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Validation of the Delirium Diagnostic Tool-Provisional in intensive care units
No full textShortcomings of intensive care units (ICU) delirium screening tools include not measuring its core features, not excluding stupor/coma and not being continuous measurement instruments. We validated the Delirium Diagnostic Tool-Provisional (DDT-Pro) that assesses all 3 core symptom domains for delirium and subsyndromal delirium (SSD) in the ICU. This is a multicenter validation following STARD guideline. Delirium reference standards were DSM-5 criteria, cluster analysis (CA) of the DDT-Pro scores and clinical validators for preestablished DDT-Pro ≤ 6 and ≤ 7 cutoffs (0-9 range) for delirium and SSD. DDT-Pro dimensionality and internal consistency reliability are reported. Of 127 patients, 29 (22.8%) had DSM-5 delirium. The area under the receiver-operator DDT-Pro curve was 90% with the ≤ 7 and ≤ 6 DDT-Pro cutoffs which had 82.7% and 80.3% accuracy at the most balanced sensitivity-specificity. The ≤ 6 cutoff specificity (85.7%) was higher, while ≤ 7 cutoff sensitivity (89.7%) was higher with NPV = 96.2%. According to CA, ≤7 cutoff differentiated 100% of nondelirium patients from SSD and delirium, whereas DSM-5 misattributed SSD. Validation of an SSD group was supported by delirium severity gradients and various clinical validators. Cases in this CA also coincided exactly with nondelirium, SSD and delirium groups prespecified by DDT-Pro cutoffs from non-ICU samples. One factor explained 69.9% of the DDT-Pro variance, Cronbach α = 0.79 (cohesive delirium dimension). Our findings indicate that the DDT-Pro has very good construct validity and discriminates ICU delirium against DSM-5, performing even more cleanly using agnostic CA for SSD and delirium diagnosis. Its continuous score structure discernment of SSD was supported by clinical validators. ICU cutoffs were the same as in previous inpatient samples
Reduction of NFX1-123 and HPV 16 E6 and E7 Decreased Telomerase and CENP-F in Cervical Cancer Cell Lines
No full textBackground: Telomerase activity is a cancer hallmark, and hTERT is the rate-limiting catalytic subunit of telomerase. In human papillomavirus type 16 E6 (16E6)-expressing epithelial cells, NFX1-123 augments and is required for full hTERT expression, leading to a growth advantage. However, no studies have investigated the role of NFX1-123 in telomerase activity regulation in HPV-associated cancers.
Methods: We knocked out NFX1-123 in CaSki cells (CaSki KO) and performed single-cell RNA sequencing to determine mRNA alterations affected by reduced NFX1-123.
Results: In CaSki KO cells, there were three cell clusters based on gene expression, each associated with different enriched biological processes. When pooled and compared with control cells, CaSki KO cells had 1661 decreased and 565 increased mRNAs involving RNA regulation, cell cycle and division, chromatin regulation, and carcinogenesis processes and pathways. CENP-F, a cell cycle and chromosome segregation gene increased in cervical cancers, was among 10 genes with the greatest decrease in mRNA expression in CaSki KO cells. CaSki and SiHa cells with either reduced NFX1-123 or knocked down HPV 16 E6 and E7, demonstrated reduced hTERT, CENP-F, and telomerase activity, and when both NFX1-123 and HPV 16 E6 and E7 were decreased, hTERT and telomerase activity fell further. Finally, hTERT and CENP-F were increased in cervical cancer primary tumors and in HPV-positive head and neck cancer primary tumors in the TCGA database.
Conclusions: These findings highlight the shared role that NFX1-123 has with HPV 16 oncogenes in driving and maintaining RNA, cell cycle, and carcinogenesis pathways, and specifically regulating hTERT, telomerase, and CENP-F
Effect of Phosphorylation Barcodes on Arrestin Binding to a Chemokine Receptor
No full textUnique phosphorylation 'barcodes' installed in different regions of an active seven-transmembrane receptor by different G-protein-coupled receptor (GPCR) kinases (GRKs) have been proposed to promote distinct cellular outcomes1, but it is unclear whether or how arrestins differentially engage these barcodes. Here, to address this, we developed an antigen-binding fragment (Fab7) that recognizes both active arrestin2 (β-arrestin1) and arrestin3 (β-arrestin2) without interacting with bound receptor polypeptides. We used Fab7 to determine the structures of both arrestins in complex with atypical chemokine receptor 3 (ACKR3) phosphorylated in different regions of its C-terminal tail by either GRK2 or GRK5 (ref. 2). The GRK2-phosphorylated ACKR3 resulted in more heterogeneous 'tail-mode' assemblies, whereas phosphorylation by GRK5 resulted in more rigid 'ACKR3-adjacent' assemblies. Unexpectedly, the finger loops of both arrestins engaged the micelle surface rather than the receptor intracellular pocket, with arrestin3 being more dynamic, partly because of its lack of a membrane-anchoring motif. Thus, both the region of the barcode and the arrestin isoform involved can alter the structure and dynamics of GPCR-arrestin complexes, providing a possible mechanistic basis for unique downstream cellular effects, such as the efficiency of chemokine scavenging and the robustness of arrestin binding in ACKR3
Proinflammatory Stress Activates Neutral Sphingomyelinase 2-Based Generation of a Ceramide-Enriched β-Cell EV Subpopulation
No full textβ-Cell extracellular vesicles (EVs) play a role as paracrine effectors in islet health, yet mechanisms connecting β-cell stress to changes in EV cargo and potential impacts on diabetes remain poorly defined. We hypothesized that β-cell inflammatory stress engages neutral sphingomyelinase 2 (nSMase2)-dependent EV formation pathways, generating ceramide-enriched small EVs that could impact surrounding β-cells. Consistent with this, proinflammatory cytokine treatment of INS-1 β-cells and human islets concurrently increased β-cell nSMase2 and ceramide abundance, as well as small EV ceramide species. Direct chemical activation or genetic knockdown of nSMase2, chemical treatment to inhibit cell death pathways, or treatment with a glucagon-like peptide-1 (GLP-1) receptor agonist also modulated β-cell EV ceramide. RNA sequencing of ceramide-enriched EVs identified a distinct set of miRNAs linked to β-cell function and identity. EV treatment from cytokine-exposed parent cells inhibited peak glucose-stimulated insulin secretion in wild-type recipient cells; this effect was abrogated when using EVs from nSMase2 knockdown parent cells. Finally, plasma EVs in children with recent-onset type 1 diabetes showed increases in multiple ceramide species. These findings highlight nSMase2 as a regulator of β-cell EV cargo and identify ceramide-enriched EV populations as a contributor to EV-related paracrine signaling under conditions of β-cell inflammatory stress and death.
Article highlights: Mechanisms connecting β-cell stress to extracellular vesicle (EV) cargo and diabetes are poorly defined. Does β-cell inflammatory stress engage neutral sphingomyelinase 2 (nSMase2)-dependent EV formation to generate ceramide-enriched small EVs? Proinflammatory cytokines increased β-cell small EV ceramide via increases in nSMase2. Ceramide-enriched EVs housed distinct cargo linked to insulin signaling, and ceramide species were enriched in plasma EVs from individuals with type 1 diabetes. Ceramide-enriched EV populations are a potential contributor to β-cell EV-related paracrine signaling
Multi-site video-based assessment with the NACC UDS-Version 3 battery: Design and participant experience in the V-Cog study
No full textIntroduction: Video-based cognitive testing is becoming more common. The 'VCog' study aims to evaluate the validity, feasibility, and acceptability of a standardized video-administered cognitive research battery.
Methods: Twelve Alzheimer's Disease Research Centers (ADRCs) administered, in randomized order, an in-person and video-adapted Uniform Data Set Version 3 (UDSv3) cognitive assessment battery to participants with normal cognition (n = 156), mild cognitive impairment (MCI; n = 230), and dementia (n = 77). Acceptability of technology and participant experience completing video testing was assessed.
Results: Of 463 participants (mean age 75.1 years, 52.5% female, and 25.5% non-White individuals), most rated video testing as easy (90.0%), convenient (95.3%) and would accept it in future visits (75.5%). Greater cognitive impairment was associated with more difficulty with setup and use of video. Staff rated most administrations as producing valid data (94.8%).
Discussion: Video-based research cognitive testing with an adapted UDSv3 battery is feasible and well-accepted among older adults with and without cognitive impairment.
Highlights: The COVID-19 pandemic increased the need for video-administered cognitive assessments in Alzheimer's Disease Research Centers (ADRCs) . Feasibility, acceptability, and validity of video cognitive testing were evaluated. Participant experience and examiner evaluations of data validity were positive. Video assessment may extend the ADRCs reach to otherwise unreachable participants
Impact of Elevated Serum Triglycerides on Children with Acute Recurrent or Chronic Pancreatitis from INSPPIRE-2
No full textObjective: To determine if mild-moderate hypertriglyceridemia (HTG) is associated with increased development of chronic pancreatitis (CP) or pancreatitis-associated complications in children with acute recurrent or CP.
Study design: Longitudinal data from the INternational Study group of Pediatric Pancreatitis: In search for a cuRE-2 (INSPPIRE-2) cohort of children with acute recurrent or CP (n = 559) were analyzed. Subjects were divided into normal triglycerides (<150 mg/dL; 1.7 mmol/L), any HTG (≥150 mg/dL; ≥1.7 mmol/L), mild-moderate HTG (150-499 mg/dL; 1.7-5.6 mmol/L), moderate HTG (500-999 mg/dL; 5.6-11.3 mmol/L), and severe HTG groups (≥1000 mg/dL; ≥11.3 mmol/L), based on highest serum triglyceride value. Laboratory, imaging, pancreatitis and hospital events, complications, and quality of life data were analyzed.
Results: In children with acute recurrent or CP and HTG, there was no increase in the number of pancreatitis attacks per person-years, nor an increase in CP prevalence. However, HTG severity was associated with increased pancreatic inflammation, pancreatic cysts, pain, hospital days, number of hospitalizations, intensive care, and missed school days.
Conclusions: Mild-moderate HTG in children with acute recurrent or CP was not associated with increased pancreatitis frequency, nor increased development of CP, but was associated with increased pancreatitis complications and disease burden. As a treatable condition, treatment of mild-moderate HTG may be considered to reduce pancreatitis-associated complications and medical burden in children with acute recurrent or CP
Tau uptake by human neurons depends on receptor LRP1 and kinase LRRK2
No full textExtracellular release and uptake of pathogenic forms of the microtubule-associated protein tau contribute to the pathogenesis of several neurodegenerative diseases, including Alzheimer's disease. Defining the cellular mechanisms and pathways for tau entry to human neurons is essential to understanding tauopathy pathogenesis and enabling the rational design of disease-modifying therapeutics. Here, whole-genome, loss-of-function CRISPR screens in human iPSC-derived excitatory neurons, the major neuronal cell type affected in these diseases, provide insights into the different cellular pathways for uptake of extracellular monomeric and fibrillar tau. Monomeric and fibrillar tau are both taken up by human neurons by receptor-mediated endocytosis, but involve different routes of entry at the neuronal surface: the low-density lipoprotein LRP1 is the primary receptor for monomeric tau, but contributes less to fibrillar tau entry. Similarly, endocytosis of monomeric tau is dependent on the familial Parkinson's disease gene LRRK2, but not required for endocytosis of fibrillar tau. These findings implicate LRP1 and LRRK2 in the pathogenesis of tauopathies and Parkinson's disease, and identify LRRK2 as a potential therapeutic target for altering progression of these diseases
Scene as Occupancy and Reconstruction: A Comprehensive Dataset for Unstructured Scene Understanding
No full textAs autonomous driving technology steps into the phase of large-scale commercialization, safety and comfort have become key indicators for measuring its performance. Currently, some studies have begun to focus on improving the safety and comfort of urban driving by paying attention to irregular surface regions. However, datasets and studies for unstructured scenes, which are characterized by numerous irregular obstacles and road surface undulations, remain exceedingly rare. To expand the scope of autonomous driving applications, a perception dataset, which focuses on irregular obstacles and road surface vibrations in unstructured scenes, has been built. It takes into consideration the fact that the detection of various irregular obstacles in unstructured scenes plays a key role in trajectory planning, while the recognition of undulating road surface conditions in these scenes is crucial for speed planning. Therefore, we investigate unstructured scene understanding through 3D semantic occupancy prediction, which is used to detect irregular obstacles in unstructured scenes, and road surface elevation reconstruction, which characterizes the bumpy and uneven conditions of road surfaces. The dataset provides detailed annotations for 3D semantic occupancy prediction and road surface elevation reconstruction, offering a comprehensive representation of unstructured scenes. In addition, trajectory and speed planning information is provided to explore the relationship between perception and planning in unstructured scenes. Natural language descriptions of scenes are also provided to explore the interpretability of autonomous driving decision-making. Experiments have been conducted with various state-of-the-art methods to demonstrate the effectiveness of our dataset and the challenges posed by these tasks. To the best of our knowledge, this is the world's first comprehensive benchmark for perception in unstructured scenes, which serves as a valuable resource for extending autonomous driving technology from urban to unstructured scenes
Strategies for Acid and Amine Cross-linking and Labeling for Protein Structural Characterization Using Mass Spectrometry
No full textCross-linking and covalent labeling are common tools for protein structural elucidation by mass spectrometry. However, despite the importance of electrostatic interactions in proteins, there are not many tools for probing both acids and amines. Therefore, we introduce novel solution-phase amine-to-acid cross-linkers and single reagent amine and acid covalent labels utilizing carbodiimide chemistry. Cross-linking and labeling sites were determined using top-down and enzymatic digestion approaches. Ubiquitin was chosen as a model protein for isotopically labeled 13C-15N-glycine cross-linking/covalent labeling and glycyl-l-proline cross-linking/covalent labeling with top-down mass spectrometry as a proof of concept. Alpha synuclein, an intrinsically disordered protein that plays a role in multiple neurological disorders, was also cross-linked/covalent labeled with these methods/reagents. We expect that these structural characterization techniques will play a role in gaining insight into functionally and pathologically important structural tendencies of disordered proteins