Digital Commons Network^™

Brain Connectivity And Socioeconomic Status At Birth And Externalizing Symptoms At Age 2 Years, Bruce Ramphal, Diana J Whalen, Jeanette K Kenley, Qiongru Yu, Christopher D Smyser, Cynthia E Rogers, Chad M Sylvester

Open Access Publications

Low childhood socioeconomic status (SES) predisposes individuals to altered trajectories of brain development and increased rates of mental illness. Brain connectivity at birth is associated with psychiatric outcomes. We sought to investigate whether SES at birth is associated with neonatal brain connectivity and if these differences account for socioeconomic disparities in infant symptoms at age 2 years that are predictive of psychopathology. Resting state functional MRI was performed on 75 full-term and 37 term-equivalent preterm newborns (n = 112). SES was characterized by insurance type, the Area Deprivation Index, and a composite score. Seed-based voxelwise linear regression related SES to …

Robust Principal Component Analysis For Accurate Outlier Sample Detection In Rna-Seq Data, Xiaoying Chen, Bo Zhang, Ting Wang, Azad Bonni, Guoyan Zhao

Open Access Publications

BACKGROUND: High throughput RNA sequencing is a powerful approach to study gene expression. Due to the complex multiple-steps protocols in data acquisition, extreme deviation of a sample from samples of the same treatment group may occur due to technical variation or true biological differences. The high-dimensionality of the data with few biological replicates make it challenging to accurately detect those samples, and this issue is not well studied in the literature currently. Robust statistics is a family of theories and techniques aim to detect the outliers by first fitting the majority of the data and then flagging data points that …

Parkinson Disease Clinical Subtypes: Key Features & Clinical Milestones, Meghan C Campbell, Peter S Myers, Alexandra J Weigand, Erin R Foster, Nigel J Cairns, Joshua J Jackson, Christina N Lessov-Schlaggar, Joel S Perlmutter

Open Access Publications

OBJECTIVES: Based on multi-domain classification of Parkinson disease (PD) subtypes, we sought to determine the key features that best differentiate subtypes and the utility of PD subtypes to predict clinical milestones.

METHODS: Prospective cohort of 162 PD participants with ongoing, longitudinal follow-up. Latent class analysis (LCA) delineated subtypes based on score patterns across baseline motor, cognitive, and psychiatric measures. Discriminant analyses identified key features that distinguish subtypes at baseline. Cox regression models tested PD subtype differences in longitudinal conversion to clinical milestones, including deep brain stimulation (DBS), dementia, and mortality.

RESULTS: LCA identified distinct subtypes: "motor only" (N = 63) …

Phenotypic Profiling In Subjects Heterozygous For 1 Of 2 Rare Variants In The Hypophosphatasia Gene (Alpl), Daniel R Tilden, Jonathan H Sheehan, John H Newman, Jens Meiler, John A Capra, Andrea Ramirez, Jill Simmons, Kathryn Dahir

Open Access Publications

CONTEXT: Hypophosphatasia (HPP) is a syndrome marked by low serum alkaline phosphatase (AlkP) activity as well as musculoskeletal and/or dental disease. While the majority of subjects with HPP carry a pathogenic variant in the

METHODS: Through BioVU, a DNA biobank that pairs individuals' genetic information with their de-identified medical records, we identified subjects with 2 rare variants with distinct reported clinical phenotypes (p.D294A and p.T273M). We then performed a manual review of these subjects' de-identified medical records along with computational modeling of protein structure to construct a genetic, biochemical and clinical phenotype for each subject and variant.

RESULTS: Twenty subjects …

Behavioral And Neural Signatures Of Working Memory In Childhood, Monica D. Rosenberg, Deanna M. Barch, Et Al

Open Access Publications

Working memory function changes across development and varies across individuals. The patterns of behavior and brain function that track individual differences in working memory during human development, however, are not well understood. Here, we establish associations between working memory, other cognitive abilities, and functional MRI (fMRI) activation in data from over 11,500 9- to 10-year-old children (both sexes) enrolled in the Adolescent Brain Cognitive Development (ABCD) Study, an ongoing longitudinal study in the United States. Behavioral analyses reveal robust relationships between working memory, short-term memory, language skills, and fluid intelligence. Analyses relating out-of-scanner working memory performance to memory-related fMRI activation …

Development Of A Single-Stranded Dna-Binding Protein Fluorescent Fusion Toolbox, Katarzyna Dubiel, Camille Henry, Lisanne M. Spenkelink, Alexander G. Kozlov, Elizabeth A. Wood, Slobodan Jergic, Nicholas E. Dixon, Antoine M. Van Oijen, Michael M. Cox, Timothy M. Lohman, Steven J. Sandler, James L. Keck

Open Access Publications

Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein …

Pet Radiopharmaceuticals For Imaging Chemotherapy-Induced Cardiotoxicity, Jothilingam Sivapackiam, Monica Sharma, Thomas H Schindler, Vijay Sharma

Open Access Publications

PURPOSE OF REVIEW: Currently, cardiotoxicity is monitored through echocardiography or multigated acquisition scanning and is defined as 10% or higher LVEF reduction. The latter stage may represent irreversible myocardium injury and limits modification of therapeutic paradigms at earliest stages. To stratify patients for anthracycline-related heart failure, highly sensitive and molecularly specific probes capable of interrogating cardiac damage at the subcellular levels have been sought.

RECENT FINDINGS: PET tracers may provide noninvasive assessment of earliest changes within myocardium. These tracers are at nascent stages of development and belong primarily to (a) mitochondrial potential-targeted and (b) general ROS (reactive oxygen species)-targeted radiotracers. …

Oligomerization Of The Vesicular Stomatitis Virus Phosphoprotein Is Dispensable For Mrna Synthesis But Facilitates Rna Replication, Louis-Marie Bloyet, Benjamin Morin, Vesna Brusic, Erica Gardner, Robin A. Ross, Tegy Vadakkan, Tomas Kirchhausen, Sean P. J. Whelan

Open Access Publications

Nonsegmented negative-strand (NNS) RNA viruses possess a ribonucleoprotein template in which the genomic RNA is sequestered within a homopolymer of nucleocapsid protein (N). The viral RNA-dependent RNA polymerase (RdRP) resides within an approximately 250-kDa large protein (L), along with unconventional mRNA capping enzymes: a GDP:polyribonucleotidyltransferase (PRNT) and a dual-specificity mRNA cap methylase (MT). To gain access to the N-RNA template and orchestrate the L

Validation Of Parkinson's Disease-Related Questionnaires In South Africa, Gill Nelson, Ntombizodwa Ndlovu, Nicola Christofides, Tintswalo M Hlungwani, Irene Faust, Brad A Racette

Open Access Publications

Background: There are very few epidemiological studies investigating Parkinson's disease (PD) in Africa. The hundreds of local languages and dialects make traditional screening and clinical evaluation tools difficult to use.

Objective: The objective of the study was to validate two commonly used PD questionnaires in an African population.

Methods: The PD Screening Questionnaire (PDSQ) and Parkinson's Disease Questionnaire (PDQ-39) were modified and translated into Afrikaans, Setswana, and isiZulu and administered to a sample of healthy local residents. We assessed the internal consistencies and cluster characteristics of the questionnaires, using a Cronbach's alpha test and exploratory factor analysis. The questionnaires were …

Replication Of Associations With Psychotic-Like Experiences In Middle Childhood From The Adolescent Brain Cognitive Development (Abcd) Study, Nicole R Karcher, Rachel L Loewy, Mark Savill, Shelli Avenevoli, Rebekah S Huber, Tony J Simon, Ingrid N Leckliter, Kenneth J Sher, Deanna M Barch

Open Access Publications

The fields of psychology and psychiatry are increasingly recognizing the importance of replication efforts. The current study aimed to replicate previous findings examining the construct validity and psychometric properties of a psychotic-like experiences (PLEs) measure in middle childhood using an independent subset of the baseline Adolescent Brain Cognitive Development (ABCD) sample. Using a remainder baseline sample of 7013 nine- to eleven-year-old children with complete data, we examined measurement invariance across race/ethnicity and sex, and examined the associations between the Prodromal Questionnaire Brief-Child Version (PQ-BC) and other measures of PLEs, internalizing symptoms, neuropsychological test performance, and developmental milestones, to determine whether …

A Survey-Based Analysis Of The Academic Job Market, Jason D. Fernandes, Sarvenaz Sarabipour, Christopher T. Smith, Natalie M. Niemi, Nafisa M. Jadavji, Ariangela J. Kozik, Alex S. Holehouse, Vikas Pejaver, Orsolya Symmons, Alexandre W. Bisson Filho, Amanda Haage

Open Access Publications

Many postdoctoral researchers apply for faculty positions knowing relatively little about the hiring process or what is needed to secure a job offer. To address this lack of knowledge about the hiring process we conducted a survey of applicants for faculty positions: the survey ran between May 2018 and May 2019, and received 317 responses. We analyzed the responses to explore the interplay between various scholarly metrics and hiring outcomes. We concluded that, above a certain threshold, the benchmarks traditionally used to measure research success - including funding, number of publications or journals published in - were unable to completely …

The Neurons That Mistook A Hat For A Face, Michael J. Arcaro, Carlos Ponce, Margaret Livingstone

Open Access Publications

Despite evidence that context promotes the visual recognition of objects, decades of research have led to the pervasive notion that the object processing pathway in primate cortex consists of multiple areas that each process the intrinsic features of a few particular categories (e.g. faces, bodies, hands, objects, and scenes). Here we report that such category-selective neurons do not in fact code individual categories in isolation but are also sensitive to object relationships that reflect statistical regularities of the experienced environment. We show by direct neuronal recording that face-selective neurons respond not just to an image of a face, but also …

Safety And Efficacy Of Co-Administered Diethylcarbamazine, Albendazole And Ivermectin During Mass Drug Administration For Lymphatic Filariasis In Haiti: Results From A Two-Armed, Open-Label, Cluster-Randomized, Community Study, Christine Dubray, Anita Sircar, Madsen Beau De Rochars, Joshua Bogus, Abdel N. Direny, Jean Romuald Ernest, Carl Fayette, Charles W. Goss, Marisa Hast, Katiuscia O'Brian, Guy Emmanuel Pavilus, Daniel Frantz Sabin, Ryan Wiegand, Gary J. Weil, Jean Frantz Lemoine

Open Access Publications

No abstract provided.

Cd56 Regulates Human Nk Cell Cytotoxicity Through Pyk2, Justin T. Gunesch, Amera L. Dixon, Tasneem A.M. Ebrahim, Melissa M. Berrien-Elliott, Swetha Tatineni, Tejas Kumar, Everardo Hegewisch-Solloa, Todd A. Fehniger, Emily M. Mace

Open Access Publications

Human natural killer (NK) cells are defined as CD56

Multidimensional Imaging Of Liver Injury Repair In Mice Reveals Fundamental Role Of The Ductular Reaction, Kenji Kamimoto, Yasuhiro Nakano, Kota Kaneko, Atsushi Miyajima, Tohru Itoh

Open Access Publications

Upon severe and/or chronic liver injury, ectopic emergence and expansion of atypical biliary epithelial-like cells in the liver parenchyma, known as the ductular reaction, is typically induced and implicated in organ regeneration. Although this phenomenon has long been postulated to represent activation of facultative liver stem/progenitor cells that give rise to new hepatocytes, recent lineage-tracing analyses have challenged this notion, thereby leaving the pro-regenerative role of the ductular reaction enigmatic. Here, we show that the expanded and remodelled intrahepatic biliary epithelia in the ductular reaction constituted functional and complementary bile-excreting conduit systems in injured parenchyma where hepatocyte bile canalicular networks …

Assessment Of Antibiotic-Resistant Organism Transmission Among Rooms Of Hospitalized Patients, Healthcare Personnel, And The Hospital Environment Utilizing Surrogate Markers And Selective Bacterial Cultures, Jennie H Kwon, Kimberly Reske, Caroline A O'Neil, Candice Cass, Sondra Seiler, Meghan A Wallace, Tiffany Hink, Stephen Y Liang, Victoria J Fraser, Carey-Ann D Burnham, Erik R Dubberke

Open Access Publications

OBJECTIVE: To assess potential transmission of antibiotic-resistant organisms (AROs) using surrogate markers and bacterial cultures.

DESIGN: Pilot study.

SETTING: A 1,260-bed tertiary-care academic medical center.

PARTICIPANTS: The study included 25 patients (17 of whom were on contact precautions for AROs) and 77 healthcare personnel (HCP).

METHODS: Fluorescent powder (FP) and MS2 bacteriophage were applied in patient rooms. HCP visits to each room were observed for 2-4 hours; hand hygiene (HH) compliance was recorded. Surfaces inside and outside the room and HCP skin and clothing were assessed for fluorescence, and swabs were collected for MS2 detection by polymerase chain reaction (PCR) …

Wide Complex Tachycardia Differentiation: A Reappraisal Of The State-Of-The-Art, Anthony H Kashou, Peter A Noseworthy, Christopher V Desimone, Abhishek J Deshmukh, Samuel J Asirvatham, Adam M May

Open Access Publications

The primary goal of the initial ECG evaluation of every wide complex tachycardia is to determine whether the tachyarrhythmia has a ventricular or supraventricular origin. The answer to this question drives immediate patient care decisions, ensuing clinical workup, and long-term management strategies. Thus, the importance of arriving at the correct diagnosis cannot be understated and has naturally spurred rigorous research, which has brought forth an ever-expanding abundance of manually applied and automated methods to differentiate wide complex tachycardias. In this review, we provide an in-depth analysis of traditional and more contemporary methods to differentiate ventricular tachycardia and supraventricular wide complex …

Growth Velocity In Children With Environmental Enteric Dysfunction Is Associated With Specific Bacterial And Viral Taxa Of The Gastrointestinal Tract In Malawian Children, Chandni Desai, Scott A. Handley, Rachel Rodgers, Cynthia Rodriguez, Maria I. Ordiz, Mark J. Manary, Lori R. Holtz

Open Access Publications

Environmental enteric dysfunction (EED) is characterized by diffuse villous atrophy of the small bowel. EED is strongly associated with stunting, a major public health problem linked to increased childhood morbidity and mortality. EED and subsequent stunting of linear growth are surmised to have microbial origins. To interrogate this relationship, we defined the comprehensive virome (eukaryotic virus and bacteriophage) and bacterial microbiome of a longitudinal cohort of rural Malawian children with extensive metadata and intestinal permeability testing at each time point. We found thirty bacterial taxa differentially associated with linear growth. We detected many eukaryotic viruses. Neither the total number of …

Evaluating The Use Of Rcbv As A Tumor Grade And Treatment Response Classifier Across Nci Quantitative Imaging Network Sites: Part Ii Of The Dsc-Mri Digital Reference Object (Dro) Challenge, Laura C. Bell, Hongyu An, Cihat Eldeniz, Richard Wahl, Et Al

Open Access Publications

We have previously characterized the reproducibility of brain tumor relative cerebral blood volume (rCBV) using a dynamic susceptibility contrast magnetic resonance imaging digital reference object across 12 sites using a range of imaging protocols and software platforms. As expected, reproducibility was highest when imaging protocols and software were consistent, but decreased when they were variable. Our goal in this study was to determine the impact of rCBV reproducibility for tumor grade and treatment response classification. We found that varying imaging protocols and software platforms produced a range of optimal thresholds for both tumor grading and treatment response, but the performance …

Conformational Distributions Of Isolated Myosin Motor Domains Encode Their Mechanochemical Properties, Justin R. Porter, Artur Meller, Maxwell I. Zimmerman, Michael J. Greenberg, Gregory R. Bowman

Open Access Publications

Myosin motor domains perform an extraordinary diversity of biological functions despite sharing a common mechanochemical cycle. Motors are adapted to their function, in part, by tuning the thermodynamics and kinetics of steps in this cycle. However, it remains unclear how sequence encodes these differences, since biochemically distinct motors often have nearly indistinguishable crystal structures. We hypothesized that sequences produce distinct biochemical phenotypes by modulating the relative probabilities of an ensemble of conformations primed for different functional roles. To test this hypothesis, we modeled the distribution of conformations for 12 myosin motor domains by building Markov state models (MSMs) from an …

Plasmodium Falciparum Translational Machinery Condones Polyadenosine Repeats, Slavica Pavlovic Djuranovic, Jessey Erath, Ryan J. Andrews, Peter O. Bayguinov, Joyce J. Chung, Douglas L. Chalker, James A.J. Fitzpatrick, Walter N. Moss, Pawel Szczesny, Sergej Djuranovic

Open Access Publications

No abstract provided.

Consensus Guideline For The Diagnosis And Treatment Of Tetrahydrobiopterin (Bh4) Deficiencies, Thomas Opladen, Toni S Pearson, Et Al

Open Access Publications

BACKGROUND: Tetrahydrobiopterin (BH

CONCLUSION: Although the total body of evidence in the literature was mainly rated as low or very low, these consensus guidelines will help to harmonize clinical practice and to standardize and improve care for BH

Study Protocol: Improving Cognition In People With Progressive Multiple Sclerosis: A Multi-Arm, Randomized, Blinded, Sham-Controlled Trial Of Cognitive Rehabilitation And Aerobic Exercise (Cogex), Anthony Feinstein, Amber Salter, Et Al

Open Access Publications

BACKGROUND: Cognitive dysfunction affects up to 70% of people with progressive MS (PMS). It can exert a deleterious effect on activities of daily living, employment and relationships. Preliminary evidence suggests that performance can improve with cognitive rehabilitation (CR) and aerobic exercise (EX), but existing data are predominantly from people with relapsing-remitting MS without cognitive impairment. There is therefore a need to investigate whether this is also the case in people with progressive forms of the disease who have objectively identified cognitive impairment. It is hypothesized that CR and EX are effective treatments for people with PMS who have cognitive impairment, …

Nadph And Glutathione Redox Link Tca Cycle Activity To Endoplasmic Reticulum Homeostasis, Erica R Gansemer, Kyle S Mccommis, Michael Martino, Abdul Qaadir King-Mcalpin, Matthew J Potthoff, Brian N Finck, Eric B Taylor, D Thomas Rutkowski

Open Access Publications

Many metabolic diseases disrupt endoplasmic reticulum (ER) homeostasis, but little is known about how metabolic activity is communicated to the ER. Here, we show in hepatocytes and other metabolically active cells that decreasing the availability of substrate for the tricarboxylic acid (TCA) cycle diminished NADPH production, elevated glutathione oxidation, led to altered oxidative maturation of ER client proteins, and attenuated ER stress. This attenuation was prevented when glutathione oxidation was disfavored. ER stress was also alleviated by inhibiting either TCA-dependent NADPH production or Glutathione Reductase. Conversely, stimulating TCA activity increased NADPH production, glutathione reduction, and ER stress. Validating these findings, …

In Utero Exposure To Transient Ischemia-Hypoxemia Promotes Long-Term Neurodevelopmental Abnormalities In Male Rat Offspring, Arvind Palanisamy, Tusar Giri, Jia Jiang, Annie Bice, James D Quirk, Sara B Conyers, Susan E Maloney, Nandini Raghuraman, Adam Q Bauer, Joel R Garbow, David F Wozniak

Open Access Publications

The impact of transient ischemic-hypoxemic insults on the developing fetal brain is poorly understood despite evidence suggesting an association with neurodevelopmental disorders such as schizophrenia and autism. To address this, we designed an aberrant uterine hypercontractility paradigm with oxytocin to better assess the consequences of acute, but transient, placental ischemia-hypoxemia in term pregnant rats. Using MRI, we confirmed that oxytocin-induced aberrant uterine hypercontractility substantially compromised uteroplacental perfusion. This was supported by the observation of oxidative stress and increased lactate concentration in the fetal brain. Genes related to oxidative stress pathways were significantly upregulated in male, but not female, offspring 1 …

Quantitative Analysis Of Transcription Factor Binding And Expression Using Calling Cards Reporter Arrays, Jiayue Liu, Christian A. Shively, Robi D. Mitra

Open Access Publications

We report a tool, Calling Cards Reporter Arrays (CCRA), that measures transcription factor (TF) binding and the consequences on gene expression for hundreds of synthetic promoters in yeast. Using Cbf1p and MAX, we demonstrate that the CCRA method is able to detect small changes in binding free energy with a sensitivity comparable to in vitro methods, enabling the measurement of energy landscapes in vivo. We then demonstrate the quantitative analysis of cooperative interactions by measuring Cbf1p binding at synthetic promoters with multiple sites. We find that the cooperativity between Cbf1p dimers varies sinusoidally with a period of 10.65 bp and …

Sex-Specific Impact Of Patterns Of Imageable Tumor Growth On Survival Of Primary Glioblastoma Patients, Paula Whitmire, Joshua B Rubin, Et Al

Open Access Publications

BACKGROUND: Sex is recognized as a significant determinant of outcome among glioblastoma patients, but the relative prognostic importance of glioblastoma features has not been thoroughly explored for sex differences.

METHODS: Combining multi-modal MR images, biomathematical models, and patient clinical information, this investigation assesses which pretreatment variables have a sex-specific impact on the survival of glioblastoma patients (299 males and 195 females).

RESULTS: Among males, tumor (T1Gd) radius was a predictor of overall survival (HR = 1.027, p = 0.044). Among females, higher tumor cell net invasion rate was a significant detriment to overall survival (HR = 1.011, p < 0.001). Female extreme survivors had significantly smaller tumors (T1Gd) (p = 0.010 t-test), but tumor size was not correlated with female overall survival (p = 0.955 CPH). Both male and female extreme survivors had significantly lower tumor cell net proliferation rates than other patients (M p = 0.004, F p = 0.001, t-test).

CONCLUSION: Despite …

A New Gorilla Adenoviral Vector With Natural Lung Tropism Avoids Liver Toxicity And Is Amenable To Capsid Engineering And Vector Retargeting, Zhi Hong Lu, Igor P. Dmitriev, Douglas E. Brough, Elena A. Kashentseva, Jie Li, David T. Curiel

Open Access Publications

Human adenoviruses have many attractive features for gene therapy applications. However, the high prevalence of preexisting immunity against these viruses in general populations worldwide has greatly limited their clinical utility. In addition, the most commonly used human adenovirus, human adenovirus subgroup C serotype 5 (HAd5), when systemically administered, triggers systemic inflammation and toxicity, with the liver being the most severely affected organ. Here, we evaluated the utility and safety of a new low-seroprevalence gorilla adenovirus (GAd; GC46) as a gene transfer vector in mice. Biodistribution studies revealed that systemically administered GAd had a selective and robust lung endothelial cell (EC) …

Objective Quantification Of Image Quality And Optical Scatter Before And After Nd:Yag Capsulotomy Using A Double-Pass Technique, Abu-Bakar Zafar, Richard C Chu, Miranda N Bishara, Mujtaba A Qazi, Ashraf M Mahmoud, Jay S Pepose

Open Access Publications

Purpose: The purpose of this study is to evaluate and compare the correlation between changes in vision and HD Analyzer dual-pass metrics versus changes in vision and conventional subjective slit lamp gradings in pseudophakic patients with posterior capsular opacity undergoing neodymium:yttrium-aluminum-garnet (Nd:YAG) capsulotomy.

Patients and Methods: High contrast (HC) and low contrast (LC) best spectacle-corrected distance visual acuity (BCVA) and HD Analyzer evaluation were prospectively performed on patients with mild-to-moderate posterior capsular opacification (PCO) and monofocal and accommodating intraocular lens implants. Differences between pre- and post-operative measurements were calculated, along with the correlation of HD Analyzer metrics and slit lamp …

Effects Of Dopaminergic Drugs On Cognitive Control Processes Vary By Genotype, Daniella J Furman, Robert L White Iii, Jenna Naskolnakorn, Jean Ye, Andrew Kayser, Mark D'Esposito

Open Access Publications

Dopamine (DA) has been implicated in modulating multiple cognitive control processes, including the robust maintenance of task sets and memoranda in the face of distractors (cognitive stability) and, conversely, the ability to switch task sets or update the contents of working memory when it is advantageous to do so (cognitive flexibility). In humans, the limited specificity of available pharmacological probes has posed a challenge for understanding the mechanisms by which DA, acting on multiple receptor families across the PFC and striatum, differentially influences these cognitive processes. Using a within-subject, placebo-controlled design, we contrasted the impact of two mechanistically distinct DA …