Citations

AbstractBackgroundTMEM106B, a lysosomal trafficking protein, is an important genetic susceptibility risk factor for frontotemporal lobar degeneration (FTLD). In GRN mutation carriers, minor allele homozygosity in the TMEM106B rs1990622 SNP modulates the risk of FTLD. TMEM106B is often the main component of amyloid fibrils in FTLD with TDP43 aggregates. TMEM106B has been quantified in human brain specimens, but its role as a fluid biomarker is unknown. Here, we investigate the clinical value of CSF TMEM106B in FTLD.MethodsCSF TMEM106B was quantified with SOMAmer proteomics (v3.0, SomaLogic®) in an original cohort of C9orf72, GRN and MAPT symptomatic and asymptomatic mutation carriers and family non‐carrier controls, recruited through ALLFTD (n = 182), and a validation cohort of sporadic neuropathology‐confirmed FTLD‐tau and FTLD‐TDP43, clinically‐diagnosed progressive supranuclear palsy – Richardson syndrome (PSP‐RS) and age‐matched controls (n = 96). CSF TMEM106B was correlated with disease severity, measured by the Frontotemporal Lobar Degeneration module global scores (CDR+NACC/FTLD); brain volume, measured with Bayesian linear mixed‐effect modeling, using age, sex and total‐intracranial volume‐corrected permutations with threshold‐free cluster enhancement; and CSF neurofilament light chain (NfL) measured with Simoa. TMEM106B SOMAmer specificity was validated in cell culture overexpression systems and human brain specimens with high TMEM106B burden.ResultsIn both cohorts, there were no differences in CSF TMEM106B by sex, phenotype or neuropathological diagnosis, except for lower levels in PSP‐RS compared to controls (‐0.31 Log2‐fold change, p < 0.001). CSF TMEM106B did not correlate with age. Regardless of disease‐causing mutation, lower CSF TMEM106B was associated with worse disease severity (Figure 1 and 2). CSF TMEM106B was lower in homozygous TMEM106B rs1990622 protective allelein C9orf2 and MAPT, but not in GRN mutation carriers (Figures 3). CSF TMEM106B showed a trend for inverse correlation with NfL in the whole sample (r = ‐0.135, p = 0.052), and correlated positively with left frontal volume in symptomatic FTLD mutation gene carriers (p < 0.05).ConclusionCSF TMEM106B is quantifiable in CSF in vivo. CSF TMEM106B levels are affected by an interaction of disease severity and FTLD and TMEM106B genotypes. Further work is needed to determine its value as a clinical biomarker.

AbstractBackgroundAlzheimer’s disease (AD) is characterized by the accumulation of neurofibrillary tangles (NFTs), comprised primarily of microtubule‐associated protein tau. NFTs are defining hallmarks of AD and its accumulation into pathological oligomers and fibrils has been associated with cognitive decline in AD and other tauopathies. Despite its pivotal role in the disease process, we still have a poor understanding of how their formation, toxicity, and spread across brain regions is regulated via tau‐associated proteins that regulate the oligomerization, pathological accumulation, and seeding of tau in affected cell types and vulnerable brain regions.MethodWe have established two proximity‐dependent biotin‐identification (BioID) models as novel approaches to identify the composition and proximal molecular environment of tau aggregates. Using an in vitro and ex vivo model approach coupled with mass spectrometry, we have identified proteins that were primarily associated with mutant tau aggregates vs. soluble wildtype tau and stratified them based on significance, molecular pathway and druggability. Immunoblotting and immunofluorescence were performed to assess the interaction of endogenous and overexpressed top candidates with tau, along with visualization with NFTs in the context of human AD pathology to assess their impact on tau‐aggregate formation and toxicity.ResultTo determine the physiological and aggregate specific interacting partners of tau, we are comparing wild type tau (TauWT) with mutant tau (Tau3xMUT) carrying three tauopathy‐associated mutations (A152T/P301L/S320F). The proteomic analysis from our combined proteomic datasets revealed that significantly changed proteins included candidates involved in RNA processing, as well as protein ubiquitination and proteasome degradation. We have begun validating proteins that are enriched in mutant tau in human tissue of AD, as well as primary tauopathies, that may be druggable targets for tauopathies.ConclusionThis study provides novel insights into cellular pathways and molecular mechanisms of neurodegeneration, by identifying in the context of living neurons and brain tissue different functional classes of tau‐associated proteins with relevance for AD pathophysiology. These are expected to include proteins that may mediate the toxic effect of NFTs, facilitate the formation or degradation of pathological tau aggregates, and catalyze posttranslational modifications of tau oligomers and associated proteins.

Abstract Transactive response DNA-binding protein 43 (TDP-43) pathology is categorized as type A-E in frontotemporal lobar degeneration and as type α-β in Alzheimer disease (AD) based on inclusion type. We screened amygdala slides of 131 cases with varying ages at death, clinical/neuroimaging findings, and AD neuropathologic changes for TDP-43 pathology using anti-phospho-TDP-43 antibodies. Seven cases (5%) only showed atypical TDP-43 inclusions that could not be typed. Immunohistochemistry and immunofluorescence assessed the atypical star-shaped TDP-43 pathology including its distribution, species, cellular localization, and colocalization with tau. All 7 had died at an extremely old age (median: 100 years [IQR: 94–101]) from nonneurological causes and none had dementia (4 cognitively unimpaired, 3 with amnestic mild cognitive impairment). Neuroimaging showed mild medial temporal involvement. Pathologically, the star-shaped TDP-43-positive inclusions were found in medial (subpial) amygdala and, occasionally, in basolateral regions. Hippocampus only showed TDP-43-positive neurites in the fimbria and subiculum while the frontal lobe was free of TDP-43 inclusions. The star-shaped inclusions were better detected with antibodies against N-terminal than C-terminal TDP-43. Double-labeling studies confirmed deposition of TDP-43 within astrocytes and colocalization with tau. We have identified a novel TDP-43 pathology with star-shaped morphology associated with superaging, with a homogeneous clinicopathologic picture, possibly representing a novel, true aging-related TDP-43 pathology.

Background: Oral decitabine/cedazuridine (ASTX727) is a fixed dose combination of decitabine (35 mg) and the cytidine deaminase inhibitor cedazuridine (100 mg) given once daily X 5 days on a 28-day cycle producing pharmacokinetic (PK) exposure equivalent to the standard intravenous (IV) decitabine regimen of 20 mg/m 2 daily X 5 days on a 28-day cycle. This was demonstrated in the pivotal ASCERTAIN study (Garcia-Manero, et al, ASH 2019) as it provided a PK bridge to existing decitabine data and demonstrated median OS was 31.7 mo. (Savona,et al. Intl. MDSF International Congress on MDS, 2021). Subjects were initially classified by the International Prognosis Scoring System (IPSS) for historical reasons, however IPSS-R and IPSS-M are enhancements to IPSS that provide dynamic risk assessment for predicting clinical outcomes in MDS. Here, the objective was to re-classify the MDS subjects enrolled on the ASCERTAIN study by IPSS-R and IPSS-M and measure the impact of informing patient outcomes based on re-calculated risk assessment. Methods: One hundred thirty-three subjects with MDS/CMML (chronic myelomonocytic leukemia) were enrolled in ASCERTAIN and were randomly assigned either IV decitabine for cycle 1 and oral decitabine/cedazuridine for cycle 2 or the opposite treatment sequence. All subjects continuing beyond cycle 2 received oral decitabine/cedazuridine for all subsequent cycles until treatment discontinuation for disease progression, toxicity, patient's decision, or HSCT. Whole blood collected prior to treatment was used for DNA isolation and molecular abnormalities identified using next-generation sequencing (NGS) hematologic malignancy panel of 179 genes including all genes commonly mutated in MDS. In the initial analysis of clinical outcomes, subjects were classified by IPSS with response assessment by IWG 2006 IPSS low and Int-1 risk levels were categorized as lower-risk MDS (LR-MDS), whereas IPSS Int-2 and high-risk categories were categorized as higher-risk MDS (HR-MDS). Subjects with sufficient data based on (e.g., available heme parameters, cytogenetics, NGS etc.) were reclassified by IPSS-R and IPSS-M. Subjects with IPSS-R score of ≤3.5 or IPSS-M of either very low, low, or moderate low were categorized as LR-MDS. Similarly subjects with IPSS-R score of &gt;3.5 or an IPSS-M categorization of either moderate high, high, or very high were categorized as HR-MDS. Reclassified subjects were reassessed for CR (Complete Response), OS, and LFS and Harrell's concordance index (c-index) was used to describe the level of agreement between each scale and outcomes. Results: Based on the available data, the number of MDS subjects in the different risk classifications were the following: IPSS: 117, IPSS-R: 104, and IPSS-M: 105. Thirteen and 12 subjects on the IPSS could not be reclassified in the IPSS-R and IPSS-M, respectively, including 5 Int-1 and 2 low-risk MDS cases. CMML subjects were excluded from these analyses. Re-classification generally resulted in the upgrade of the subjects from LR-MDS to HR-MDS (Fig. 1). Thirty-one (26.5%) subjects from IPSS were reallocated in the IPSS-R to different risk categories; 3 (9.7%) were downgraded and 28 (90.3%) were upgraded. Similarly on reclassification with IPSS-M 34 (32.4%) of the patients reclassified; 5 (14.8%) were downgraded and 29 (85.3%) were upgraded. For IPSS LR-MDS, 23.2% of patients achieved CR, and 22.9% in the IPSS HR-MDS. Similarly, 21.6% patients achieved CR in the IPSS-R LR-MDS, and 23.9% in the IPSS-R HR-MDS; 26.3% patients achieved CR in the IPSS-M LR-MDS, and 20.9% in the IPSS-M HR-MDS. The c-index for the IPSS was .64 (OS) and .67 (LFS), IPSS-R was .70 (OS) and .71 (LFS), and .75 (OS) and .78 (LFS) for the IPSS-M (Table1). Conclusion: Reclassification from IPSS to IPSS-R or IPSS-M upgraded multiple subjects from a LR to a HR category, describing the ASCERTAIN patient population as a majority higher risk population with worse prognosis than previously assumed based on the IPSS. The efficacy as measured by the CR rates did not change when the LR and HR categories were defined by the different risk stratification systems. In contrast, the c-index improves with migration from IPSS to IPSS-R to IPSS-M, indicating an increased discriminatory ability of IPSS-M score in comparison to IPSS and IPSS-R, to predict patient outcomes.

Abstract Objective Given the importance AI in genomics and its potential impact on human health, the American Medical Informatics Association—Genomics and Translational Biomedical Informatics (GenTBI) Workgroup developed this assessment of factors that can further enable the clinical application of AI in this space. Process A list of relevant factors was developed through GenTBI workgroup discussions in multiple in-person and online meetings, along with review of pertinent publications. This list was then summarized and reviewed to achieve consensus among the group members. Conclusions Substantial informatics research and development are needed to fully realize the clinical potential of such technologies. The development of larger datasets is crucial to emulating the success AI is achieving in other domains. It is important that AI methods do not exacerbate existing socio-economic, racial, and ethnic disparities. Genomic data standards are critical to effectively scale such technologies across institutions. With so much uncertainty, complexity and novelty in genomics and medicine, and with an evolving regulatory environment, the current focus should be on using these technologies in an interface with clinicians that emphasizes the value each brings to clinical decision-making.

Abstract Uncontrolled hemorrhage accounts for up to 40% of trauma related mortality, and approximately 30% of trauma related deaths occur in the hospital within the first 1-4 hours of injury. The Advanced Trauma Life Support (ATLS) guidelines still recommend initial resuscitation with crystalloid fluid as an integral part of maintaining or restoring hemodynamic stability, but are starting to recognize the role of early resuscitation with blood products in patients with evidence of Class III and IV hemorrhage, in line with recent clinical trial and observational data. Although there is a growing body of evidence that early transfusion with blood products may be associated with improved survival compared with crystalloid infusion, the optimal transfusion strategy still remains unclear and is being actively investigated in clinical trials. In silico models of resuscitation offer a means to evaluate the effectiveness and safety of resuscitation protocols, including effects on body fluid compartment volumes, tissue oxygenation, and hemostatic factors. Here we describe our work extending earlier deterministic models of trauma resuscitation to incorporate hemodynamic, hemostatic, resuscitation, biomarker, and tissue-oxygenation domains into a single comprehensive model. Additionally, this model expands upon previous deterministic models to create a stochastic computational model of hemostatic resuscitation that accounts for variations in blood component composition and temporal features of transfusion therapy. Using this stochastic model, an in silico trial was piloted to compare conventional component therapy, i.e. red cell, platelet and plasma components, to cold-stored whole blood using the following starting assumptions: patient weight of 81.3±21.8 kg, hematocrit of 41.8±4.3%, initial bleeding rate between 50 and 150 mL/min, systolic blood pressure of 125±18 mmHg, fibrinogen of 293±88 mg/dL, international normalized ratio of 1.0±0.1 and platelet count of 239±62x109/L, with blood component and whole blood constituent distributions based on laboratory quality control data. This in silico trial demonstrated that resuscitation with whole blood was associated with a 28-minute reduction in the time spent in the critical window defined by platelet count &lt;50x109/L, INR≥2, hemoglobin &lt;9 g/dL and fibrinogen &lt;150 mg/dL compared with conventional component resuscitation over the course of a 4 hour simulation (p&lt;0.001 based on median regression). We demonstrate how stochastic models of hemostatic resuscitation can be used to perform in silico trials of resuscitation strategies. Our work can guide inclusion and exclusion criteria for future clinical trials and facilitate identification of patient subgroups most likely to benefit from certain resuscitation strategies. This work was partially funded by the AABB Foundation (formerly the National Blood Foundation).

Peripheral chemoreceptors located within the carotid bodies activate cardiorespiratory reflexes in response to hypoxia. Carotid body chemoreceptor activity has been shown to be enhanced in conditions such as congestive heart failure, hypertension, and possibly aging. Nitric oxide (NO) has been proposed as an inhibitory modulator of carotid body chemosensitivity in response to hypoxia. Additionally, NO bioavailability is often reduced with cardiovascular risk factors such as aging. In the present study, we tested the hypothesis that increasing NO bioavailability via dietary nitrate (NO 3 ) supplementation would reduce cardiorespiratory responses to hypoxia in older adults. A double‐blind, randomized, placebo‐controlled, cross‐over study design was used to evaluate minute ventilation (V E ) and heart rate (HR) responses to acute hypoxia in 7 older adults (66±2 years; 5M/2F) before and after 4 weeks of NO 3 supplementation (beetroot powder) and placebo (beetroot powder devoid of NO 3 ). On each study visit, subjects underwent two separate isocapnic hypoxic tests (self‐regulating, partial rebreathing system with 10% O 2 ). Subjects remained on 10% O 2 until SpO 2 reached ≤80%. The hypoxic exposures were separated by a 15‐minute washout period with the subjects breathing room air. V E , HR, and SpO 2 were continuously monitored throughout each hypoxic exposure. Data were grouped together using a 7‐breath average with the slope of the linear regression line for each dependent variable (V E and HR) versus SpO 2 serving as a measure of peripheral chemosensitivity. The slopes for the two hypoxia tests on each study visit were averaged and used for statistical comparisons. On study day one (true baseline) acute hypoxia increased both V E and HR ( P <0.05 for both). Four weeks of NO 3 supplementation reduced the hypoxic ventilatory response (−0.16 ± −0.05 vs. −0.06 ± −0.03 L/minute/%SpO 2 , P <0.05), whereas no change was observed following the placebo phase of the study (−0.14 ± −0.07 vs. −0.14 ± −0.08 L/minute/%SpO 2 , P=0.67). Additionally, the heart rate responses to acute hypoxia were also reduced following NO 3 supplementation (−0.49 ± −0.08 vs. −0.30 ± −0.05 beats/minute/%SpO 2 , P <0.05), but not with placebo (−0.54 ± −0.11 vs. −0.45 ± −0.08 beats/minute/%SpO 2 , P=0.21). Our preliminary findings suggest that supplementation of NO 3 to boost NO bioavailability in older adults blunts the cardiorespiratory responses to hypoxia. Future studies should examine the therapeutic potential of NO 3 in patient populations exhibiting exaggerated peripheral chemosensitivity. Support or Funding Information Research supported by Neogenis Inc.

Steatotic hepatocytes accumulate profound numbers of large neutral lipid‐storage organelles called lipid droplets (LDs). The process by which the formation or utilization of these organelles are perturbed during NAFLD and NASH is poorly defined. EtOH consumption can also exacerbate these conditions markedly by yet unknown mechanisms. Thus, there is a critical need to understand lipolytic mechanisms for LD catabolism in hepatocytes. In adipocytes, catecholamines induce lipolysis through a β‐adrenergic/cAMP pathway that activates cytosolic lipases ATGL and HSL to catabolize LD triglycerides. Importantly, β‐adrenergic induction of LD catabolism has not been demonstrated in hepatocytes. Thus, the GOAL of this study was to determine whether hepatocytes, like adipocytes, also undergo cAMP‐mediated lipolysis in response to β‐adrenergic (β‐AR) stimulation. First, treatment of primary rat hepatocytes or Hep3B human hepatoma cells with the β‐AR agonist isoproterenol caused a 20–40% reduction in total cellular LD content within 24 hours. β‐AR induced LD loss was mimicked by the cAMP agonist forskolin/IBMX and was abrogated by the cAMP‐dependent protein kinase (PKA) inhibitor H89. In correlation, PKA activation following β‐AR stimulation was confirmed by FRET biosensors and biochemical analysis. β‐AR stimulation directly activated the lipolytic pathway, as evidenced by cAMP/PKA‐mediated phosphorylation of hormone sensitive lipase (HSL) and adipose triglyceide lipase (ATGL). In addition, pharmacological inhibitors against ATGL or HSL each blocked β‐AR induced LD loss, suggesting that lipase activation is critical for this process in stimulated cells. To examine whether β‐AR/cAMP stimulation triggered lipase recruitment directly to the LD, live‐cell imaging of Hep3B cells expressing either ATGL‐EGFP or HSL‐sfGFP showed that within 60 min of stimulation, each lipase was significantly displaced from the cytosol and recruited to the LD surface. This recruitment was blocked by the PKA inhibitor H89. Finally, to test if EtOH exposure attenuates the activation of lipolysis in hepatocytes, we isolated primary hepatocytes from rats fed a 6‐week ethanol‐containing Lieber‐DeCarli diet. Compared with controls, ethanol‐exposed hepatocytes showed a drastic inhibition in lipase activation and LD breakdown following β‐AR/cAMP stimulation. Remarkably, in ethanol‐metabolizing VA13 hepatocytes, ethanol insult completely prevented isoproterenol‐induced translocation of ATGL‐EGFP to the LD. In summary , these results show that β‐adrenergic stimulation mobilizes cytosolic lipases for LD breakdown in hepatocytes, and perturbation of this pathway could be a major consequence of chronic ethanol insult leading to fatty liver. Manipulation of this regulatory cascade could be useful in developing new therapies to attenuate fatty liver disease. Support or Funding Information This study was generously supported by funding from NIAAA 5R01AA020735 (MAM and CAC) and NIDDK 5R37DK044650 (MAM), T32DK007352‐36 (MBS), and P30DK084567 (Mayo Clinic Center for Cell Signaling in Gastroenterology).