79-8.65, p < 0.01, respectively). Access of femoral artery < 3 mm and OD/AD ratio > 50% are associated with increased incidence of LOP. Avoidance of these factors may help decrease complications in infants undergoing cardiac catheterizations.

Construction of Femoral Vessel Nomograms for Planning Cardiac Interventional Procedures in Children 0-4 Years Old.

The objectives of this study were to construct femoral artery (FA) and femoral vein (FV) nomograms in children aged 0-4 years and to construct probability curves for the occurrence of arterial access complications based on the size of the FA. The FV and FA are commonly accessed during cardiac catheterizations in children with congenital heart diseases (CHD). However, nomograms for vessel dimensions based on child's age or size are not available. This knowledge may be helpful for interventional planning. A prospective study was performed on 400 children (age 0-4 years) with CHD undergoing cardiac catheterizations over a 3-year period. Ultrasound evaluation of the right and left FA and FV was performed under anesthesia prior to vascular access. Regression modeling was applied to derive nomograms based on quantile polynomial regression, which yielded good fit to the data judged by R-squared. GAMLSS transformation method was used to formulate smoothed percentiles. A separate prospective evaluation of FA to determine the size below which loss of pulse (LOP) are likely to occur was performed. Nomograms for FA and FV diameter and cross-sectional area against age and body surface area and probability curves for FA LOP were constructed. It is now possible to examine ultrasound-based normal sizes of femoral vein and artery in children 0-4 years of age. Femoral vessel nomograms and LOP probability curves may help with interventional planning. Future studies with larger sample size, including children of other ages may be useful.

Utility of the Medtronic microvascular plug™ as a transcatheter implantable and explantable pulmonary artery flow restrictor in a swine model.

BACKGROUND: A surgical pulmonary artery band (PAB) is used to control excessive pulmonary blood flow for certain congenital heart diseases. Previous attempts have been made to develop a transcatheter, implantable pulmonary flow restrictor (PFR) without great success. We modified a microvascular plug (MVP) to be used as a PFR. The objectives of this study were to demonstrate feasibility of transcatheter implantation and retrieval of the modified MVP as a PFR, and compare PA growth while using the PFR versus PAB. METHODS AND RESULTS: The PFR was implanted in eight newborn piglets in bilateral branch pulmonary arteries (PAs). Immediately post-PFR implantation, the right ventricular systolic pressure increased from a median of 20-51 mmHg. Transcatheter retrieval of PFR was 100% successful at 3, 6, and 9 weeks and 50% at 12-weeks post-implant. A left PAB was placed via thoracotomy in four other newborn piglets. Debanding was performed 6-weeks later via balloon angioplasty. On follow-up, the proximal left PA diameters in the PFR and the PAB groups were similar (median 8 vs. 7.1 mm; p = 0.11); albeit the surgical band sites required repeat balloon angioplasty secondary to recurrent stenosis. By histopathology, there was grade II vessel injury in two pigs immediately post-retrieval of PFR that healed by 12 weeks. CONCLUSIONS: Transcatheter implantation and retrieval of the MVP as a PFR is feasible. PA growth is comparable to surgical PAB, which is likely to require reinterventions. The use of the MVP as a PFR in humans has to be trialed before recommending its routine use.

Ultrasound-guided femoral arterial access in pediatric cardiac catheterizations: A prospective evaluation of the prevalence, risk factors, and mechanism for acute loss of arterial pulse.

OBJECTIVES: The objectives of this study were to describe the prevalence, mechanisms, and identify risk factors for acute loss of arterial pulse (LOP) in children who had ultrasound-guided femoral arterial access (UGFAA) during cardiac catheterization. BACKGROUND: LOP is a known complication in children following femoral arterial (FA) access for cardiac catheterization. The prevalence of LOP requiring treatment ranges between 4% and 8%. METHODS: A prospective study was performed including 486 cardiac catheterizations using UGFAA in children ≤18 years over a 3 years period. Ultrasound and Doppler evaluations were performed prior to and at the end of the procedure. RESULTS: LOP was identified in 33 cases (6.8%) with 23 (4.7%) requiring treatment. For children ≤6 months, the prevalence of LOP requiring treatment was 13.6%. FA diameter <3 mm was the only significant independent predictor for LOP (OR: 8.44, 95% CI: 2.07-34.5, P < 0.001). Smaller patient size, number of access attempts, time required for access, operator experience, sheath size, and length of procedure were not found to be significant predictors. Children with LOP had a greater percentage decrease in vessel diameter (median 62% vs 18%, P < 0.001) compared to those without LOP. FA thrombus was diagnosed only in 9 patients (27% of those with LOP). CONCLUSIONS: The prevalence of LOP requiring treatment is 4.7% when UGFAA is used during pediatric cardiac catheterizations. Arterial spasm was more common than thrombus as a cause of LOP. FA diameter <3 mm was the only independent predictor for LOP in this carefully designed prospective study. © 2016 Wiley Periodicals, Inc.

Genesis and regulation of C-terminal cyclic imides from protein damage.

C-Terminal cyclic imides are post-translational modifications (PTMs) that can arise from spontaneous intramolecular cleavage of asparagine or glutamine residues resulting in a form of irreversible protein damage. These protein damage events are recognized and removed by the E3 ligase substrate adapter cereblon (CRBN), indicating that these aging-related modifications may require cellular quality control mechanisms to prevent deleterious effects. However, the factors that determine protein or peptide susceptibility to C-terminal cyclic imide formation or their effect on protein stability have not been explored in detail. Here, we characterize the primary and secondary structures of peptides and proteins that promote intrinsic formation of C-terminal cyclic imides in comparison to deamidation, a related form of protein damage. Extrinsic effects from solution properties and stressors on the cellular proteome additionally promote C-terminal cyclic imide formation on proteins like glutathione synthetase (GSS) that are susceptible to aggregation if the protein damage products are not removed by CRBN. This systematic investigation provides insight to the regions of the proteome that are prone to these unexpectedly frequent modifications, the effects of this form of protein damage on protein stability, and the biological role of CRBN.

COCCIDIOSIS IN GREEN TURTLES (CHELONIA MYDAS) IN AUSTRALIA: PATHOGENESIS, SPATIAL AND TEMPORAL DISTRIBUTION, AND CLIMATE-RELATED DETERMINANTS OF DISEASE OUTBREAKS.

An epizootic of coccidiosis in free-ranging green turtles (Chelonia mydas) occurred in Australia in 1991 and the parasites were thought to be Caryospora cheloniae. Recurring outbreaks over an increased geographic range followed. We used medical records and temporal and spatial data of turtles diagnosed with coccidiosis between 1991 and 2014 to characterize the disease and factors associated with outbreaks. Most affected animals were subadults or older. Neurologic signs with intralesional cerebral coccidia were observed. Coccidia associated with inflammation and necrosis were predominantly found in the intestine, brain, kidney, and thyroid. Cases occurred in the spring and summer. Three major outbreaks (1991, 2002, and 2014) were concentrated in Port Stephens, New South Wales (NSW) and Moreton Bay, Queensland, but cases occurred as far south as Sydney, NSW. Coccidiosis cases were more likely during, or 1 mo prior to, El Niño-like events. Molecular characterization of the 18S rRNA locus of coccidia from tissues of 10 green turtles collected in 2002 and 2004 in Port Stevens and Sydney imply that they were Schellackia-like organisms. Two genotypes were identified. The Genotype 3 sequence was most common (in eight of 10 turtles), with 98.8% similarity to the 18S sequence of Schellackia orientalis. The Genotype 4 sequence was less common (in two of 10 turtles) with 99.7% similarity to the 18S sequence of the most common genotype (Genotype 1) detected in turtles from the 2014 Moreton Bay outbreak. Our study will help with the identification and management of future outbreaks and provide tools for identification of additional disease patterns in green turtles.

A new approach to understanding structure-function relationships in cytochromes P450 by targeting terpene metabolism in the wild.

A strategy for elucidating sequence determinants of function in the class of cytochrome P450 (CYP) enzymes that catalyze the first steps of terpene metabolism in wild microbiomes is described. Wild organisms that can use camphor, terpineol, pinene and limonene were isolated from soils rich in coniferous waste. Cell free extracts and growth beers were analyzed by gas chromatography/mass spectrometry to identify primary oxidative metabolites. For one organism, Pseudomonas nitroreducens TPJM, a cytochrome P450 (CYP108B1) isolated from cell free extracts was demonstrated to catalyze the oxidation of α-terpineol in assays combining the native ferredoxin and putidaredoxin reductase, and the resulting oxidation products identified by gas chromatography/mass spectrometry. Shotgun sequencing of PnTPJM identified four candidate P450 genes, including an apparently fragmentary gene with a high degree of homology with the known enzyme CYP108 (P450terp).