Single-cell atlas reveals correlates of high cognitive function, dementia, and resilience to Alzheimer's disease pathology.

Alzheimer's disease (AD) is the most common cause of dementia worldwide, but the molecular and cellular mechanisms underlying cognitive impairment remain poorly understood. To address this, we generated a single-cell transcriptomic atlas of the aged human prefrontal cortex covering 2.3 million cells from postmortem human brain samples of 427 individuals with varying degrees of AD pathology and cognitive impairment. Our analyses identified AD-pathology-associated alterations shared between excitatory neuron subtypes, revealed a coordinated increase of the cohesin complex and DNA damage response factors in excitatory neurons and in oligodendrocytes, and uncovered genes and pathways associated with high cognitive function, dementia, and resilience to AD pathology. Furthermore, we identified selectively vulnerable somatostatin inhibitory neuron subtypes depleted in AD, discovered two distinct groups of inhibitory neurons that were more abundant in individuals with preserved high cognitive function late in life, and uncovered a link between inhibitory neurons and resilience to AD pathology.

Guam ALS-PDC is a distinct double-prion disorder featuring both tau and Aß prions.

The amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC) of Guam is an endemic neurodegenerative disease that features widespread tau tangles, occasional α-synuclein Lewy bodies, and sparse ß-amyloid (Aß) plaques distributed in the central nervous system. Extensive studies of genetic or environmental factors have failed to identify a cause of ALS-PDC. Building on prior work describing the detection of tau and Aß prions in Alzheimer's disease (AD) and Down syndrome brains, we investigated ALS-PDC brain samples for the presence of prions. We obtained postmortem frozen brain tissue from 26 donors from Guam with ALS-PDC or no neurological impairment and 71 non-Guamanian donors with AD or no neurological impairment. We employed cellular bioassays to detect the prion conformers of tau, α-synuclein, and Aß proteins in brain extracts. In ALS-PDC brain samples, we detected high titers of tau and Aß prions, but we did not detect α-synuclein prions in either cohort. The specific activity of tau and Aß prions was increased in Guam ALS-PDC compared with sporadic AD. Applying partial least squares regression to all biochemical and prion infectivity measurements, we demonstrated that the ALS-PDC cohort has a unique molecular signature distinguishable from AD. Our findings argue that Guam ALS-PDC is a distinct double-prion disorder featuring both tau and Aß prions.

Biases in ARG-Based Inference of Historical Population Size in Populations Experiencing Selection.

Inferring the demographic history of populations provides fundamental insights into species dynamics and is essential for developing a null model to accurately study selective processes. However, background selection and selective sweeps can produce genomic signatures at linked sites that mimic or mask signals associated with historical population size change. While the theoretical biases introduced by the linked effects of selection have been well established, it is unclear whether ancestral recombination graph (ARG)-based approaches to demographic inference in typical empirical analyses are susceptible to misinference due to these effects. To address this, we developed highly realistic forward simulations of human and Drosophila melanogaster populations, including empirically estimated variability of gene density, mutation rates, recombination rates, purifying, and positive selection, across different historical demographic scenarios, to broadly assess the impact of selection on demographic inference using a genealogy-based approach. Our results indicate that the linked effects of selection minimally impact demographic inference for human populations, although it could cause misinference in populations with similar genome architecture and population parameters experiencing more frequent recurrent sweeps. We found that accurate demographic inference of D. melanogaster populations by ARG-based methods is compromised by the presence of pervasive background selection alone, leading to spurious inferences of recent population expansion, which may be further worsened by recurrent sweeps, depending on the proportion and strength of beneficial mutations. Caution and additional testing with species-specific simulations are needed when inferring population history with non-human populations using ARG-based approaches to avoid misinference due to the linked effects of selection.

SVision: a deep learning approach to resolve complex structural variants.

Complex structural variants (CSVs) encompass multiple breakpoints and are often missed or misinterpreted. We developed SVision, a deep-learning-based multi-object-recognition framework, to automatically detect and haracterize CSVs from long-read sequencing data. SVision outperforms current callers at identifying the internal structure of complex events and has revealed 80 high-quality CSVs with 25 distinct structures from an individual genome. SVision directly detects CSVs without matching known structures, allowing sensitive detection of both common and previously uncharacterized complex rearrangements.

Incorporating temporal information during feature engineering bolsters emulation of spatio-temporal emergence.

MOTIVATION: Emergent biological dynamics derive from the evolution of lower-level spatial and temporal processes. A long-standing challenge for scientists and engineers is identifying simple low-level rules that give rise to complex higher-level dynamics. High-resolution biological data acquisition enables this identification and has evolved at a rapid pace for both experimental and computational approaches. Simultaneously harnessing the resolution and managing the expense of emerging technologies-e.g. live cell imaging, scRNAseq, agent-based models-requires a deeper understanding of how spatial and temporal axes impact biological systems. Effective emulation is a promising solution to manage the expense of increasingly complex high-resolution computational models. In this research, we focus on the emulation of a tumor microenvironment agent-based model to examine the relationship between spatial and temporal environment features, and emergent tumor properties. RESULTS: Despite significant feature engineering, we find limited predictive capacity of tumor properties from initial system representations. However, incorporating temporal information derived from intermediate simulation states dramatically improves the predictive performance of machine learning models. We train a deep-learning emulator on intermediate simulation states and observe promising enhancements over emulators trained solely on initial conditions. Our results underscore the importance of incorporating temporal information in the evaluation of spatio-temporal emergent behavior. Nevertheless, the emulators exhibit inconsistent performance, suggesting that the underlying model characterizes unique cell populations dynamics that are not easily replaced. AVAILABILITY AND IMPLEMENTATION: All source codes for the agent-based model, emulation, and analyses are publicly available at the corresponding DOIs: 10.5281/zenodo.10622155, 10.5281/zenodo.10611675, 10.5281/zenodo.10621244, respectively.

Suppression of inflammation and acute lung injury by Miz1 via repression of C/EBP-δ.

Inflammation is essential for host defense but can cause tissue damage and organ failure if unchecked. How the inflammation is resolved remains elusive. Here we report that the transcription factor Miz1 was required for terminating lipopolysaccharide (LPS)-induced inflammation. Genetic disruption of the Miz1 POZ domain, which is essential for the transactivation or repression activity of Miz1, resulted in hyperinflammation, lung injury and greater mortality in LPS-treated mice but a lower bacterial load and mortality in mice with Pseudomonas aeruginosa pneumonia. Loss of the Miz1 POZ domain prolonged the expression of proinflammatory cytokines. After stimulation, Miz1 was phosphorylated at Ser178, which was required for recruitment of the histone deacetylase HDAC1 to repress transcription of the gene encoding C/EBP-δ, an amplifier of inflammation. Our data provide a long-sought mechanism underlying the resolution of LPS-induced inflammation.

Different α-synuclein prion strains cause dementia with Lewy bodies and multiple system atrophy.

The α-synuclein protein can adopt several different conformations that cause neurodegeneration. Different α-synuclein conformers cause at least three distinct α-synucleinopathies: multiple system atrophy (MSA), dementia with Lewy bodies (DLB), and Parkinson's disease (PD). In earlier studies, we transmitted MSA to transgenic (Tg) mice and cultured HEK cells both expressing mutant α-synuclein (A53T) but not to cells expressing α-synuclein (E46K). Now, we report that DLB is caused by a strain of α-synuclein prions that is distinct from MSA. Using cultured HEK cells expressing mutant α-synuclein (E46K), we found that DLB prions could be transmitted to these HEK cells. Our results argue that a third strain of α-synuclein prions likely causes PD, but further studies are needed to identify cells and/or Tg mice that express a mutant α-synuclein protein that is permissive for PD prion replication. Our findings suggest that other α-synuclein mutants should give further insights into α-synuclein prion replication, strain formation, and disease pathogenesis, all of which are likely required to discover effective drugs for the treatment of PD as well as the other α-synucleinopathies.

Legume-wide comparative analysis of pod shatter locus PDH1 reveals phaseoloid specificity, high cowpea expression, and stress responsive genomic context.

Pod dehiscence is a major source of yield loss in legumes, which is exacerbated by aridity. Disruptive mutations in "Pod indehiscent 1" (PDH1), a pod sclerenchyma-specific lignin biosynthesis gene, has been linked to significant reductions in dehiscence in several legume species. We compared syntenic PDH1 regions across 12 legumes and two outgroups to uncover key historical evolutionary trends at this important locus. Our results clarified the extent to which PDH1 orthologs are present in legumes, showing the typical genomic context surrounding PDH1 has only arisen relatively recently in certain phaseoloid species (Vigna, Phaseolus, Glycine). The notable absence of PDH1 in Cajanus cajan may be a major contributor to its indehiscent phenotype compared with other phaseoloids. In addition, we identified a novel PDH1 ortholog in Vigna angularis and detected remarkable increases in PDH1 transcript abundance during Vigna unguiculata pod development. Investigation of the shared genomic context of PDH1 revealed it lies in a hotspot of transcription factors and signaling gene families that respond to abscisic acid and drought stress, which we hypothesize may be an additional factor influencing expression of PDH1 under specific environmental conditions. Our findings provide key insights into the evolutionary history of PDH1 and lay the foundation for optimizing the pod dehiscence role of PDH1 in major and understudied legume species.

Competition for time: Evidence for an overlooked, diversity-maintaining competitive mechanism.

Understanding how diversity is maintained in plant communities requires that we first understand the mechanisms of competition for limiting resources. In ecology, there is an underappreciated but fundamental distinction between systems in which the depletion of limiting resources reduces the growth rates of competitors and systems in which resource depletion reduces the time available for competitors to grow, a mechanism we call 'competition for time'. Importantly, modern community ecology and our framing of the coexistence problem are built on the implicit assumption that competition reduces the growth rate. However, recent theoretical work suggests competition for time may be the predominant competitive mechanism in a broad array of natural communities, a significant advance given that when species compete for time, diversity-maintaining trade-offs emerge organically. In this study, we first introduce competition for time conceptually using a simple model of interacting species. Then, we perform an experiment in a Mediterranean annual grassland to determine whether competition for time is an important competitive mechanism in a field system. Indeed, we find that species respond to increased competition through reductions in their lifespan rather than their rate of growth. In total, our study suggests competition for time may be overlooked as a mechanism of biodiversity maintenance.

crosshap: R package for local haplotype visualization for trait association analysis.

SUMMARY: Genome-wide association studies (GWAS) excels at harnessing dense genomic variant datasets to identify candidate regions responsible for producing a given phenotype. However, GWAS and traditional fine-mapping methods do not provide insight into the complex local landscape of linkage that contains and has been shaped by the causal variant(s). Here, we present crosshap, an R package that performs robust density-based clustering of variants based on their linkage profiles to capture haplotype structures in a local genomic region of interest. Following this, crosshap is equipped with visualization tools for choosing optimal clustering parameters (ɛ) before producing an intuitive figure that provides an overview of the complex relationships between linked variants, haplotype combinations, phenotype, and metadata traits. AVAILABILITY AND IMPLEMENTATION: The crosshap package is freely available under the MIT license and can be downloaded directly from CRAN with R >4.0.0. The development version is available on GitHub alongside issue support (https://github.com/jacobimarsh/crosshap). Tutorial vignettes and documentation are available (https://jacobimarsh.github.io/crosshap/).

Long-term outcomes of bladder-sparing therapy vs radical cystectomy in BCG-unresponsive non-muscle-invasive bladder cancer.

OBJECTIVE: To quantify the oncological risks of bladder-sparing therapy (BST) in patients with Bacillus Calmette-Guérin (BCG)-unresponsive non-muscle-invasive bladder cancer (NMIBC) compared to upfront radical cystectomy (RC). PATIENTS AND METHODS: Pre-specified data elements were collected from retrospective cohorts of patients with BCG-unresponsive NMIBC from 10 international sites. After Institutional Review Board approval, patients were included if they had BCG-unresponsive NMIBC meeting United States Food and Drug Administration criteria. Oncological outcomes were collected following upfront RC or BST. BST regimens included re-resection or surveillance only, repeat BCG, intravesical chemotherapy, systemic immunotherapy, and clinical trials. RESULTS: Among 578 patients, 28% underwent upfront RC and 72% received BST. The median (interquartile range) follow-up was 50 (20-69) months. There were no statistically significant differences in metastasis-free survival, cancer-specific survival, or overall survival between treatment groups. In the BST group, high-grade recurrence rates were 37% and 52% at 12 and 24 months and progression to MIBC was observed in 7% and 13% at 12 and 24 months, respectively. RC was performed in 31.7% in the BST group and nodal disease was found in 13% compared with 4% in upfront RC (P = 0.030). CONCLUSION: In a selected cohort of patients, initial BST offers comparable survival outcomes to upfront RC in the intermediate term. Rates of recurrence and progression increase over time especially in patients treated with additional lines of BST.

Ammonium-Binding Bifunctional Aza-Crown Ether Catalysts for Substrate-Selective Hydroxyl Functionalization.

Herein, we describe a new bifunctional macrocyclic catalyst that employs multiple weak noncovalent interactions to enable substrate-selective O-silylation of ammonium alcohols over more reactive aliphatic alcohols with up to >20:1 substrate selectivity. Our catalytic strategy merges (i) the use of crown ethers as ammonium-binding receptors and (ii) the exploitation of N-methyl imidazole as a catalytic motif. Our collective mechanistic studies reveal the importance of receptor size, conformational preorganization, and the number of hydrogen-bonding acceptor units needed to achieve high selectivity within the macrocyclic binding pocket.

Scrutinizing the use of contrasted chest CTs in extremity sarcoma staging and surveillance.

BACKGROUND: Since 2015, the American College of Radiology (ACR) has recommended staging for lung metastasis via chest computed tomography (CT) without contrast for extremity sarcoma staging and surveillance. The purpose of this study was to determine our institutional compliance with this recommendation. METHODS: This was a retrospective chart review of patients diagnosed with sarcoma in the extremities who received CT imaging of the chest for pulmonary staging and surveillance at our institution from 2005 to 2023. A total of 1916 CT studies were included for analysis. We scrutinized ordering patterns before and after 2015 based on the ACR-published metastasis staging and screening guidelines. An institutional and patient cost analysis was performed between CT modalities. RESULTS: The prevalence of CT scans ordered and performed with contrast was greater than those without contrast both prior and post-ACR 2015 guidelines. Furthermore, 79.2% of patient's final surveillance CTs after 2015 were performed with contrast. A cost analysis was performed and demonstrated an additional $297 704 in patient and institutional costs. CONCLUSIONS: At our institution, upon review of CT chest imaging for pulmonary staging and surveillance in patients with extremity sarcoma the use of contrast has been routinely utilized despite a lack of evidence for its necessity and contrary to ACR guidelines.

Remote Microphone Systems for Autistic and Nonautistic Youth: Effects on Audiovisual Task Engagement.

OBJECTIVES: A recent study has provided empirical support for the use of remote microphone (RM) systems to improve listening-in-noise performance of autistic youth. It has been proposed that RM system effects might be achieved by boosting engagement in this population. The present study used behavioral coding to test this hypothesis in autistic and nonautistic youth listening in an ecologically valid, noisy environment. DESIGN: We drew on extant data from a recent experimental study in which 56 youth (32 autistic, 24 nonautistic) matched at the group level on age and biological sex completed listening-in-noise tasks wherein they reported their perception of audiovisual syllables, words, sentences, and passages with and without an RM system; conditions were counter-balanced across participants. As previously reported, perceptual accuracy varied with stimulus complexity and overall improved with the RM system, with improvements not significantly different between groups. Video recordings of participants completing listening-in-noise tasks in both conditions were coded via a 5-second, partial-interval coding system by naive coders for (a) engagement in the task (indexed via proportion of intervals in which participants displayed on-task behaviors) and (b) verbal, stimulus-specific protesting in the task (indexed via proportion of intervals in which participants displayed verbal, stimulus-specific protesting behaviors). Examples of on-task behaviors included attending to the screen and completing task activities. Examples of protesting behaviors included complaining about stimuli volume or the inability to hear. Chronological age, autism features, language ability, audiovisual speech integration as measured by psychophysical tasks, tactile responsiveness, and nonverbal intelligence quotient were evaluated as putative predictors and/or moderators of effects on behaviors of interest. RESULTS: In general, participants were highly engaged in the task, and there were few protests, reflecting more than 90% and fewer than 0.5% of coded intervals, respectively. We did not detect any statistically significant effects of group or RM system use on task engagement. Nonautistic youth were engaged in the listening-in-noise task for an average of 97.45% of intervals, whereas autistic youth were engaged in the listening-in-noise task for an average of 94.25% of intervals. In contrast, verbal, stimulus-specific protesting in the listening-in-noise task was significantly reduced, on average, in the RM (0.04% of intervals) versus the No RM (0.2% of intervals) conditions. There were no effects related to group for this behaviorally coded outcome. In addition, select participant characteristics predicted engagement within conditions across participants. Greater language ability and nonverbal intelligence quotient predicted increased engagement when not using an RM system. Increased features of autism and wider temporal binding windows for audiovisual speech predicted reduced engagement while using an RM system, and greater audiovisual integration predicted increased engagement while using an RM system. CONCLUSIONS: The results of this study suggest that RM system use reduces verbal, stimulus-specific protesting, which likely reflects difficulty engaging when listening in noise. The present study extends our previous study to provide additional empirical support for RM system use in autistic and nonautistic youth.

Electrophysiological Measures of Listening-in-Noise With and Without Remote Microphone System Use in Autistic and Non-Autistic Youth.

OBJECTIVES: This study examined the neural mechanisms by which remote microphone (RM) systems might lead to improved behavioral performance on listening-in-noise tasks in autistic and non-autistic youth. DESIGN: Cortical auditory evoked potentials (CAEPs) were recorded in autistic (n = 25) and non-autistic (n = 22) youth who were matched at the group level on chronological age ( M = 14.21 ± 3.39 years) and biological sex. Potentials were recorded during an active syllable identification task completed in quiet and in multi-talker babble noise with and without the use of an RM system. The effects of noise and RM system use on speech-sound-evoked P1-N1-P2 responses and the associations between the cortical responses and behavioral performance on syllable identification were examined. RESULTS: No group differences were observed for behavioral or CAEP measures of speech processing in quiet or in noise. In the combined sample, syllable identification in noise was less accurate and slower than in the quiet condition. The addition of the RM system to the noise condition restored accuracy, but not the response speed, to the levels observed in quiet. The CAEP analyses noted amplitude reductions and latency delays in the noise compared with the quiet condition. The RM system use increased the N1 amplitude as well as reduced and delayed the P2 response relative to the quiet and noise conditions. Exploratory brain-behavior correlations revealed that larger N1 amplitudes in the RM condition were associated with greater behavioral accuracy of syllable identification. Reduced N1 amplitude and accelerated P2 response were associated with shorter syllable identification response times when listening with the RM system. CONCLUSIONS: Findings suggest that although listening-in-noise with an RM system might remain effortful, the improved signal to noise ratio facilitates attention to the sensory features of the stimuli and increases speech sound identification accuracy.

Affinity of small-molecule solutes to hydrophobic, hydrophilic, and chemically patterned interfaces in aqueous solution.

Performance of membranes for water purification is highly influenced by the interactions of solvated species with membrane surfaces, including surface adsorption of solutes upon fouling. Current efforts toward fouling-resistant membranes often pursue surface hydrophilization, frequently motivated by macroscopic measures of hydrophilicity, because hydrophobicity is thought to increase solute-surface affinity. While this heuristic has driven diverse membrane functionalization strategies, here we build on advances in the theory of hydrophobicity to critically examine the relevance of macroscopic characterizations of solute-surface affinity. Specifically, we use molecular simulations to quantify the affinities to model hydroxyl- and methyl-functionalized surfaces of small, chemically diverse, charge-neutral solutes represented in produced water. We show that surface affinities correlate poorly with two conventional measures of solute hydrophobicity, gas-phase water solubility and oil-water partitioning. Moreover, we find that all solutes show attraction to the hydrophobic surface and most to the hydrophilic one, in contrast to macroscopically based hydrophobicity heuristics. We explain these results by decomposing affinities into direct solute interaction energies (which dominate on hydroxyl surfaces) and water restructuring penalties (which dominate on methyl surfaces). Finally, we use an inverse design algorithm to show how heterogeneous surfaces, with multiple functional groups, can be patterned to manipulate solute affinity and selectivity. These findings, importantly based on a range of solute and surface chemistries, illustrate that conventional macroscopic hydrophobicity metrics can fail to predict solute-surface affinity, and that molecular-scale surface chemical patterning significantly influences affinity-suggesting design opportunities for water purification membranes and other engineered interfaces involving aqueous solute-surface interactions.

Resetting proteostasis with ISRIB promotes epithelial differentiation to attenuate pulmonary fibrosis.

Pulmonary fibrosis is a relentlessly progressive and often fatal disease with a paucity of available therapies. Genetic evidence implicates disordered epithelial repair, which is normally achieved by the differentiation of small cuboidal alveolar type 2 (AT2) cells into large, flattened alveolar type 1 (AT1) cells as an initiating event in pulmonary fibrosis pathogenesis. Using models of pulmonary fibrosis in young adult and old mice and a model of adult alveologenesis after pneumonectomy, we show that administration of ISRIB, a small molecule that restores protein translation by EIF2B during activation of the integrated stress response (ISR), accelerated the differentiation of AT2 into AT1 cells. Accelerated epithelial repair reduced the recruitment of profibrotic monocyte-derived alveolar macrophages and ameliorated lung fibrosis. These findings suggest a dysfunctional role for the ISR in regeneration of the alveolar epithelium after injury with implications for therapy.

Early Identification and Management of Cervical Impairments in Pediatric Patients With Concussion May Reduce Risk of Delayed Recovery.

OBJECTIVE: Previous research, including high-quality systematic reviews, has found that cervical injury, which often accompanies concussive head injury, can delay recovery from concussion. One pilot randomized controlled trial found that focused cervical assessment and appropriate intervention in children and young adults with persisting postconcussive symptoms (PPCS) improved recovery outcomes. Our sports medicine clinics adopted this approach early (within 2 weeks) in children (aged 10-18 years) after concussion. This study describes our clinical management protocol and compares the recovery trajectories in children after concussion with and without a concomitant cervical injury. DESIGN: Prospective cohort study. SETTING: Three university-affiliated outpatient sports medicine clinics from September 2016 to December 2019. PATIENTS: One-hundred thirty-four concussed children with cervical impairment (mean age 14.9 years, 65% male, and 6.2 days since concussion) were compared with 130 concussed children without cervical impairment (mean age 14.9 years, 57% male, and 6.0 days since concussion). INDEPENDENT VARIABLES: Examination findings related to the cervical spine (range of motion, cervical spasm, and cervical tenderness). MAIN OUTCOME MEASURES: Recovery time (measured in days), concussion symptom burden (Postconcussion Symptom Scale), and incidence of PPCS. RESULTS: Children with cervical impairment reported a higher initial symptom burden; however, there were no differences in recovery time (33.65 [28.20-39.09] days vs 35.98 [27.50-44.45] days, P = 0.651) or incidence of PPCS (40.0% vs 34.3%, P = 0.340). CONCLUSIONS: We conclude that within this pediatric population, early identification and management of cervical injuries concomitant with concussion may reduce the risk of delayed recovery.

An Exploratory Analysis of Physical Examination Subtypes in Pediatric Athletes With Concussion.

OBJECTIVE: Pediatric athletes with concussion present with a variety of impairments on clinical assessment and require individualized treatment. The Buffalo Concussion Physical Examination is a brief, pertinent clinical assessment for individuals with concussion. The purpose of this study was to identify physical examination subtypes in pediatric athletes with concussion within 2 weeks of injury that are relevant to diagnosis and treatment. DESIGN: Secondary analysis of a published cohort study and clinician consensus. SETTING: Three university-affiliated sports medicine centers. PARTICIPANTS: Two hundred seventy children (14.9 ± 1.9 years). INDEPENDENT VARIABLES: Orthostatic intolerance, horizontal and vertical saccades, smooth pursuits, vestibulo-ocular reflex, near-point convergence, complex tandem gait, neck range of motion, neck tenderness, and neck spasm. MAIN OUTCOME MEASURES: Correlations between independent variables were calculated, and network graphs were made. k -means and hierarchical clustering were used to identify clusters of impairments. Optimal number of clusters was assessed. Results were reviewed by experienced clinicians and consensus was reached on proposed subtypes. RESULTS: Physical examination clusters overlapped with each other, and no optimal number of clusters was identified. Clinician consensus suggested 3 possible subtypes: (1) visio-vestibular (horizontal and vertical saccades, smooth pursuits, and vestibulo-ocular reflex), (2) cervicogenic (neck range of motion and spasm), and (3) autonomic/balance (orthostatic intolerance and complex tandem gait). CONCLUSIONS: Although we identified 3 physical examination subtypes, it seemed that physical examination findings alone are not enough to define subtypes that are both statistically supported and clinically relevant, likely because they do not include symptoms, assessment of mood or cognitive problems, or graded exertion testing.