Assaying Lysosomal Enzyme Activity in Dictyostelium discoideum.

Lysosomes are membrane-enclosed organelles that digest intracellular material. They contain more than 50 different enzymes that can degrade a variety of macromolecules including nucleic acids, proteins, polysaccharides, and lipids. In addition to functioning within lysosomes, lysosomal enzymes are also secreted. Alterations in the levels and activities of lysosomal enzymes dysregulates lysosomes, which can lead to the intralysosomal accumulation of biological material and the development of lysosomal storage diseases (LSDs) in humans. Dictyostelium discoideum has a long history of being used to study the trafficking and functions of lysosomal enzymes. More recently, it has been used as a model system to study several LSDs. In this chapter, we outline the methods for assessing the activity of several lysosomal enzymes in D. discoideum (α-galactosidase, ß-galactosidase, α-glucosidase, ß-glucosidase, ß-N-acetylglucosaminidase, α-mannosidase, cathepsin B, cathepsin D, cathepsin F, palmitoyl protein thioesterase 1, and tripeptidyl peptidase 1).

Mechanisms regulating the intracellular trafficking and release of CLN5 and CTSD.

Ceroid lipofuscinosis neuronal 5 (CLN5) and cathepsin D (CTSD) are soluble lysosomal enzymes that also localize extracellularly. In humans, homozygous mutations in CLN5 and CTSD cause CLN5 disease and CLN10 disease, respectively, which are two subtypes of neuronal ceroid lipofuscinosis (commonly known as Batten disease). The mechanisms regulating the intracellular trafficking of CLN5 and CTSD and their release from cells are not well understood. Here, we used the social amoeba Dictyostelium discoideum as a model system to examine the pathways and cellular components that regulate the intracellular trafficking and release of the D. discoideum homologs of human CLN5 (Cln5) and CTSD (CtsD). We show that both Cln5 and CtsD contain signal peptides for secretion that facilitate their release from cells. Like Cln5, extracellular CtsD is glycosylated. In addition, Cln5 release is regulated by the amount of extracellular CtsD. Autophagy induction promotes the release of Cln5, and to a lesser extent CtsD. Release of Cln5 requires the autophagy proteins Atg1, Atg5, and Atg9, as well as autophagosomal-lysosomal fusion. Atg1 and Atg5 are required for the release of CtsD. Together, these data support a model where Cln5 and CtsD are actively released from cells via their signal peptides for secretion and pathways linked to autophagy. The release of Cln5 and CtsD from cells also requires microfilaments and the D. discoideum homologs of human AP-3 complex mu subunit, the lysosomal-trafficking regulator LYST, mucopilin-1, and the Wiskott-Aldrich syndrome-associated protein WASH, which all regulate lysosomal exocytosis in this model organism. These findings suggest that lysosomal exocytosis also facilitates the release of Cln5 and CtsD from cells. In addition, we report the roles of ABC transporters, microtubules, osmotic stress, and the putative D. discoideum homologs of human sortilin and cation-independent mannose-6-phosphate receptor in regulating the intracellular/extracellular distribution of Cln5 and CtsD. In total, this study identifies the cellular mechanisms regulating the release of Cln5 and CtsD from D. discoideum cells and provides insight into how altered trafficking of CLN5 and CTSD causes disease in humans.

Loss of mfsd8 alters the secretome during Dictyostelium aggregation.

Major facilitator superfamily domain-containing protein 8 (MFSD8) is a transmembrane protein that has been reported to function as a lysosomal chloride channel. In humans, homozygous mutations in MFSD8 cause a late-infantile form of neuronal ceroid lipofuscinosis (NCL) called CLN7 disease. In the social amoeba Dictyostelium discoideum, Mfsd8 localizes to cytoplasmic puncta and vesicles, and regulates conserved processes during the organism's life cycle. Here, we used D. discoideum to examine the effect of mfsd8-deficiency on the secretome during the early stages of multicellular development. Mass spectrometry revealed 61 proteins that were differentially released by cells after 4 and 8 h of starvation. Most proteins were present in increased amounts in mfsd8- conditioned buffer compared to WT indicating that loss of mfsd8 deregulates protein secretion and/or causes the release of proteins not normally secreted by WT cells. GO term enrichment analyses showed that many of the proteins aberrantly released by mfsd8- cells localize to compartments and regions of the cell associated with the endo-lysosomal and secretory pathways. Mass spectrometry also revealed proteins previously known to be impacted by the loss of mfsd8 (e.g., cathepsin D), as well as proteins that may underlie mfsd8-deficiency phenotypes during aggregation. Finally, we show that mfsd8-deficiency reduces intracellular proteasome 20S activity due to the abnormal release of at least one proteasomal subunit. Together, this study reveals the impact of mfsd8 loss on the secretome during D. discoideum aggregation and lays the foundation for follow up work that investigates the role of altered protein release in CLN7 disease.

Cardiovascular Outcomes of Coronary Computed Tomography Angiography Versus Functional Testing in Suspected Coronary Syndromes: Real-World Evidence From the Nationwide Cohort.

Background Real-world evidence for the selection of gatekeeping studies in patients with suspected coronary syndromes is limited. Methods and Results We identified 27 036 patients who underwent coronary computed tomography angiography (CCTA), single-photon emission computed tomography, and the treadmill test for suspected coronary syndromes from the Korean National Health Insurance Service-National Sample Cohort between 2006 and 2014. The primary end point was a composite of cardiac death and myocardial infarction, and the secondary end point was a composite of the primary end point and revascularization. During a median follow-up of 5.4 years, the risk of both primary and secondary end points was significantly higher in the single-photon emission computed tomography group (hazard ratio [HR], 1.81 [95% CI, 1.34-2.45]; and HR, 1.42 [95% CI, 1.22-1.66]), but significantly lower in the treadmill test group (HR, 0.53 [95% CI, 0.42-0.67]; and HR, 0.69 [95% CI, 0.62-0.76]) compared with the CCTA group. After balancing baseline risk factors, there was no significant difference in the primary end point in those with single-photon emission computed tomography (HR, 1.11 [95% CI, 0.78-1.57]; P=0.58) or treadmill test (HR, 0.84 [95% CI, 0.65-1.08]; P=0.18) groups, compared with the CCTA group. The event rate of the secondary end point was significantly lower in the treadmill test group than in the CCTA group (HR, 0.87 [95% CI, 0.78-0.96]; P=0.008). Conclusions Compared with functional testing, initial CCTA was not associated with a lower rate of cardiac death or myocardial infarction when used as an initial diagnostic test for patients with suspected coronary syndromes.

Reclassification of moderate aortic stenosis based on data-driven phenotyping of hemodynamic progression.

The management and follow-up of moderate aortic stenosis (AS) lacks consensus as the progression patterns are not well understood. This study aimed to identify the hemodynamic progression of AS, and associated risk factors and outcomes. We included patients with moderate AS with at least three transthoracic echocardiography (TTE) studies performed between 2010 and 2021. Latent class trajectory modeling was used to classify AS groups with distinctive hemodynamic trajectories, which were determined by serial systolic mean pressure gradient (MPG) measurements. Outcomes were defined as all-cause mortality and aortic valve replacement (AVR). A total of 686 patients with 3093 TTE studies were included in the analysis. Latent class model identified two distinct AS trajectory groups based on their MPG: a slow progression group (44.6%) and a rapid progression group (55.4%). Initial MPG was significantly higher in the rapid progression group (28.2 ± 5.6 mmHg vs. 22.9 ± 2.8 mmHg, P < 0.001). The prevalence of atrial fibrillation was higher in the slow progression group; there was no significant between-group difference in the prevalence of other comorbidities. The rapid progression group had a significantly higher AVR rate (HR 3.4 [2.4-4.8], P < 0.001); there was no between-group difference in mortality (HR 0.7 [0.5-1.0]; P = 0.079). Leveraging longitudinal echocardiographic data, we identified two distinct groups of patients with moderate AS: slow and rapid progression. A higher initial MPG (≥ 24 mmHg) was associated with more rapid progression of AS and higher rates of AVR, thus indicating the predictive value of MPG in management of the disease.

Robotic Resection of Pulmonary Sequestration With Fluorescence Image Guidance.

We present the case of a 21-year-old woman with intralobar pulmonary sequestration in the right lower lobe. Minimally invasive robotic resection of the sequestration was performed to limit the loss of normal-functioning lung. This case also demonstrates the use of indocyanine green fluorescence guidance for demarcation of the sequestration.

An altered transcriptome underlies cln5-deficiency phenotypes in Dictyostelium discoideum.

Mutations in CLN5 cause a subtype of neuronal ceroid lipofuscinosis (NCL) called CLN5 disease. The NCLs, commonly referred to as Batten disease, are a family of neurodegenerative lysosomal storage diseases that affect all ages and ethnicities globally. Previous research showed that CLN5 participates in a variety of cellular processes. However, the precise function of CLN5 in the cell and the pathway(s) regulating its function are not well understood. In the model organism Dictyostelium discoideum, loss of the CLN5 homolog, cln5, impacts various cellular and developmental processes including cell proliferation, cytokinesis, aggregation, cell adhesion, and terminal differentiation. In this study, we used comparative transcriptomics to identify differentially expressed genes underlying cln5-deficiency phenotypes during growth and the early stages of multicellular development. During growth, genes associated with protein ubiquitination/deubiquitination, cell cycle progression, and proteasomal degradation were affected, while genes linked to protein and carbohydrate catabolism were affected during early development. We followed up this analysis by showing that loss of cln5 alters the intracellular and extracellular amounts of proliferation repressors during growth and increases the extracellular amount of conditioned medium factor, which regulates cAMP signalling during the early stages of development. Additionally, cln5 - cells displayed increased intracellular and extracellular amounts of discoidin, which is involved in cell-substrate adhesion and migration. Previous work in mammalian models reported altered lysosomal enzyme activity due to mutation or loss of CLN5. Here, we detected altered intracellular activities of various carbohydrate enzymes and cathepsins during cln5 - growth and starvation. Notably, cln5 - cells displayed reduced ß-hexosaminidase activity, which aligns with previous work showing that D. discoideum Cln5 and human CLN5 can cleave the substrate acted upon by ß-hexosaminidase. Finally, consistent with the differential expression of genes associated with proteasomal degradation in cln5 - cells, we also observed elevated amounts of a proteasome subunit and reduced proteasome 20S activity during cln5 - growth and starvation. Overall, this study reveals the impact of cln5-deficiency on gene expression in D. discoideum, provides insight on the genes and proteins that play a role in regulating Cln5-dependent processes, and sheds light on the molecular mechanisms underlying CLN5 disease.

Validation of deep learning-based fully automated coronary artery calcium scoring using non-ECG-gated chest CT in patients with cancer.

This study aimed to demonstrate clinical feasibility of deep learning (DL)-based fully automated coronary artery calcium (CAC) scoring software using non-electrocardiogram (ECG)-gated chest computed tomography (CT) from patients with cancer. Overall, 913 patients with colorectal or gastric cancer who underwent non-contrast-enhanced chest CT between 2013 and 2015 were included. Agatston scores obtained by manual segmentation of CAC on chest CT were used as reference. Reliability of automated CAC score acquisition was evaluated using intraclass correlation coefficients (ICCs). The agreement for cardiovascular disease (CVD) risk stratification was assessed with linearly weighted k statistics. ICCs between the manual and automated CAC scores were 0.992 (95% CI, 0.991 and 0.993, p<0.001) for total Agatston scores, 0.863 (95% CI, 0.844 and 0.880, p<0.001) for the left main, 0.964 (95% CI, 0.959 and 0.968, p<0.001) for the left anterior descending, 0.962 (95% CI, 0.956 and 0.966, p<0.001) for the left circumflex, and 0.980 (95% CI, 0.978 and 0.983, p<0.001) for the right coronary arteries. The agreement for cardiovascular risk was excellent (k=0.946, p<0.001). Current DL-based automated CAC software showed excellent reliability for Agatston score and CVD risk stratification using non-ECG gated CT scans and might allow the identification of high-risk cancer patients for CVD.

Nationwide Trends of Gatekeeper to Invasive Coronary Angiography in Suspected Coronary Artery Disease.

BACKGROUND AND OBJECTIVES: Real-world trends in the utility and type of gatekeeping studies in invasive coronary angiography (ICA) requires further investigation. METHODS: We identified outpatients who underwent noninvasive cardiac tests or directly ICA for suspected coronary artery disease (CAD) from the nationwide Korea Health Insurance Review and Assessment Service-National Patient Sample database between 2012 and 2018. RESULTS: Among 71,401 patients, the percentage of patients who were evaluated for suspected CAD was 34.7% for treadmill test (TMT), 4.2% for single-photon emission computed tomography (SPECT), 24.2% for coronary computed tomography angiography (CCTA), 1.6% for multiple gatekeepers, and 32.3% for directly ICA without noninvasive studies. The proportion of CCTA as a gatekeeper showed linear increase, (18.6% in 2012 and 28.8% in 2018; p<0.001), while those of TMT, SPECT, and direct ICA have decreased (p<0.001, p=0.03, and p<0.001, respectively). The overall incidence of downstream ICA after gatekeeper was 13.8% (6,662/48,346), and SPECT showed higher ICA rate in pairwise comparison with TMT and CCTA (p<0.001). Patients who performed gatekeepers before ICA showed higher rate of subsequent PCI (34.7% vs. 32.3%; p<0.001) and CABG (3.5% vs. 1.0%; p<0.001), compared to those who directly underwent ICA, and CCTA was associated with higher revascularization rate after ICA in pairwise comparison with TMT and SPECT (p<0.001). CONCLUSIONS: Nationwide database demonstrated that CCTA is utilized increasingly as a gatekeeper for ICA and is associated with high revascularization rate after ICA in outpatients with suspected CAD.

Promoting cultural competency and osteopathic medicine awareness among premedical students through a summer premedical rural enrichment program.

CONTEXT: Premedical preparatory programs at osteopathic medical schools that recruit students from medically underserved areas (MUAs) may promote interest in practicing osteopathic medicine in underserved or rural areas. In these programs, emphasis on cultural competency may increase diversity among medical school applicants and decrease healthcare disparities in the future. OBJECTIVES: The goal of this study is to determine whether a summer premedical rural enrichment program (PREP) held at an osteopathic medical school located in a MUA will foster greater prioritization of cultural competency in medicine, enhance interest in practicing in rural or underserved areas, and increase familiarity with osteopathic medicine. METHODS: An eight-week summer PREP was hosted at the California Health Sciences University College of Osteopathic Medicine (CHSU-COM) in Clovis, California. Seventy-eight diverse participants were recruited from the Central Valley, an underserved region of California. Attendees were required to finish the formal application process and were recommended to have completed medical school prerequisite courses. The curriculum included Medical College Admission Test (MCAT) preparation through team-based learning sessions, introduction to the osteopathic medical school curriculum, osteopathic philosophy, and osteopathic manipulative medicine, as well as integrated anatomy and physiology sessions, medical school application workshops, mock interviews, simulation workshops, and sociology and cultural competency sessions. Data were collected via a voluntary and anonymous survey administered before and after the program with questions about familiarity with osteopathy, interest in practicing in underserved areas, medical school preparedness, and a post-course survey about cultural competency. The surveys had students rate statements on a Likert scale. RESULTS: Seventy-four of the 78 premedical students (95%) completed the pre-and postsurvey. There was a significant increase in agreement to statements evaluating medical school preparedness, osteopathic familiarity, and desire to practice medicine locally in the postprogram survey, compared to the preprogram survey. In the cultural competency postsurvey, 75.0% of the responses to questions that evaluated the positive effect of the course were "Agree" or "Strongly Agree." Of the reported postcourse outcomes, the average MCAT score was 504 ± 6.2 (38 students reported, 50.7%). Of the 27 participants who reported matriculation, 16 (59.2%) were admitted to osteopathic medical schools, 9 (33.3%) to allopathic medical schools, and 2 to other health programs. CONCLUSIONS: After completing the PREP program, premedical participants reported that they have better understanding of cultural competency and improvement in preparation for medical school, including familiarity with osteopathic medicine, and interest in serving MUAs. These findings indicate that similar programs may have a positive impact on MUAs. These programs may help create diverse and culturally competent osteopathic physicians.

Mfsd8 Modulates Growth and the Early Stages of Multicellular Development in Dictyostelium discoideum.

MFSD8 is a transmembrane protein that has been reported to transport chloride ions across the lysosomal membrane. Mutations in MFSD8 are associated with a subtype of Batten disease called CLN7 disease. Batten disease encompasses a family of 13 inherited neurodegenerative lysosomal storage diseases collectively referred to as the neuronal ceroid lipofuscinoses (NCLs). Previous work identified an ortholog of human MFSD8 in the social amoeba D. discoideum (gene: mfsd8, protein: Mfsd8), reported its localization to endocytic compartments, and demonstrated its involvement in protein secretion. In this study, we further characterized the effects of mfsd8 loss during D. discoideum growth and early stages of multicellular development. During growth, mfsd8 - cells displayed increased rates of proliferation, pinocytosis, and expansion on bacterial lawns. Loss of mfsd8 also increased cell size, inhibited cytokinesis, affected the intracellular and extracellular levels of the quorum-sensing protein autocrine proliferation repressor A, and altered lysosomal enzyme activity. During the early stages of development, loss of mfsd8 delayed aggregation, which we determined was at least partly due to impaired cell-substrate adhesion, defects in protein secretion, and alterations in lysosomal enzyme activity. Overall, these results show that Mfsd8 plays an important role in modulating a variety of processes during the growth and early development of D. discoideum.

Cancer Therapy-Related Cardiac Dysfunction in Patients Treated with a Combination of an Immune Checkpoint Inhibitor and Doxorubicin.

Backgrounds: There are scarce data on whether immune checkpoint inhibitors (ICIs) increase the risk of cardiac dysfunction when used with cardiotoxic agents. Thus, we evaluated cardiac dysfunction in patients with sarcoma receiving doxorubicin with or without ICI using echocardiography and left ventricular global longitudinal strain (LVGLS). Methods: A total of 95 patients were included in this study. Echocardiography and LVGLS were evaluated at baseline and follow-up (at 3 and 6 months of chemotherapy) and compared with the doxorubicin (Dox; n = 73) and concomitant ICI with doxorubicin (Dox-ICI; n = 22) groups. Cancer therapy-related cardiac dysfunction (CTRCD) was defined as a left ventricular ejection fraction (LVEF) drop of >10% and LVEF of <50% (definite CTRCD), LVEF drop of >10%, LVEF of ≥50%, and LVGLS relative reduction of >15% (probable CTRCD) at six months. Results: There were no significant differences in age, cumulative dose of doxorubicin, and cardiovascular risk factors between the two groups. At baseline, the LVEF was similar in the Dox and Dox-ICI groups (p = 0.493). In the Dox group, LVEF decreased to 59 ± 6% (Δ −7 ± 1.3%, p < 0.001) and LVGLS decreased from −17.3 ± 3.2% to −15.4 ± 3.2% (Δ −10.1 ± −1.9%, p < 0.001) at six months. In the Dox-ICI group, LVEF decreased to 55 ± 9% (Δ −9 ± 2.1%, p < 0.001), along with a significant decrease in LVGLS (from −18.6 ± 1.9% to −15.3 ± 3.6%, Δ −12.4 ± −2.4%, p < 0.001). Over a median follow-up of 192 days, there were no cases with clinical manifestations of fulminant myocarditis. In the Dox group, definite and probable CTRCD were observed in seven (10.1%) and five (7.4%) patients, respectively. In the Dox-ICI group, definite and probable CTRCD were observed in four (19%) and four (19%) patients, respectively. The total number of patients who developed CTRCD was significantly higher in the Dox-ICI group than in the Dox group (38.1% vs. 17.4%, p = 0.042). Serum troponin-T level was significantly higher in the Dox-ICI group than in the Dox group (53.3 vs. 27.5 pg/mL, p = 0.023). Conclusions: ICIs may increase the risk of CTRCD when used with cardiotoxic agents. CTRCD should be monitored in patients treated with ICIs by cardiac biomarkers and echocardiography, including LV-GLS.

The Cellular and Developmental Roles of Cullins, Neddylation, and the COP9 Signalosome in Dictyostelium discoideum.

Cullins (CULs) are a core component of cullin-RING E3 ubiquitin ligases (CRLs), which regulate the degradation, function, and subcellular trafficking of proteins. CULs are post-translationally regulated through neddylation, a process that conjugates the ubiquitin-like modifier protein neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) to target cullins, as well as non-cullin proteins. Counteracting neddylation is the deneddylase, COP9 signalosome (CSN), which removes NEDD8 from target proteins. Recent comparative genomics studies revealed that CRLs and the CSN are highly conserved in Amoebozoa. A well-studied representative of Amoebozoa, the social amoeba Dictyostelium discoideum, has been used for close to 100 years as a model organism for studying conserved cellular and developmental processes owing to its unique life cycle comprised of unicellular and multicellular phases. The organism is also recognized as an exceptional model system for studying cellular processes impacted by human diseases, including but not limited to, cancer and neurodegeneration. Recent work shows that the neddylation inhibitor, MLN4924 (Pevonedistat), inhibits growth and multicellular development in D. discoideum, which supports previous work that revealed the cullin interactome in D. discoideum and the roles of cullins and the CSN in regulating cellular and developmental processes during the D. discoideum life cycle. Here, we review the roles of cullins, neddylation, and the CSN in D. discoideum to guide future work on using this biomedical model system to further explore the evolutionarily conserved functions of cullins and neddylation.

Autophagy in the Neuronal Ceroid Lipofuscinoses (Batten Disease).

The neuronal ceroid lipofuscinoses (NCLs), also referred to as Batten disease, are a family of neurodegenerative diseases that affect all age groups and ethnicities around the globe. At least a dozen NCL subtypes have been identified that are each linked to a mutation in a distinct ceroid lipofuscinosis neuronal (CLN) gene. Mutations in CLN genes cause the accumulation of autofluorescent lipoprotein aggregates, called ceroid lipofuscin, in neurons and other cell types outside the central nervous system. The mechanisms regulating the accumulation of this material are not entirely known. The CLN genes encode cytosolic, lysosomal, and integral membrane proteins that are associated with a variety of cellular processes, and accumulated evidence suggests they participate in shared or convergent biological pathways. Research across a variety of non-mammalian and mammalian model systems clearly supports an effect of CLN gene mutations on autophagy, suggesting that autophagy plays an essential role in the development and progression of the NCLs. In this review, we summarize research linking the autophagy pathway to the NCLs to guide future work that further elucidates the contribution of altered autophagy to NCL pathology.

Improving Left Atrial Appendage Occlusion Device Size Determination by Three-Dimensional Printing-Based Preprocedural Simulation.

BACKGROUND: The two-dimensional (2D)-based left atrial appendage (LAA) occluder (LAAO) size determination by using transesophageal echocardiography (TEE) is limited by the structural complexity and wide anatomical variation of the LAA. OBJECTIVE: This study aimed to assess the accuracy of the LAAO size determination by implantation simulation by using a three-dimensional (3D)-printed model compared with the conventional method based on TEE. METHODS: We retrospectively reviewed patients with anatomically and physiologically properly implanted the Amplatzer Cardiac Plug and Amulet LAAO devices between January 2014 and December 2018 by using the final size of the implanted devices as a standard for size prediction accuracy. The use of 3D-printed model simulations in device sizing was compared with the conventional TEE-based method. RESULTS: A total of 28 cases with the percutaneous LAA occlusion were reviewed. There was a minimal difference [-0.11 mm; 95% CI (-0.93, 0.72 mm); P = 0.359] between CT-based reconstructed 3D images and 3D-printed left atrium (LA) models. Device size prediction based on TEE measurements showed poor agreement (32.1%), with a mean difference of 2.3 ± 3.2 mm [95% CI (-4.4, 9.0)]. The LAAO sizing by implantation simulation with 3D-printed models showed excellent correlation with the actually implanted LAAO size (r = 0.927; bias = 0.7 ± 2.5). The agreement between the 3D-printed and the implanted size was 67.9%, with a mean difference of 0.6 mm [95% CI (-1.9, 3.2)]. CONCLUSION: The use of 3D-printed LA models in the LAAO size determination showed improvement in comparison with conventional 2D TEE method.

Pre-procedural determination of device size in left atrial appendage occlusion using three-dimensional cardiac computed tomography.

The complex structure of the left atrial appendage (LAA) brings limitations to the two-dimensional-based LAA occlusion (LAAO) size prediction system using transesophageal echocardiography. The LAA anatomy can be evaluated more precisely using three-dimensional images from cardiac computed tomography (CT); however, there is lack of data regarding which parameter to choose from CT-based images during pre-procedural planning of LAAO. We aimed to assess the accuracy of measurements derived from cardiac CT images for selecting LAAO devices. We retrospectively reviewed 62 patients with Amplatzer Cardiac Plug and Amulet LAAO devices who underwent implantation from 2017 to 2020. The minimal, maximal, average, area-derived, and perimeter-derived diameters of the LAA landing zone were measured using CT-based images. Predicted device sizes using sizing charts were compared with actual successfully implanted device sizes. The mean size of implanted devices was 27.1 ± 3.7 mm. The perimeter-derived diameter predicted device size most accurately (mean error = - 0.8 ± 2.4 mm). All other parameters showed significantly larger error (mean error; minimal diameter = - 4.9 ± 3.3 mm, maximal diameter = 1.0 ± 2.9 mm, average diameter = - 1.6 ± 2.6 mm, area-derived diameter = - 2.0 ± 2.6 mm) than the perimeter-derived diameter (all p for difference < 0.05). The error for other parameters were larger in cases with more eccentrically-shaped landing zones, while the perimeter-derived diameter had minor error regardless of eccentricity. When oversizing was used, all parameters showed significant disagreement. The perimeter-derived diameter on cardiac CT images provided the most accurate estimation of LAAO device size regardless of landing zone eccentricity. Oversizing was unnecessary when using cardiac CT to predict an accurate LAAO size.

Aberrant Autophagy Impacts Growth and Multicellular Development in a Dictyostelium Knockout Model of CLN5 Disease.

Mutations in CLN5 cause a subtype of neuronal ceroid lipofuscinosis (NCL) called CLN5 disease. While the precise role of CLN5 in NCL pathogenesis is not known, recent work revealed that the protein has glycoside hydrolase activity. Previous work on the Dictyostelium discoideum homolog of human CLN5, Cln5, revealed its secretion during the early stages of development and its role in regulating cell adhesion and cAMP-mediated chemotaxis. Here, we used Dictyostelium to examine the effect of cln5-deficiency on various growth and developmental processes during the life cycle. During growth, cln5 - cells displayed reduced cell proliferation, cytokinesis, viability, and folic acid-mediated chemotaxis. In addition, the growth of cln5 - cells was severely impaired in nutrient-limiting media. Based on these findings, we assessed autophagic flux in growth-phase cells and observed that loss of cln5 increased the number of autophagosomes suggesting that the basal level of autophagy was increased in cln5 - cells. Similarly, loss of cln5 increased the amounts of ubiquitin-positive proteins. During the early stages of multicellular development, the aggregation of cln5 - cells was delayed and loss of the autophagy genes, atg1 and atg9, reduced the extracellular amount of Cln5. We also observed an increased amount of intracellular Cln5 in cells lacking the Dictyostelium homolog of the human glycoside hydrolase, hexosaminidase A (HEXA), further supporting the glycoside hydrolase activity of Cln5. This observation was also supported by our finding that CLN5 and HEXA expression are highly correlated in human tissues. Following mound formation, cln5 - development was precocious and loss of cln5 affected spore morphology, germination, and viability. When cln5 - cells were developed in the presence of the autophagy inhibitor ammonium chloride, the formation of multicellular structures was impaired, and the size of cln5 - slugs was reduced relative to WT slugs. These results, coupled with the aberrant autophagic flux observed in cln5 - cells during growth, support a role for Cln5 in autophagy during the Dictyostelium life cycle. In total, this study highlights the multifaceted role of Cln5 in Dictyostelium and provides insight into the pathological mechanisms that may underlie CLN5 disease.

Inhibiting Neddylation with MLN4924 Suppresses Growth and Delays Multicellular Development in Dictyostelium discoideum.

Neddylation is a post-translational modification that is essential for a variety of cellular processes and is linked to many human diseases including cancer, neurodegeneration, and autoimmune disorders. Neddylation involves the conjugation of the ubiquitin-like modifier neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) to target proteins, and has been studied extensively in various eukaryotes including fungi, plants, and metazoans. Here, we examine the biological processes influenced by neddylation in the social amoeba, Dictyostelium discoideum, using a well-established inhibitor of neddylation, MLN4924 (pevonedistat). NEDD8, and the target of MLN4924 inhibition, NEDD8-activating enzyme E1 (NAE1), are highly conserved in D. discoideum (Nedd8 and Nae1, respectively). Treatment of D. discoideum cells with MLN4924 increased the amount of free Nedd8, suggesting that MLN4924 inhibited neddylation. During growth, MLN4924 suppressed cell proliferation and folic acid-mediated chemotaxis. During multicellular development, MLN4924 inhibited cyclic adenosine monophosphate (cAMP)-mediated chemotaxis, delayed aggregation, and suppressed fruiting body formation. Together, these findings indicate that neddylation plays an important role in regulating cellular and developmental events during the D. discoideum life cycle and that this organism can be used as a model system to better understand the essential roles of neddylation in eukaryotes, and consequently, its involvement in human disease.

A Proteomics Analysis of Calmodulin-Binding Proteins in Dictyostelium discoideum during the Transition from Unicellular Growth to Multicellular Development.

Calmodulin (CaM) is an essential calcium-binding protein within eukaryotes. CaM binds to calmodulin-binding proteins (CaMBPs) and influences a variety of cellular and developmental processes. In this study, we used immunoprecipitation coupled with mass spectrometry (LC-MS/MS) to reveal over 500 putative CaM interactors in the model organism Dictyostelium discoideum. Our analysis revealed several known CaMBPs in Dictyostelium and mammalian cells (e.g., myosin, calcineurin), as well as many novel interactors (e.g., cathepsin D). Gene ontology (GO) term enrichment and Search Tool for the Retrieval of Interacting proteins (STRING) analyses linked the CaM interactors to several cellular and developmental processes in Dictyostelium including cytokinesis, gene expression, endocytosis, and metabolism. The primary localizations of the CaM interactors include the nucleus, ribosomes, vesicles, mitochondria, cytoskeleton, and extracellular space. These findings are not only consistent with previous work on CaM and CaMBPs in Dictyostelium, but they also provide new insight on their diverse cellular and developmental roles in this model organism. In total, this study provides the first in vivo catalogue of putative CaM interactors in Dictyostelium and sheds additional light on the essential roles of CaM and CaMBPs in eukaryotes.