Publications

IMPORTANCE: Restless legs syndrome (RLS) is a sleep-related movement disorder that affects approximately 3% of US adults to a clinically significant extent and can cause substantial sleep disturbance.

OBSERVATIONS: Restless legs syndrome is characterized by an overwhelming urge to move the limbs, typically the legs, often accompanied by unpleasant limb sensations (eg, achiness, tingling). Symptoms, provoked by immobility, are relieved while moving and are typically present or most severe in the evening or at night. Restless legs syndrome symptoms may lead to difficulty falling asleep, staying asleep, or returning to sleep. According to population-based studies, approximately 8% of US adults experience RLS symptoms of any frequency annually and 3% experience moderately or severely distressing symptoms at least twice weekly. Patients with RLS have impaired quality of life and elevated rates of cardiovascular disease (29.6% with coronary artery disease, stroke, or heart failure), depression (30.4%), and suicidal ideation or self-harm (0.35 cases/1000 person-years). Restless legs syndrome is common among patients with multiple sclerosis (27.5%), end-stage kidney disease (24%), and iron deficiency anemia (23.9%); during pregnancy and especially in the third trimester (22%); with peripheral neuropathy (eg, diabetic, idiopathic; 21.5%); and with Parkinson disease (20%). Other risk factors include family history of RLS, northern European descent, female sex (2:1 vs male sex), and older age (RLS prevalence of 10% in adults ≥65 years). Restless legs syndrome is diagnosed based on clinical history; polysomnography is not recommended for diagnosis. Iron supplementation with ferrous sulfate (325-650 mg daily or every other day) or intravenous iron (1000 mg) should be initiated for serum ferritin level less than or equal to 100 ng/mL or transferrin saturation less than 20%. If possible, medications associated with RLS, including serotonergic antidepressants, dopamine antagonists, and centrally acting H1 antihistamines (eg, diphenhydramine), should be discontinued. Gabapentinoids (eg, gabapentin, gabapentin enacarbil, pregabalin) are first-line pharmacologic therapy. In randomized clinical trials, approximately 70% of patients treated with gabapentinoids had much or very much improved RLS symptoms vs approximately 40% with placebo (P < .001). Dopamine agonists (eg, ropinirole, pramipexole, rotigotine) are no longer recommended as first-line medications due to the risk of augmentation, an iatrogenic worsening of RLS symptoms, which has an annual incidence of 7% to 10% with these medications. Patients who do not improve with first-line treatment or have augmented RLS often benefit from low-dose opioids (eg, methadone 5-10 mg daily).

CONCLUSIONS AND RELEVANCE: Restless legs syndrome affects approximately 3% of adults and can have negative effects on sleep and quality of life. Initial management includes cessation of exacerbating medications, as well as iron supplementation for patients with low-normal iron indices. If medication therapy is indicated, gabapentinoids are first-line treatment.

Selective degradation of a disease-associated protein of interest (POI) is a powerful therapeutic strategy. FDA-approved and investigational glue and degrader drugs function by recruiting a POI to an E3 ubiquitin ligase that mediates POI polyubiquitination and triggers proteasomal degradation. However, using E3 ligases as an intermediary and requiring POI polyubiquitination makes this mechanism of action complex and difficult to rationalize and optimize. These issues have led to interest in evaluating whether direct recruitment of non-ubiquitinated POIs to the proteasome might achieve the same pharmacological outcome. Here, we examined the potential of direct-to-proteasome non-ubiquitinated POI recruitment. Using a tag strategy, we first demonstrated that the proteasomal 19S cap region proteins, RPN13 and RPN1, can recruit non-ubiquitinated POI model proteins, such as BRD4, to the proteasome and induce degradation. Subsequently, we developed small molecule-based bifunctional recruiter molecules (Proteasome Cap Targeting Chimeras, CAP-TACs) and showed that they recruit several distinct POIs, including BRD4, PRMT5, and FKBP12, to specific subunits in the 19S cap region and induce their ubiquitination-independent, proteasome-dependent degradation. This study provides further evidence that bifunctional small molecules can re-localize POIs to the proteasome and induce their degradation in the absence of ubiquitination, which broadens the capabilities of targeted protein degradation.

Multiplexed fluorescent imaging methods are essential for studying cellular function by visualization of biomolecules in cells and tissues with high-resolution spatial information but usually suffer from limitations of 3-5 multiplexity because of spectral overlap between the used fluorophores. We introduce a method using scaled-up sequential DNA displacement reactions to enable highly multiplexed fluorescent imaging. The fluorescent signals of targets are encoded in different DNA probes in a single step. Each DNA probe's fluorescent signal can be activated for imaging and removed to avoid signal overlap with targets in the next round by two sequential, independent DNA displacement reactions. Massive targets in situ can be imaged when a scaled-up DNA displacement reaction is applied sequentially. We experimentally screened a set of rapid and orthogonal DNA displacement sequences from 144 different in situ reactions and developed 25 DNA probes with 50 selected displacement sequences for multiplexed imaging. We demonstrated 25-plex RNA imaging in a single fluorophore channel in fixed cells within 20 min, using the 50 sequential displacement reactions that consisted of 100 DNA strands simultaneously. To further demonstrate the robustness and practical usage, we showed 24-plex RNA imaging with the method in retinal tissues and resolved different cell types. Because of the vast sequence design space of DNA probes, theoretically unlimited multiplexity can be achieved. This method significantly simplifies the high-plex fluorescent imaging process with preprogrammed complex dynamic DNA nanotechnology and has broad biotechnical applications for future medicine and diagnostics.

BACKGROUND: The COVID-19 pandemic presented unprecedented challenges to hospital system and critical care resources, leading to significant changes to operations and patient care. There are limited national data on these changes and instances of unsanctioned deviations from patient care, yet understanding the COVID response is key to future preparedness efforts. We sought to understand how hospitals and states navigated scarcity during COVID-19, particularly in the absence of a declaration of crisis standards of care.

METHODS: Between February 2022 and September 2022 we conducted 34 interviews with 36 leaders of U.S. states' COVID-19 planning and response efforts. Interviews were transcribed verbatim and verified. We analyzed interviews using iterative inductive thematic analysis for descriptions of resource scarcity and changes to policies and procedures to prevent rationing lifesaving care.

RESULTS: Nearly all participants described equipment and personnel scarcity in their home institution or state during COVID-19. Hospitals across regions and states developed formal and informal coordination processes for load and resource sharing in response to influxes of high-acuity patients, avoiding formal rationing of lifesaving resources in many regions. Participants also described unsanctioned patient triage, early discharge, and patients counseled to accept less aggressive care (e.g., premature transition to hospice) in states that had not declared crisis standards of care.

CONCLUSIONS: Extending limited resources and inter-institutional collaboration helped avoid formal rationing. Yet, patient care was unquestionably impacted due to scarcity, both real and perceived. Reports of using hospital triage protocols to deny patients lifesaving care outside of formally recognized crisis conditions and attempts to nudge patients to accept less-resource-intensive care are concerning. This may have had disproportionate effects on older adults, individuals with disabilities, and racial and ethnic minoritized groups. To avoid unsanctioned deviations from standard practice in future health emergencies, we recommend that transparent and equitable triage protocols are implemented with robust oversight.

The airway epithelium, a primary target for viral infection, plays a critical role in disease response-particularly in individuals with preexisting airway conditions such as cystic fibrosis (CF). At the onset of the SARS-CoV-2 pandemic, individuals with CF were expected to have severe outcomes based on earlier viral outbreaks; however, those on effective CF transmembrane conductance regulator (CFTR) modulators showed milder disease. Patients with CF on the CFTR modulator combination elexacaftor/tezacaftor/ivacaftor (ETI) combination therapy showed attenuated viral infection and reduced airway epithelial damage. To investigate how this is accomplished, we used an induced pluripotent stem cells (iPSC)-derived airway epithelium model of CF and syngeneic CFTR-corrected cells to examine responses to SARS-CoV-2 infection. CF iPSC-airways were significantly more susceptible to viral infection and epithelial injury compared with their corrected counterparts, despite comparable expression of viral entry factors. Strikingly, pretreatment with ETI conferred significant protection in CFTR-corrected and non-CF, wild-type (WT) airway epithelia, as well as in iPSC-derived and primary epithelia. Single-cell RNA sequencing analysis confirmed a heightened infection and proinflammatory response in CF iPSC-airways, whereas ETI treatment significantly reduced these responses in both CF and CFTR-corrected iPSC-airways. Mechanistically, ETI treatment led to increased type I interferon signaling and induction of antiviral genes, whereas expression of many other proinflammatory genes was suppressed in both CF and non-CF iPSC-airways. These results underscore the therapeutic promise of CFTR-modulators such as ETI in mitigating SARS-CoV-2 infection and inflammation, not only in CF airways but also in non-CF airways, highlighting the broad applicability of CFTR-modulators as a therapeutic strategy in viral pneumonia and inflammatory lung disease.NEW & NOTEWORTHY Using rigorously controlled iPSC-derived airway models, the study shows that CFTR-deficient cells are more vulnerable to SARS-CoV-2 and display stronger inflammation than syngeneic CFTR-corrected controls. The CFTR modulator ETI reduces viral injury and boosts antiviral pathways in both CF and non-CF cells. Even at baseline, CFTR modulation enhances antiviral responses and lowers inflammation. Overall, the findings reveal a broad protective antiviral effect of CFTR modulators and highlight their therapeutic promise in inflammatory lung disease.

OBJECTIVE: To measure the relative levels of signal and noise in expert diagnosis of epilepsy.

METHODS: Twenty multinational epileptologists independently reviewed 50 vignettes of adult and pediatric patients presenting with suspected seizure(s) on two separate occasions with a ≥30-day washout period. Experts provided a diagnosis of epilepsy or non-epilepsy based on clinical information and, if requested, routine EEG and neuroimaging data. Cases had an established clinical diagnosis of epilepsy or non-epilepsy based on capture of habitual paroxysmal events on video-EEG or long-term clinical follow-up. Experts' judgments were analyzed to decompose variability into different sources: signal (objective differences between cases), level noise (experts' bias toward over/under-diagnosis), pattern noise (experts' idiosyncratic reactions to specific case features), and occasion noise (inconsistency across occasions).

RESULTS: The probability of an expert making a different diagnosis for a given case on two different occasions was 16%. The probability of two different experts making a different diagnosis for the same case was 26%. Signal (case "difficulty") accounted for 66-69% of total variation, with 31-34% attributable to noise. Level noise was the largest contributor in the absence of EEG/neuroimaging results (23%), while pattern noise dominated when test results were available (24%). Occasion noise contributed relatively little (1%) but was still sufficient to cause diagnostic reversals in 16-22% between occasions.

SIGNIFICANCE: The degree of noise in expert diagnosis of epilepsy is substantial, stemming primarily from physicians' idiosyncratic interpretations of case features and variable dispositions toward over- or under-diagnosis. Strategies to improve reliability are needed, including standardized data collection protocols and structured decision algorithms. For "difficult cases," where expert reliability and accuracy are lowest, our findings support current clinical practice which favors early referral for video-EEG monitoring over reliance on diagnostic anchoring. This diagnostic pathway may become more accessible with advances in EEG technology (e.g., wearable devices) and artificial intelligence.