A Versatile Virus-Mimetic Engineering Approach for Concurrent Protein Nanocage Surface-Functionalization and Cargo Encapsulation.

Naturally occurring protein nanocages like ferritin are self-assembled from multiple subunits. Because of their unique cage-like structure and biocompatibility, there is a growing interest in their biomedical use. A multipurpose and straightforward engineering approach does not exist for using nanocages to make drug-delivery systems by encapsulating hydrophilic or hydrophobic drugs and developing vaccines by surface functionalization with a protein like an antigen. Here, a versatile engineering approach is described by mimicking the HIV-1 Gap polyprotein precursor. Various PREcursors of nanoCages (PREC) are designed and created by linking two ferritin subunits via a flexible linker peptide containing a protease cleavage site. These precursors can have additional proteins at their N-terminus, and their protease cleavage generates ferritin-like nanocages named protease-induced nanocages (PINCs). It is demonstrated that PINC formation allows concurrent surface decoration with a protein and hydrophilic or hydrophobic drug encapsulation up to fourfold more than the amount achieved using other methods. The PINCs/Drug complex is stable and efficiently kills cancer cells. This work provides insight into the precursors' design rules and the mechanism of PINCs formation. The engineering approach and mechanistic insight described here will facilitate nanocages' applications in drug delivery or as a platform for making multifunctional therapeutics like mosaic vaccines.

Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages.

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.

Antibiotic resistance monitoring in wastewater in the Nordic countries: A systematic review.

The Nordic countries (Denmark, Finland, Iceland, Norway, and Sweden) have effectively kept lower antibiotic-resistant bacterial (ARB) pathogen rates than many other countries. However, in recent years, these five countries have encountered a rise in ARB cases and challenges in treating infections due to the growing prevalence of ARB pathogens. Wastewater-based surveillance (WBS) is a valuable supplement to clinical methods for ARB surveillance, but there is a lack of comprehensive understanding of WBS application for ARB in the Nordic countries. This review aims to compile the latest state-of-the-art developments in WBS for ARB monitoring in the Nordic countries and compare them with clinical surveillance practices. After reviewing 1480 papers from the primary search, 54 were found relevant, and 15 additional WBS-related papers were included. Among 69 studies analyzed, 42 dedicated clinical epidemiology, while 27 focused on wastewater monitoring. The PRISMA review of the literature revealed that Nordic countries focus on four major WBS objectives of ARB: assessing ARB in the human population, identifying ARB evading wastewater treatment, quantifying removal rates, and evaluating potential ARB evolution during the treatment process. In both clinical and wastewater contexts, the most studied targets were pathogens producing carbapenemase and extended-spectrum beta-lactamase (ESBL), primarily Escherichia coli and Klebsiella spp. However, vancomycin-resistant Enterococcus (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) have received more attention in clinical epidemiology than in wastewater studies, probably due to their lower detection rates in wastewater. Clinical surveillance has mostly used culturing, antibiotic susceptibility testing, and genotyping, but WBS employed PCR-based and metagenomics alongside culture-based techniques. Imported cases resulting from international travel and hospitalization abroad appear to have frequently contributed to the rise in ARB pathogen cases in these countries. The many similarities between the Nordic countries (e.g., knowledge exchange practices, antibiotic usage patterns, and the current ARB landscape) could facilitate collaborative efforts in developing and implementing WBS for ARB in population-level screening.

Perceptions of exercise behavior and well-being in anomalous aortic origin of coronary arteries.

Anomalous aortic origin of a coronary artery (AAOCA) is associated with sudden death in the young. Risk stratification and management decision-making remain challenging. Data addressing post-diagnosis perceptions of exercise behavior and safety are lacking. We aimed to determine how AAOCA affects exercise behaviors, safety perceptions, and emotional well-being of patients/parents. Qualitative and quantitative analysis of AAOCA patient-/parent-specific survey was conducted to examine exercise frequency/restrictions, perceived safety of competitive/recreational exercise, and psychosocial well-being. Subgroups stratified by AAOCA subtype, surgical intervention, and physician-driven restrictions were compared using chi-squared and Fisher's exact tests. Cohen's kappa determined agreement in parent/child responses. AAOCA subtypes included 13 (24%) left AAOCA, 36 (67%) right AAOCA, and 5 (9%) other/unknown. Of 54 parents and 41 paired child responses, 22% of patients were physician-restricted from exercise. Parents imposed restrictions on competitive/recreational exercise 34%/26% of the time, respectively. Children without physician restrictions still self-restricted exercise 35% of the time. Parents reported feeling their child was unsafe exercising 61% competitively and 33% recreationally. Twenty-two percent of children reported feeling unsafe exercising, with good agreement to parental perceptions of competitive exercise safety (kappa = 0.779, p < 0.001). One-third of parents and children reported feeling sad, angry, or lonely, and about half reported feeling different. Importantly, 47% of children desired to exercise more. No difference was seen across restriction status, AAOCA subtype, or surgical management strategy. CONCLUSION: There are different perceptions of exercise behavior and safety following AAOCA evaluation, regardless of risk category or management strategy, impacting their well-being. These unmet needs should be at the forefront of care. WHAT IS KNOWN: • AAOCA is one of the leading causes of sudden cardiac death in the young. • Exercise restriction varies according to AAOCA subtype and its perceived risk of inducing myocardial ischemia. WHAT IS NEW: • There are different perceptions of exercise behavior and safety in patients and parents following a diagnosis of AAOCA, impacting their well-being. • Risk category or management strategy has no effect in patients' and parents' perception of exercise safety. • These unmet needs in this population should be at the forefront of care.

Labyrinthine cor triatriatum sinister in fetal hypoplastic left heart syndrome is associated with poor outcomes.

OBJECTIVES: A subset of hypoplastic-left-heart-syndrome (HLHS) fetuses have a complex cor-triatriatum sinister that we named "labyrinthine-cor (L-cor)". We sought to determine the prevalence of L-cor in HLHS fetuses and hypothesized that it is associated with increased mortality. METHODS: This single-center retrospective cohort study included all HLHS fetuses from January 2010-December 2020. Fetuses with other hypoplastic-left-heart variants, inadequate images, lack of follow-up and fetal atrial-septal interventions were excluded. RAS was defined as the ratio of pulmonary-vein forward-to-reverse velocity-time-integral (VTI) ≤ 5 and severe-RAS defined as VTI-ratio <3. Kaplan-Meier survival-analysis was performed for the primary outcome of transplant-free survival for 62 weeks after gestational-age of 30 weeks (∼1 year). RESULTS: Of the 156 consecutive fetuses with HLHS, 11 (7.7%) had L-cor and 8/11 (72.7%) of these had RAS. When compared to HLHS-RAS without L-cor, fetuses with HLHS-RAS and L-cor were less likely to survive to 28 days (87% vs. 62.5%, p = 0.017) and to 1 year (69.6% vs. 25%, p = 0.029). When comparing by survival analysis, fetuses with severe-RAS with L-cor had lower survival compared severe-RAS without L-cor (p = 0.020). CONCLUSION: L-cor in fetal HLHS is associated with increased mortality. Recognition of this finding is important for prognostication and atrial-septal-intervention planning.

Creating a flow restrictor in ductal stenting: a novel technique.

Ductal stenting has transformed the care of neonates with ductal-dependent critical CHD, especially in low-income countries. In small infants, a 3.5- or 4-mm stent may lead to too much pulmonary blood flow resulting in pulmonary oedema. We herein presented a novel technique to restrict ductal stent flow in a premature neonate with pulmonary atresia and intact ventricular septum following radiofrequency perforation of the pulmonary valve.

Validation of diagnostic coding for chronic obstructive pulmonary disease in an electronic health record system in Hong Kong.

INTRODUCTION: Electronic health record databases can facilitate epidemiology research regarding diseases such as chronic obstructive pulmonary disease (COPD), a common medical condition worldwide. We aimed to assess the validity of International Classification of Diseases, 9th Revision (ICD-9) code algorithms for identifying COPD in Hong Kong's territory-wide electronic health record system, the Clinical Data Analysis and Reporting System (CDARS). METHODS: Adult patients diagnosed with COPD at all public hospitals in Hong Kong and specifically at Queen Mary Hospital from 2011 to 2020 were identified using the ICD-9 code 496 (Chronic airway obstruction, not elsewhere classified) within the CDARS. Two respiratory specialists reviewed clinical records and spirometry results to confirm the presence of COPD in a randomly selected group of cases. RESULTS: During the study period, 93 971 and 2479 patients had the diagnostic code for COPD at all public hospitals in Hong Kong and specifically at Queen Mary Hospital, respectively. Two hundred cases were randomly selected from Queen Mary Hospital for validation using medical records and spirometry results. The overall positive predictive value was 81.5% (95% confidence interval=76.1%-86.9%). We also developed an algorithm to identify COPD cases in our cohort. CONCLUSION: This study represents the first validation of ICD-9 coding for COPD in the CDARS. Our findings demonstrated that the ICD-9 code 496 is a reliable indicator for identifying COPD cases, supporting the use of the CDARS database for further clinical research concerning COPD.

Beneficial and detrimental effects of non-specific binding during DNA hybridization.

DNA strands have to sample numerous states to find the alignment that maximizes Watson-Crick-Franklin base pairing. This process depends strongly on sequence, which affects the stability of the native duplex as well as the prevalence of non-native inter- and intramolecular helices. We present a theory that describes DNA hybridization as a three-stage process: diffusion, registry search, and zipping. We find that non-specific binding affects each of these stages in different ways. Mis-registered intermolecular binding in the registry search stage helps DNA strands sample different alignments and accelerates the hybridization rate. Non-native intramolecular structure affects all three stages by rendering portions of the molecule inert to intermolecular association, limiting mis-registered alignments to be sampled, and impeding the zipping process. Once in-register base pairs are formed, the stability of the native structure is important to hold the molecules together long enough for non-native contacts to break.

Reemergence of Cosmopolitan Genotype Dengue Virus Serotype 2, Southern Vietnam.

We performed phylogenetic analysis on dengue virus serotype 2 Cosmopolitan genotype in Ho Chi Minh City, Vietnam. We document virus emergence, probable routes of introduction, and timeline of events. Our findings highlight the need for continuous, systematic genomic surveillance to manage outbreaks and forecast future epidemics.

Good outcomes for arteriovenous fistula with buttonhole cannulation for chronic hemodialysis in children and adolescents.

BACKGROUND: Arteriovenous fistula (AVF) is the preferred access for chronic hemodialysis (HD) in children and adolescents, but central venous catheter use is still high. METHODS: Retrospective chart review of children and adolescents with AVF created between January 2003 and December 2015 was performed to assess primary failure (PF), maturation time, functional primary and functional cumulative patency, and potential risk factors for AVF dysfunction. RESULTS: Ninety-nine AVF were created in 79 patients (54% male; 7-24 years; 16-147 kg) by experienced surgeons. Duplex ultrasonography vein mapping was used to assist with site selection. PF occurred in 17 AVF (17%) in 14 patients. Patient age, gender, ethnicity, underlying disease, time on dialysis, and AVF site were not associated with PF or patency. Coagulation abnormality was positively associated with PF (p = 0.03). Function was achieved in 82 AVF (83%) in 77 patients (97%). Median maturation time was 83 days (range 32-271). AVF were accessed via buttonholes. Functional primary patency was 95%, 84%, and 53% at 1, 2, and 5 years. Overall 1- and 2-year functional cumulative patency was 95%, but lower for small patients 16-30 kg (88%) and those greater than 80 kg (91%). The 5-year patency rate was 80%, but significantly lower for 16-30 kg (59%) and greater than 80 kg (55%). Risk analysis showed significantly better patency for 31-45 kg and 46-80 kg groups (p < 0.01), non-obese BMI (p = 0.01), and buttonhole self-cannulation (p = 0.03). CONCLUSIONS: This study provides more information about successful AVF with buttonhole cannulation in pediatric hemodialysis patients lending additional support for AVF use in pediatrics. A higher resolution version of the Graphical abstract is available as Supplementary information.

Potential selection and maintenance of manure-originated multi-drug resistant plasmids at sub-clinical concentrations for tetracycline family antibiotics.

The goal of this study was to (a) determine the minimum selection concentrations of tetracycline family antibiotics necessary to maintain plasmids carrying tetracycline-resistant genes and (b) correlate these results to environmental hotspot concentrations reported in previous studies. This study used two plasmids (pT295A and pT413A) originating from dairy manure in a surrogate Escherichia coli host CV601. The minimum selection concentrations of antibiotics tested in nutrient-rich medium were determined as follows: 0.1 mg/L for oxytetracycline, 0.45 mg/L for chlortetracycline, and 0.13-0.25 mg/L for tetracycline. Mixing oxytetracycline and chlortetracycline had minimum selection concentration values increased 2-fold compared to those in single antibiotic tests. Minimum selection concentrations found in this study were lower than reported environmental hotspot concentrations, suggesting that tetracycline family antibiotics were likely to be the driver for the selection and maintenance of these plasmids. Relatively high plasmid loss rates (>90%) were observed when culturing a strain carrying a tetracycline-resistant plasmid in antibiotic-free nutrient-rich and nutrient-defined media. Overall, results suggested that these plasmids can be maintained at concentrations environmentally relevant in wastewater treatment plants, sewage, manure, and manured soil; however, they are unstable and easily lost in the absence of antibiotics.

Abnormal Left Ventricular Strain Correlates with Left Ventricular Dysfunction but not Aortic Pathology in Marfan Syndrome in Children.

Cardiomyopathy is a complication in adults with Marfan syndrome (MFS). Early recognition of MFS patients at high risk of cardiomyopathy could impact monitoring and treatment. Abnormal ventricular strain has been associated with impaired ventricular function among adults with MFS but remains understudied in children. We retrospectively analyzed a cohort of patients with MFS undergoing cardiac magnetic resonance imaging (CMR) performed in 2003-2018 at age < 19 years. Correlations were evaluated between initial global circumferential strain (GCS) and global longitudinal strain (GLS) and the outcomes of left ventricular ejection fraction (LVEF), aortic root z-score, and vertebral artery tortuosity index corrected for height (VTI-h), all measured from CMR, using Spearman correlation. In those with serial CMR, the ability of ventricular strain to predict development of abnormal LVEF within a 5-year period was assessed. A total of 31 subjects were included (median age at initial CMR 13.5 years, Q1Q3 10.7-16.2 years), with 48% (n = 15) having LVEF < 55%. Worse GCS and worse GLS were associated with lower LVEF (ρ = - 0.629, p < 0.001 and ρ = - 0.411, p = 0.030, respectively). A clinical cutoff of GCS = - 34% predicted LVEF < 55% with sensitivity = 80% and specificity = 50%. Neither GCS nor GLS was associated with aortic root z-score (GCS: p = 0.524; GLS: p = 0.624) nor VTI-h (GCS: p = 0.949; GLS: p = 0.593). Of those with LVEF ≥ 55%, initial GCS and GLS did not differ between those with later normal versus abnormal LVEF (GCS: p = 0.505; GLS: p = 0.232). In this cohort, abnormal LV strain was associated with abnormal LVEF, but not with aortic dilation or low LVEF within the 5 years post-CMR.

Perforated posterior mitral valve leaflet in mitral annular disjunction and infective endocarditis.

We herein presented a 17-year-old female with history of mild mitral valve prolapse who was admitted for methicillin-sensitive Staphylococcus aureus endocarditis and diagnosed with mitral annular disjunction and perforated posterior mitral valve leaflet on two-dimensional and three-dimensional echocardiography (P1-P2). A perforation in the posterior leaflet was confirmed and repaired during surgical intervention. This is a rare presentation of leaflet perforation in the area of mitral annular disjunction.

Intramural course of an anomalous left coronary artery is not always associated with a slit-like ostium.

Anomalous aortic origin of the left coronary artery from an incorrect aortic sinus has been reported as the second most common causes of sudden cardiac arrest in young athletes. Intramural course of the proximal left coronary artery is considered a high-risk morphology. It is associated with a slit-like ostium and elliptical shape of the proximal artery. In this case, all pre-operative cardiac images demonstrated a round ostium and round luminal shape of the left coronary artery, which suggested no evidence of intramural course. On intraoperative inspection, although the ostium and proximal left coronary artery appeared round, the patient had a long intramural course of the left coronary artery which our surgeons performed a successful unroofing procedure. The post-operative images showed a patent new ostium from the correct aortic sinus. These findings are very unusual and proved that we should not exclude intramural course even in the absence of a slit-like ostium and elliptical shape of the proximal left coronary artery.

Transection and reimplantation of anomalous right coronary artery from single left coronary artery in a collegiate athlete.

Coronary artery anomalies may induce myocardial ischaemia and sudden cardiac death when associated with areas of compression or stenosis. We present a case of transection and reimplantation of an anomalous interarterial right coronary artery arising from a single left main coronary artery. The 18-year-old collegiate athlete had exertional chest pain and haemodynamically significant compromise to coronary blood flow.

Evolution of surgical repair of intraseptal anomalous left coronary artery with myocardial ischaemia.

BACKGROUND: Anomalous aortic origin of the left coronary artery with intraseptal course is a rare coronary anomaly associated with an increased risk of myocardial ischaemia. The role and techniques for surgical intervention are evolving, with numerous novel surgical techniques for this challenging anatomy reported in the last 5 years. We report our single-centre experience with surgical repair of intraseptal anomalous left coronary artery in the paediatric population, including clinical presentation, evaluation, and short- to mid-term outcomes. METHODS: All patients with coronary anomalies presenting to our institution undergo standardised clinical evaluation. Five patients aged 4 to 17 years underwent surgical intervention for intraseptal anomalous aortic origin of the left coronary artery between 2012 and 2022. Surgical techniques included coronary artery bypass grafting (n = 1), direct reimplantation with limited supra-arterial myotomy via right ventriculotomy (n = 1), and transconal supra-arterial myotomy with right ventricular outflow tract patch reconstruction (n = 3). RESULTS: All patients had evidence of haemodynamically significant coronary compression, and three had evidence of inducible myocardial ischaemia pre-operatively. There were no deaths or major complications. Median follow-up was 6.1 months (range 3.1-33.4 months). Patients who underwent supra-arterial myotomy (with or without reimplantation) had improved coronary flow and perfusion based on stress imaging and catheterisation data. CONCLUSIONS: Surgical approaches to intraseptal anomalous left coronary artery with evidence of myocardial ischaemia continue to evolve, with new techniques demonstrating promising improvement in coronary perfusion. Further studies are warranted to determine long-term outcomes and refine indications for repair.

Type IV pili facilitated natural competence in Fusobacterium nucleatum.

OBJECTIVES: Many bacterial species naturally take up DNA from their surroundings and recombine it into their chromosome through homologous gene transfer (HGT) to aid in survival and gain advantageous functions. Herein we present the first characterization of Type IV pili facilitated natural competence in Fusobacterium nucleatum, which is a Gram-negative, anaerobic bacterium that participates in a range of infections and diseases including periodontitis, preterm birth, and cancer. METHODS: Here we used bioinformatics on multiple Fusobacterium species, as well as molecular genetics to characterize natural competence in strain F. nucleatum subsp. nucleatum ATCC 23726. RESULTS: We bioinformatically identified components of the Type IV conjugal pilus machinery and show this is a conserved system within the Fusobacterium genus. We next validate Type IV pili in natural competence in F. nucleatum ATCC 23726 and show that gene deletions in key components of pilus deployment (pilQ) and cytoplasmic DNA import (comEC) abolish DNA uptake and chromosomal incorporation. We next show that natural competence may require native F. nucleatum DNA methylation to bypass restriction modification systems and allow subsequent genomic homologous recombination. CONCLUSIONS: In summary, this proof of principle study provides the first characterization of natural competence in Fusobacterium nucleatum and highlights the potential to exploit this DNA import mechanism as a genetic tool to characterize virulence mechanisms of an opportunistic oral pathogen.

Enhanced Fusobacterium nucleatum Genetics Using Host DNA Methyltransferases To Bypass Restriction-Modification Systems.

Bacterial restriction-modification (R-M) systems are a first-line immune defense against foreign DNA from viruses and other bacteria. While R-M systems are critical in maintaining genome integrity, R-M nucleases unfortunately present significant barriers to targeted genetic modification. Bacteria of the genus Fusobacterium are oral, Gram-negative, anaerobic, opportunistic pathogens that are implicated in the progression and severity of multiple cancers and tissue infections, yet our understanding of their direct roles in disease have been severely hindered by their genetic recalcitrance. Here, we demonstrate a path to overcome these barriers in Fusobacterium by using native DNA methylation as a host mimicry strategy to bypass R-M system cleavage of transformed plasmid DNA. We report the identification, characterization, and successful use of Fusobacterium nucleatum type II and III DNA methyltransferase (MTase) enzymes to produce a multifold increase in gene knockout efficiency in the strain Fusobacterium nucleatum subsp. nucleatum 23726, as well as the first system for efficient gene knockouts and complementations in F. nucleatum subsp. nucleatum 25586. We show plasmid protection can be accomplished in vitro with purified enzymes, as well as in vivo in an Escherichia coli host that constitutively expresses F. nucleatum subsp. nucleatum MTase enzymes. In summary, this proof-of-concept study characterizes specific MTases that are critical for bypassing R-M systems and has enhanced our understanding of enzyme combinations that could be used to genetically modify clinical isolates of Fusobacterium that have thus far been inaccessible to molecular characterization. IMPORTANCE Fusobacterium nucleatum is an oral opportunistic pathogen associated with diseases that include cancer and preterm birth. Our understanding of how this bacterium modulates human disease has been hindered by a lack of genetic systems. Here, we show that F. nucleatum DNA methyltransferase-modified plasmid DNA overcomes the transformation barrier and has allowed the development of a genetic system in a previously inaccessible strain. We present a strategy that could potentially be expanded to enable the genetic modification of highly recalcitrant strains, thereby fostering investigational studies to uncover novel host-pathogen interactions in Fusobacterium.

Revisiting approaches to and considerations for urinalysis and urine culture reflexive testing.

Urinalysis is considered the world's oldest laboratory test. Today, many laboratories use macroscopic urinalysis as a screening tool to determine when to subject urine samples for a microscopic urinalysis and/or bacterial culture. While reflexive urine microscopy has been practiced for decades, and reflexive urine culture, more recently, evidence-based guidelines regarding optimal reflexive criteria and workflows are lacking. Standard approaches are hindered, in part, by a lack of harmonization of urinalysis and urine culture practices, heterogeneity in patient populations that are studied, and lack of provider adherence to recommended practices. This review summarizes studies that have evaluated the performance of reflexive urine microscopy and reflexive urine culture, particularly in the context of urinary tract infections. It also examines reported clinical outcomes from reflexive urinalysis interventions and their impact on antibiotic stewardship efforts. Finally, it discusses laboratory operational considerations for the implementation of reflexive algorithms.

Molecular Basis of the Mechanisms Controlling MASTL.

The human MASTL (Microtubule-associated serine/threonine kinase-like) gene encodes an essential protein in the cell cycle. MASTL is a key factor preventing early dephosphorylation of M-phase targets of Cdk1/CycB. Little is known about the mechanism of MASTL activation and regulation. MASTL contains a non-conserved insertion of 550 residues within its activation loop, splitting the kinase domain, and making it unique. Here, we show that this non-conserved middle region (NCMR) of the protein is crucial for target specificity and activity. We performed a phosphoproteomic assay with different MASTL constructs identifying key phosphorylation sites for its activation and determining whether they arise from autophosphorylation or exogenous kinases, thus generating an activation model. Hydrogen/deuterium exchange data complements this analysis revealing that the C-lobe in full-length MASTL forms a stable structure, whereas the N-lobe is dynamic and the NCMR and C-tail contain few localized regions with higher-order structure. Our results indicate that truncated versions of MASTL conserving a cryptic C-Lobe in the NCMR, display catalytic activity and different targets, thus establishing a possible link with truncated mutations observed in cancer-related databases.