Mitotic gene regulation by the N-MYC-WDR5-PDPK1 nexus.

BACKGROUND: During mitosis the cell depends on proper attachment and segregation of replicated chromosomes to generate two identical progeny. In cancers defined by overexpression or dysregulation of the MYC oncogene this process becomes impaired, leading to genomic instability and tumor evolution. Recently it was discovered that the chromatin regulator WDR5-a critical MYC cofactor-regulates expression of genes needed in mitosis through a direct interaction with the master kinase PDPK1. However, whether PDPK1 and WDR5 contribute to similar mitotic gene regulation in MYC-overexpressing cancers remains unclear. Therefore, to characterize the influence of WDR5 and PDPK1 on mitotic gene expression in cells with high MYC levels, we performed a comparative transcriptomic analysis in neuroblastoma cell lines defined by MYCN-amplification, which results in high cellular levels of the N-MYC protein. RESULTS: Using RNA-seq analysis, we identify the genes regulated by N-MYC and PDPK1 in multiple engineered CHP-134 neuroblastoma cell lines and compare them to previously published gene expression data collected in CHP-134 cells following inhibition of WDR5. We find that as expected N-MYC regulates a multitude of genes, including those related to mitosis, but that PDPK1 regulates specific sets of genes involved in development, signaling, and mitosis. Analysis of N-MYC- and PDPK1-regulated genes reveals a small group of commonly controlled genes associated with spindle pole formation and chromosome segregation, which overlap with genes that are also regulated by WDR5. We also find that N-MYC physically interacts with PDPK1 through the WDR5-PDPK1 interaction suggesting regulation of mitotic gene expression may be achieved through a N-MYC-WDR5-PDPK1 nexus. CONCLUSIONS: Overall, we identify a small group of genes highly enriched within functional gene categories related to mitotic processes that are commonly regulated by N-MYC, WDR5, and PDPK1 and suggest that a tripartite interaction between the three regulators may be responsible for setting the level of mitotic gene regulation in N-MYC amplified cell lines. This study provides a foundation for future studies to determine the exact mechanism by which N-MYC, WDR5, and PDPK1 converge on cell cycle related processes.

Apneic Oxygenation During Prolonged Laryngoscopy in Obese Patients: A Randomized, Controlled Trial of Buccal RAE Tube Oxygen Administration.

BACKGROUND: Despite optimal preoxygenation, obese patients undergoing induction of general anesthesia exhibit significant hypoxemia after 2 to 4 minutes of apnea. Apneic oxygenation techniques can assist airway management by extending the safe apnea time. We hypothesized that a novel method of apneic oxygenation via the oral route would effectively prolong safe apnea in an obese surgical population. METHODS: In this open-label, parallel-arm, randomized-controlled efficacy trial, 40 ASA physical status I-II obese patients with body mass index (BMI) 30-40 were randomly assigned to standard care (n = 20) or buccal oxygenation (n = 20) during induction of total IV anesthesia. Buccal oxygen was administered via a modified 3.5-mm Ring-Adair-Elwyn (RAE) tube apposed to the left internal cheek. Prolonged laryngoscopy maintained apnea with a patent airway until SpO2 dropped below 95% or 750 seconds elapsed. The primary outcome was time to reach SpO2 < 95%. RESULTS: Patient characteristics were similar in both study arms. Recipients of buccal oxygenation were less likely to exhibit SpO2 < 95% during 750 seconds of apnea; hazard ratio 0.159 (95% confidence interval 0.044-0.226, P < .0001). Median (interquartile range [IQR]) apnea times with SpO2 ≥ 95% were prolonged in this group; 750 (389-750) versus 296 (244-314) seconds, P < .0001. CONCLUSIONS: Clinically important prolongation of safe apnea times can be achieved delivering buccal oxygen to obese patients on induction of anesthesia. This novel use of apneic oxygenation via the oral route may improve management of the difficult airway and overcome some of the limitations of alternative techniques.

Reconsidering gender relative to risk of rupture in the contemporary management of abdominal aortic aneurysms.

OBJECTIVE: Abdominal aortic aneurysms (AAAs) may rupture at smaller diameters in women than in men, and women may be at higher risk and have poorer outcomes in elective and emergent interventions because of age and comorbidities. Practice guidelines recommending elective AAA repair at >5.5 cm are gender neutral and may not adequately reflect increased risks in women or the potential advantages of elective lower risk endovascular procedures. METHODS: Patients with a diagnosis of AAA discharged from a single referral hospital during a 14-year period were identified for retrospective analysis. RESULTS: A total of 2121 patients with AAAs were studied, 499 women (23.5%) and 1622 men (76.5%). Women were older and had a greater incidence of hypertension, smoking, chronic obstructive pulmonary disease, dyslipidemia, and renal insufficiency. Intact AAAs in 467 women had a mean diameter of 4.4 ± 1.3 cm compared with 1538 men at 5.0 ± 1.4 cm (P < .01). The ruptured AAAs in 32 women (6.4%) had a mean diameter of 6.1 ± 1.5 cm compared with 84 men (5.2%) at 7.7 ± 1.9 cm (P < .01). Women had a twofold increased frequency of AAA rupture than men at all size intervals (P < .01). The frequency of ruptured AAAs <5.5 cm among 10 of 32 women with ruptured AAAs was 31.3%; among 7 of 84 men with ruptured AAAs, it was 8.3% (P < .01). The frequency of ruptured AAAs <5.5 cm in all 383 women with AAAs <5.5 cm was 2.6%; in 1042 men, it was 0.6% (P < .01). Of the 1211 AAA repairs, 574 (47.4%) were open aneurysm repair (OAR) and 637 (52.6%) were endovascular aneurysm repair (EVAR). Mortality after elective OAR in 475 patients of both sexes was 5.1%; for EVAR in 676 patients, mortality was 1.6% (P < .01). No differences in mortality with respect to OAR or EVAR were found between the female and male cohorts in either intact or ruptured AAAs. CONCLUSIONS: Women with AAAs are older and have a higher frequency of cardiovascular risk factors than men. Women rupture AAAs with a greater frequency than men at all size intervals and have a fourfold increased frequency of rupture at <5.5 cm. No differences in surgical mortality between women and men were found. Current practice guidelines for elective AAA operative intervention should be reconsidered and stratified by gender.

Management of late main-body aortic endograft component uncoupling and type IIIa endoleak encountered with the Endologix Powerlink and AFX platforms.

OBJECTIVE: Junctional component separation producing type IIIa endoleak after endovascular abdominal aortic aneurysm repair (EVAR) is an uncommon but serious complication requiring unanticipated reinterventions. This retrospective study analyzed main-body EVAR component uncoupling and type IIIa endoleaks encountered with Powerlink and AFX (Endologix Inc, Irvine, Calif) endografts during an 8-year period. METHODS: Type IIIa endoleaks were identified from a database of secondary interventions and clinical surveillance. Operative reports, medical records, and computed tomography studies were reviewed. Clinical and imaging characteristics were analyzed over time, and differences were compared at appropriate follow-up intervals. RESULTS: Since 2006, 701 patients underwent primary EVAR using Endologix Powerlink (352 patients, 2006-2011) or AFX (349 patients, 2011-2014) endografts. Endoleaks required 32 secondary interventions (4.6%), including type Ia in 4 patients (1 proximal extension and 3 explants); type Ib in 8 patients (all distal extensions for enlarging iliac aneurysms); type II in 1 patient (explant); type IIIa in 17 patients (2.4%), who were the subject of this report; and type IIIb in 2 patients (both EVAR relining). The 17 patients with type IIIa endoleak were an average age of 71 years, and 14 (82%) were men. The mean preoperative abdominal aortic aneurysm (AAA) diameter was 70 ± 18 mm. The repair was elective in 16 patients and an emergency in one. Ten cases were performed with Powerlink and seven with AFX. Analysis of serial computed tomography scans found significant changes in AAA diameter; renal-to-bifurcation straight-line, centerline, and greater curvature lengths; EVAR angulation; and loss of EVAR component overlap. The average time from EVAR to reintervention was 32 months. Three patients returned with a ruptured AAA and three with AAA thrombosis, and three of these patients (18%) died ≤30 days of the emergency reintervention. Secondary procedures included EVAR relining with additional bridging components in 14 patients (82%), explant in 2, and axillobifemoral bypass in 1. No new cases of endograft uncoupling have been identified in patients treated with AFX since December 2012 after adoption of revised instructions for use. CONCLUSIONS: Although a small number of secondary interventions were needed after EVAR with the Endologix Powerlink or AFX endografts, most were undertaken for late main-body component uncoupling and type IIIa endoleak, which can occur after sideways displacement of the endograft in large and angulated AAAs. Patients treated before 2013 under the old instructions for use should be evaluated for signs of impending component separation and monitored annually, noting that expected indicators of endograft failure, such as increasing AAA diameter and endoleak, may be absent.

Super-Enhancer Dysregulation in Rhabdoid Tumor Cells Is Regulated by the SWI/SNF ATPase BRG1.

Mutations in the SWI/SNF chromatin remodeling complex occur in ~20% of cancers. In rhabdoid tumors defined by loss of the SWI/SNF subunit SMARCB1, dysregulation of enhancer-mediated gene expression is pivotal in driving oncogenesis. Enhancer dysregulation in this setting is tied to retention of the SWI/SNF ATPase BRG1-which becomes essential in the absence of SMARCB1-but precisely how BRG1 contributes to this process remains unknown. To characterize how BRG1 participates in chromatin remodeling and gene expression in SMARCB1-deficient cells, we performed a genome-wide characterization of the impact of BRG1 depletion in multiple rhabdoid tumor cell lines. We find that although BRG1-regulated open chromatin sites are distinct at the locus level, the biological characteristics of the loci are very similar, converging on a set of thematically related genes and pointing to the involvement of the AP-1 transcription factor. The open chromatin sites regulated by BRG1 colocalize with histone-marked enhancers and intriguingly include almost all super-enhancers, revealing that BRG1 plays a critical role in maintaining super-enhancer function in this setting. These studies can explain the essentiality of BRG1 to rhabdoid tumor cell identity and survival and implicate the involvement of AP-1 as a critical downstream effector of rhabdoid tumor cell transcriptional programs.

The annotation of GBA1 has been concealed by its protein-coding pseudogene GBAP1.

Mutations in GBA1 cause Gaucher disease and are the most important genetic risk factor for Parkinson's disease. However, analysis of transcription at this locus is complicated by its highly homologous pseudogene, GBAP1. We show that >50% of short RNA-sequencing reads mapping to GBA1 also map to GBAP1. Thus, we used long-read RNA sequencing in the human brain, which allowed us to accurately quantify expression from both GBA1 and GBAP1. We discovered significant differences in expression compared to short-read data and identify currently unannotated transcripts of both GBA1 and GBAP1. These included protein-coding transcripts from both genes that were translated in human brain, but without the known lysosomal function-yet accounting for almost a third of transcription. Analyzing brain-specific cell types using long-read and single-nucleus RNA sequencing revealed region-specific variations in transcript expression. Overall, these findings suggest nonlysosomal roles for GBA1 and GBAP1 with implications for our understanding of the role of GBA1 in health and disease.

Generation of TWO G51D SNCA missense mutation iPSC lines (CRICKi011-A, CRICKi012-A) from two individuals at risk of Parkinson's disease.

Mutations or multiplications of the SNCA (Synuclein Alpha) gene cause rare autosomal dominant Parkinson's disease (PD). The SNCA G51D missense mutation is associated with a synucleinopathy that shares PD and multiple system atrophy (MSA) characteristics. We generated induced pluripotent stem cell (iPSC) lines from two individuals with SNCA G51D missense mutations at risk of PD. Dermal fibroblasts were reprogrammed to pluripotency using a non-integrating mRNA-based protocol. The resulting human iPSCs displayed normal morphology, expressed markers associated with pluripotency, and differentiated into the three germ layers. The iPSC lines could facilitate disease-modelling and therapy development studies for synucleinopathies.

Prediction of mechanistic subtypes of Parkinson's using patient-derived stem cell models.

Parkinson's disease is a common, incurable neurodegenerative disorder that is clinically heterogeneous: it is likely that different cellular mechanisms drive the pathology in different individuals. So far it has not been possible to define the cellular mechanism underlying the neurodegenerative disease in life. We generated a machine learning-based model that can simultaneously predict the presence of disease and its primary mechanistic subtype in human neurons. We used stem cell technology to derive control or patient-derived neurons, and generated different disease subtypes through chemical induction or the presence of mutation. Multidimensional fluorescent labelling of organelles was performed in healthy control neurons and in four different disease subtypes, and both the quantitative single-cell fluorescence features and the images were used to independently train a series of classifiers to build deep neural networks. Quantitative cellular profile-based classifiers achieve an accuracy of 82%, whereas image-based deep neural networks predict control and four distinct disease subtypes with an accuracy of 95%. The machine learning-trained classifiers achieve their accuracy across all subtypes, using the organellar features of the mitochondria with the additional contribution of the lysosomes, confirming the biological importance of these pathways in Parkinson's. Altogether, we show that machine learning approaches applied to patient-derived cells are highly accurate at predicting disease subtypes, providing proof of concept that this approach may enable mechanistic stratification and precision medicine approaches in the future.

Will they fit? Development of a measurement device to assess body habitus compatibility with MRI bore diameter for emergency trauma imaging.

Excessive obesity poses a significant limitation to radiographic magnetic resonance imaging (MRI), particularly related to aperture or bore diameter due to the patient's girth. Determination of whether a patient will fit into the bore of the MRI scanner is currently accomplished using patient height, weight, and MRI technician experience. These simple methods have proven unreliable. We sought to develop a device and method which could accurately determine whether a patient would fit into the MRI scanner. We developed an MRI template prototype which was tested against the standard radiology methods in a pilot study (n = 6). We then performed a prospective validation study in adult human volunteers (n = 100) to assess the accuracy of the MRI template. We collected height, weight, shoulder and pelvis girth/diameter for each study participant to evaluate the body dimension measurements that would assist in determination of whether a patient would fit into the MRI scanner. Using the MRI template, we determined that 11 of the 100 study participants would not fit in the MRI scanner and 10 were confirmed to not fit into the MRI aperture [positive predictive value (PPV) 0.91 (0.58-0.99); negative predictive value (NPV) 1.00 (0.95-1.00), sensitivity 1.00 (0.69-1.00), specificity 0.99 (0.93-0.99), likelihood ratio positive test 90 (12.81-632), likelihood ratio negative test 0, accuracy 99%]. In comparison, the body measurement method did not perform as well [PPV 0.66 (0.34-0.90), NPV 0.97 (0.92-0.99), sensitivity 0.80 (0.44-0.97), specificity 0.95 (0.89-0.98), likelihood ratio positive test 17.97 (6.56-49.2), likelihood ratio negative test 0.209 (0.06-0.72), accuracy 94%]. This study confirmed that the use of an MRI template is an accurate tool in determining whether an obese patient can fit through the MRI bore and be accommodated in the MRI scanner.

Mitochondrial dysfunction is a key pathological driver of early stage Parkinson's.

BACKGROUND: The molecular drivers of early sporadic Parkinson's disease (PD) remain unclear, and the presence of widespread end stage pathology in late disease masks the distinction between primary or causal disease-specific events and late secondary consequences in stressed or dying cells. However, early and mid-stage Parkinson's brains (Braak stages 3 and 4) exhibit alpha-synuclein inclusions and neuronal loss along a regional gradient of severity, from unaffected-mild-moderate-severe. Here, we exploited this spatial pathological gradient to investigate the molecular drivers of sporadic PD. METHODS: We combined high precision tissue sampling with unbiased large-scale profiling of protein expression across 9 brain regions in Braak stage 3 and 4 PD brains, and controls, and verified these results using targeted proteomic and functional analyses. RESULTS: We demonstrate that the spatio-temporal pathology gradient in early-mid PD brains is mirrored by a biochemical gradient of a changing proteome. Importantly, we identify two key events that occur early in the disease, prior to the occurrence of alpha-synuclein inclusions and neuronal loss: (i) a metabolic switch in the utilisation of energy substrates and energy production in the brain, and (ii) perturbation of the mitochondrial redox state. These changes may contribute to the regional vulnerability of developing alpha-synuclein pathology. Later in the disease, mitochondrial function is affected more severely, whilst mitochondrial metabolism, fatty acid oxidation, and mitochondrial respiration are affected across all brain regions. CONCLUSIONS: Our study provides an in-depth regional profile of the proteome at different stages of PD, and highlights that mitochondrial dysfunction is detectable prior to neuronal loss, and alpha-synuclein fibril deposition, suggesting that mitochondrial dysfunction is one of the key drivers of early disease.

Protein aggregation and calcium dysregulation are hallmarks of familial Parkinson's disease in midbrain dopaminergic neurons.

Mutations in the SNCA gene cause autosomal dominant Parkinson's disease (PD), with loss of dopaminergic neurons in the substantia nigra, and aggregation of α-synuclein. The sequence of molecular events that proceed from an SNCA mutation during development, to end-stage pathology is unknown. Utilising human-induced pluripotent stem cells (hiPSCs), we resolved the temporal sequence of SNCA-induced pathophysiological events in order to discover early, and likely causative, events. Our small molecule-based protocol generates highly enriched midbrain dopaminergic (mDA) neurons: molecular identity was confirmed using single-cell RNA sequencing and proteomics, and functional identity was established through dopamine synthesis, and measures of electrophysiological activity. At the earliest stage of differentiation, prior to maturation to mDA neurons, we demonstrate the formation of small ß-sheet-rich oligomeric aggregates, in SNCA-mutant cultures. Aggregation persists and progresses, ultimately resulting in the accumulation of phosphorylated α-synuclein aggregates. Impaired intracellular calcium signalling, increased basal calcium, and impairments in mitochondrial calcium handling occurred early at day 34-41 post differentiation. Once midbrain identity fully developed, at day 48-62 post differentiation, SNCA-mutant neurons exhibited mitochondrial dysfunction, oxidative stress, lysosomal swelling and increased autophagy. Ultimately these multiple cellular stresses lead to abnormal excitability, altered neuronal activity, and cell death. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease.

Pathological structural conversion of α-synuclein at the mitochondria induces neuronal toxicity.

Aggregation of alpha-synuclein (α-Syn) drives Parkinson's disease (PD), although the initial stages of self-assembly and structural conversion have not been directly observed inside neurons. In this study, we tracked the intracellular conformational states of α-Syn using a single-molecule Förster resonance energy transfer (smFRET) biosensor, and we show here that α-Syn converts from a monomeric state into two distinct oligomeric states in neurons in a concentration-dependent and sequence-specific manner. Three-dimensional FRET-correlative light and electron microscopy (FRET-CLEM) revealed that intracellular seeding events occur preferentially on membrane surfaces, especially at mitochondrial membranes. The mitochondrial lipid cardiolipin triggers rapid oligomerization of A53T α-Syn, and cardiolipin is sequestered within aggregating lipid-protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial reactive oxygen species (ROS) generation, which accelerates the oligomerization of A53T α-Syn and causes permeabilization of mitochondrial membranes and cell death. These processes were also observed in induced pluripotent stem cell (iPSC)-derived neurons harboring A53T mutations from patients with PD. Our study highlights a mechanism of de novo α-Syn oligomerization at mitochondrial membranes and subsequent neuronal toxicity.

Finding solutions for bird restoration and livestock management: comparing grazing exclusion levels.

Riparian habitats in the western United States support high avian abundance and diversity and are important areas for livestock grazing, although grazing contributes to the degradation of riparian vegetation. Complete removal of livestock is generally the most beneficial for bird communities, but alternative management approaches allowing for seasonal livestock grazing may also increase avian habitat quality. Determining management approaches that integrate conservation priorities and human use has the potential to improve conditions for birds across many grazed landscapes. We anticipated that both the full and seasonal exclusion of cattle from riparian areas would result in the recovery of avian populations. To test this, we experimentally compared the effects of total cattle exclusion vs. seasonal usage on bird populations in the riparian areas of two creeks in the San Joaquin Valley, California, USA. Avian species richness and average abundance were measured over six years, from 2001 to 2006. In general, native avian abundance and richness increased in both full- and seasonal-exclosure areas, with increases compared to the pretreatment year for all years except 2005. Habitats that had complete cattle exclusion recovered more significantly, with 29% lower avian abundance in seasonally grazed habitats. There was no significant difference in avian richness between the two grazing treatments, although richness did increase over time. In addition to increases in native species, abundance of nonnative birds and brood parasites also increased significantly, with a greater avian abundance in the full-exclusion areas. A direct comparison with the Breeding Bird Survey (BBS), to further investigate annual avian population trends, explained the native species decline in 2005. This comparison revealed that observed trends were the effect of experimental treatment rather than a reflection of regional trends. Although the overall trends indicate that the full exclusion of livestock from riparian areas results in more abundant bird populations, seasonal exclusion is also a successful method for avian recovery. Land managers should consider both options in riparian areas, selecting full cattle removal for avian management only and seasonal cattle removal for situations where incorporating conservation and land use practices is advantageous.

Stress reactivity elicits a tissue-specific reduction in telomere length in aging zebrafish (Danio rerio).

Individual differences in personality are associated with variation in healthy aging. Health behaviours are often cited as the likely explanation for this association; however, an underlying biological mechanism may also exist. Accelerated leukocyte telomere shortening is implicated in multiple age-related diseases and is associated with chronic activation of the hypothalamus-pituitary-adrenal (HPA) axis, providing a link between stress-related personality differences and adverse health outcomes. However, the effects of the HPA axis are tissue specific. Thus, leukocyte telomere length may not accurately reflect telomere length in disease-relevant tissues. Here, we examined the correlation between stress reactivity and telomere length in heart and brain tissue in young (6-9 month) and aging (18 month) zebrafish. Stress reactivity was assessed by tank diving and through gene expression. Telomere length was assessed using quantitative PCR. We show that aging zebrafish have shorter telomeres in both heart and brain. Telomere length was inversely related to stress reactivity in heart but not brain of aging individuals. These data support the hypotheses that an anxious predisposition contributes to accelerated telomere shortening in heart tissue, which may have important implications for our understanding of age-related heart disease, and that stress reactivity contributes to age-related telomere shortening in a tissue-specific manner.

Relapsing polychondritis presenting as meningoencephalitis with valvular abnormality: a case report.

Relapsing polychondritis is a rare rheumatologic disorder that is characterized by recurrent inflammation of selected connective tissue sites and destruction of cartilage throughout the body. We report a case of newly diagnosed relapsing polychondritis in a 40-year-old man presenting with episcleritis, deformed "cauliflower" ears, aortic regurgitation, and aseptic meningoencephalitis. Steroid therapy was instituted with good resolution of his clinical symptoms.

Aspergillus thromboembolism from a mycotic ascending aortic pseudoaneurysm.

This case report describes an immune-competent patient with acute upper extremity ischemia caused by thromboembolism from an Aspergillus-infected ascending aortic pseudoaneurysm. Efforts to identify the source of an acute arterial thromboembolic occlusion should be made, and a high index of suspicion for mycotic infection should be maintained in patients with an atypical presentation, such as fevers of unknown origin. Additional measures, such as pathologic examination of thromboembolic debris, blood cultures, and positron emission tomography, should be performed to identify the etiology in these unexplained situations.