Utility of Genome Sequencing After Nondiagnostic Exome Sequencing in Unexplained Pediatric Epilepsy.

Importance: Epilepsy is the most common neurological disorder of childhood. Identifying genetic diagnoses underlying epilepsy is critical to developing effective therapies and improving outcomes. Most children with non-acquired (unexplained) epilepsy remain genetically unsolved, and the utility of genome sequencing after nondiagnostic exome sequencing is unknown. Objective: To determine the diagnostic (primary) and clinical (secondary) utility of genome sequencing after nondiagnostic exome sequencing in individuals with unexplained pediatric epilepsy. Design: This cohort study performed genome sequencing and comprehensive analyses for 125 participants and available biological parents enrolled from August 2018 to May 2023, with data analysis through April 2024 and clinical return of diagnostic and likely diagnostic genetic findings. Clinical utility was evaluated. Setting: Pediatric referral center. Participants: Participants with unexplained pediatric epilepsy and previous nondiagnostic exome sequencing; biological parents when available. Exposures: Short-read genome sequencing and analysis. Main Outcomes and Measures: Primary outcome measures were the diagnostic yield of genome sequencing, defined as the percentage of participants receiving a diagnostic or likely diagnostic genetic finding, and the unique diagnostic yield of genome sequencing, defined as the percentage of participants receiving a diagnostic or likely diagnostic genetic finding that required genome sequencing. The secondary outcome measure was clinical utility of genome sequencing, defined as impact on evaluation, treatment, or prognosis for the participant or their family. Results: 125 participants (58 [46%] female) were enrolled with median age at seizure onset 3 [IQR 1.25, 8] years, including 44 (35%) with developmental and epileptic encephalopathies. The diagnostic yield of genome sequencing was 7.2% (9/125), with diagnostic genetic findings in five cases and likely diagnostic genetic findings in four cases. Among the solved cases, 7/9 (78%) required genome sequencing for variant detection (small copy number variant, three noncoding variants, and three difficult to sequence small coding variants), for a unique diagnostic yield of genome sequencing of 5.6% (7/125). Clinical utility was documented for 4/9 solved cases (44%). Conclusions and Relevance: These findings suggest that genome sequencing can have diagnostic and clinical utility after nondiagnostic exome sequencing and should be considered for patients with unexplained pediatric epilepsy.

Small molecule inhibition of glycogen synthase I reduces muscle glycogen content and improves biomarkers in a mouse model of Pompe disease.

Pompe disease is a rare genetic disorder caused by a deficiency of the enzyme acid alpha-glucosidase (GAA). This enzyme is responsible for breaking down glycogen, leading to the abnormal accumulation of glycogen, which results in progressive muscle weakness and metabolic dysregulation. In this study, we investigated the hypothesis that the small molecule inhibition of glycogen synthase I (GYS1) may reduce muscle glycogen content and improve metabolic dysregulation in a mouse model of Pompe disease. To address this hypothesis, we studied four groups of male mice: a control group of wild-type B6129SF1/J mice fed either regular chow (WT) or a GYS1 inhibitor (MZ-101) diet (WT-GYS1), and Pompe model mice B6;129-Gaatm1Rabn/J fed either regular chow (GAA-KO) or MZ-101 diet (GAA-GYS1) for 7 days. Our findings revealed that GAA-KO mice exhibited abnormal glycogen accumulation in the gastrocnemius, heart, and diaphragm. In contrast, inhibiting GYS1 reduced glycogen levels in all tissues compared to GAA-KO mice. Furthermore, GAA-KO mice displayed reduced spontaneous activity during the dark cycle compared to WT mice, while GYS1 inhibition counteracted this effect. Compared to GAA-KO mice, GAA-GYS1 mice exhibited improved glucose tolerance and whole-body insulin sensitivity. These improvements in insulin sensitivity could be attributed to increased AMPK phosphorylation in the gastrocnemius of WT-GYS1 and GAA-GYS1 mice. Additionally, the GYS1 inhibitor led to a reduction in the phosphorylation of GSS641 and the LC3 autophagy marker. Together, our results suggest that targeting GYS1 could serve as a potential strategy for treating glycogen storage disorders and metabolic dysregulation.

Effect of positive allosteric modulation and orthosteric agonism of dopamine D2-like receptors on respiration in mouse models of Rett syndrome.

Rett syndrome (RTT) is an autism spectrum disorder caused by loss-of-function mutations in the methyl-CPG-binding protein 2 (Mecp2) gene. Frequent apneas and irregular breathing are prevalent in RTT, and also occur in rodent models of the disorder, including Mecp2Bird and Mecp2R168X mice. Sarizotan, a serotonin 5-HT1a and dopamine D2-like receptor agonist, reduces the incidence of apneas and irregular breathing in mouse models of RTT (Abdala et al., 2014). Targeting the 5HT1a receptor alone also improves respiration in RTT mice (Levitt et al., 2013). However, the contribution of D2-like receptors in correcting these respiratory disturbances remains untested. PAOPA, a dopamine D2-like receptor positive allosteric modulator, and quinpirole, a dopamine D2-like receptor orthosteric agonist, were used in conjunction with whole-body plethysmography to evaluate whether activation of D2-like receptors is sufficient to improve breathing disturbances in female heterozygous Mecp2Bird/+ and Mecp2R168X/+ mice. PAOPA did not significantly change apnea incidence or irregularity score in RTT mice. PAOPA also had no effect on the ventilatory response to hypercapnia (7 % CO2). In contrast, quinpirole reduced apnea incidence and irregularity scores and improved the hypercapnic ventilatory response in Mecp2R168X/+ and Mecp2Bird/+ mice, while also reducing respiratory rate. These results suggest that D2-like receptors could contribute to the positive effects of sarizotan in the correction of respiratory abnormalities in Rett syndrome. However, positive allosteric modulation of D2-like receptors alone was not sufficient to evoke these effects.

Cytosolic calcium regulates hepatic mitochondrial oxidation, intrahepatic lipolysis, and gluconeogenesis via CAMKII activation.

To examine the roles of mitochondrial calcium Ca2+ ([Ca2+]mt) and cytosolic Ca2+ ([Ca2+]cyt) in the regulation of hepatic mitochondrial fat oxidation, we studied a liver-specific mitochondrial calcium uniporter knockout (MCU KO) mouse model with reduced [Ca2+]mt and increased [Ca2+]cyt content. Despite decreased [Ca2+]mt, deletion of hepatic MCU increased rates of isocitrate dehydrogenase flux, α-ketoglutarate dehydrogenase flux, and succinate dehydrogenase flux in vivo. Rates of [14C16]palmitate oxidation and intrahepatic lipolysis were increased in MCU KO liver slices, which led to decreased hepatic triacylglycerol content. These effects were recapitulated with activation of CAMKII and abrogated with CAMKII knockdown, demonstrating that [Ca2+]cyt activation of CAMKII may be the primary mechanism by which MCU deletion promotes increased hepatic mitochondrial oxidation. Together, these data demonstrate that hepatic mitochondrial oxidation can be dissociated from [Ca2+]mt and reveal a key role for [Ca2+]cyt in the regulation of hepatic fat mitochondrial oxidation, intrahepatic lipolysis, gluconeogenesis, and lipid accumulation.

Brain state dynamics differ between eyes open and eyes closed rest.

The human brain exhibits spatio-temporally complex activity even in the absence of external stimuli, cycling through recurring patterns of activity known as brain states. Thus far, brain state analysis has primarily been restricted to unimodal neuroimaging data sets, resulting in a limited definition of state and a poor understanding of the spatial and temporal relationships between states identified from different modalities. Here, we applied hidden Markov model (HMM) to concurrent electroencephalography-functional magnetic resonance imaging (EEG-fMRI) eyes open (EO) and eyes closed (EC) resting-state data, training models on the EEG and fMRI data separately, and evaluated the models' ability to distinguish dynamics between the two rest conditions. Additionally, we employed a general linear model approach to identify the BOLD correlates of the EEG-defined states to investigate whether the fMRI data could be used to improve the spatial definition of the EEG states. Finally, we performed a sliding window-based analysis on the state time courses to identify slower changes in the temporal dynamics, and then correlated these time courses across modalities. We found that both models could identify expected changes during EC rest compared to EO rest, with the fMRI model identifying changes in the activity and functional connectivity of visual and attention resting-state networks, while the EEG model correctly identified the canonical increase in alpha upon eye closure. In addition, by using the fMRI data, it was possible to infer the spatial properties of the EEG states, resulting in BOLD correlation maps resembling canonical alpha-BOLD correlations. Finally, the sliding window analysis revealed unique fractional occupancy dynamics for states from both models, with a selection of states showing strong temporal correlations across modalities. Overall, this study highlights the efficacy of using HMMs for brain state analysis, confirms that multimodal data can be used to provide more in-depth definitions of state and demonstrates that states defined across different modalities show similar temporal dynamics.

Evolution of Whirly1 in the angiosperms: sequence, splicing, and expression in a clade of early transitional mycoheterotrophic orchids.

The plastid-targeted transcription factor Whirly1 (WHY1) has been implicated in chloroplast biogenesis, plastid genome stability, and fungal defense response, which together represent characteristics of interest for the study of autotrophic losses across the angiosperms. While gene loss in the plastid and nuclear genomes has been well studied in mycoheterotrophic plants, the evolution of the molecular mechanisms impacting genome stability is completely unknown. Here, we characterize the evolution of WHY1 in four early transitional mycoheterotrophic orchid species in the genus Corallorhiza by synthesizing the results of phylogenetic, transcriptomic, and comparative genomic analyses with WHY1 genomic sequences sampled from 21 orders of angiosperms. We found an increased number of non-canonical WHY1 isoforms assembled from all but the greenest Corallorhiza species, including intron retention in some isoforms. Within Corallorhiza, phylotranscriptomic analyses revealed the presence of tissue-specific differential expression of WHY1 in only the most photosynthetically capable species and a coincident increase in the number of non-canonical WHY1 isoforms assembled from fully mycoheterotrophic species. Gene- and codon-level tests of WHY1 selective regimes did not infer significant signal of either relaxed selection or episodic diversifying selection in Corallorhiza but did so for relaxed selection in the late-stage full mycoheterotrophic orchids Epipogium aphyllum and Gastrodia elata. Additionally, nucleotide substitutions that most likely impact the function of WHY1, such as nonsense mutations, were only observed in late-stage mycoheterotrophs. We propose that our findings suggest that splicing and expression changes may precede the selective shifts we inferred for late-stage mycoheterotrophic species, which therefore does not support a primary role for WHY1 in the transition to mycoheterotrophy in the Orchidaceae. Taken together, this study provides the most comprehensive view of WHY1 evolution across the angiosperms to date.

Handling and Tagging Techniques for Implanting Juvenile American Shad with a New Acoustic Microtransmitter.

The use of telemetry techniques to better understand the behavior and survival of juvenile American shad (Alosa sapidissima), as they migrate through hydropower systems, has been challenging because shad are widely known to be particularly sensitive to handling. The goal of this study was to develop a tagging protocol using a new, acoustic micro transmitter that minimizes the detrimental effects of the tagging process and maximizes post-tagging survival of juvenile American shad. Limiting out-of-water handling and the use of brackish saltwater (7.5 parts per thousand) before and after tagging improved survival for shad tagged using a simple pectoral implantation method. This protocol provides a detailed, step-by-step procedure for tagging juvenile shad with acoustic transmitters. Fish tagged using this procedure and held in the laboratory for 60 days had an 81.5% survival rate, compared to 70% for their untagged counterparts. The successful tagging and handling practices developed in this study could be applied to field telemetry studies of juvenile shad and other sensitive species.

Do Absorbable Sutures Work for Rectus Diastasis Repair in Abdominoplasty Patients?

Background: The standard treatment for rectus diastasis is rectus sheath plication during abdominoplasty. Lasting correction of diastasis is essential, but there is currently a debate as to whether absorbable or nonabsorbable rectus plication achieves a lower rate of recurrence. Objectives: The goal of this study is to assess long-term patient outcomes and the recurrence of rectus diastasis after plication with long-lasting absorbable sutures. Methods: A retrospective study of abdominoplasties performed by the senior author between 2018 and 2022 was performed. Only female patients with >6 months of follow-up were included. Plication of the rectus muscles was performed with a combination of interrupted, buried, figure of eight #0 polydioxanone suture and running #0 Maxon (Covidien, Mansfield, MA). Outcomes were assessed by physical examination at postoperative visits. A retrospective chart review was used to obtain demographic and perioperative information. Results: Seventy-one patients underwent abdominoplasty with an average follow-up of 21.1 months. The average age was 43 years, and the average BMI was 27 kg/m2. Correction of rectus diastasis was performed using absorbable sutures in all patients with no recurrence of diastasis in any patient (0% diastasis recurrence rate). Complications included delayed wound healing (11%), seroma (8.5%), hematoma (2.8%), and deep vein thrombosis/pulmonary embolism (2.8%). No patients needed reoperation. Conclusions: Abdominal wall plication using a double-layered, long-lasting absorbable suture closure is a safe, reliable, and effective method to address rectus diastasis during abdominoplasty. Our technique achieved no recurrence of diastasis in any patient and a low complication profile.

Musculoskeletal glomus tumor: a review of 218 lesions in 176 patients.

OBJECTIVE: To review the spectrum of clinical and imaging features of glomus tumor involving the musculoskeletal system including the typically solitary forms as well as the rarer multifocal forms (glomuvenous malformation and glomangiomatosis). MATERIALS AND METHODS: A retrospective review of our institutional pathology database from 1996 to 2023 identified 176 patients with 218 confirmed glomus tumors. Primary imaging studies included MRI (125), radiographs (100), clinical/intraoperative photos (77), and ultrasound (36). Lesions were divided into two groups: those that are typically solitary involving specific anatomic areas (finger, toe, soft tissue, coccyx, and bone), and those that are multifocal (glomuvenous malformation and glomangiomatosis). RESULTS: The finger was the most frequently involved anatomic location for the classic (sporadic) glomus tumor occurring in 51% of patients, 77% of which were women, with the nail plate involved in more of the 75% of cases. Sporadic lesions involving the skin, subcutaneous adipose tissue, and deep soft tissue were termed "soft tissue," and were identified in 39% of patients, 90% of which were in the extremities and in men in 81% of cases. The multifocal syndromic forms of glomus disease occurred in younger individuals and involved less than 6% of the study group. Patients with glomuvenous malformation presented early with predominantly cutaneous involvement, while those with glomangiomatosis present later, often with both superficial and deep involvement, and a high rate of local tumor recurrence. CONCLUSION: While glomus tumor is generally uncommon, it frequently involves the musculoskeletal extremities. Knowledge of the spectrum of characteristic locations and appearances will facilitate definitive diagnosis.

Impact of Bariatric Surgery on Asthma Severity and Medication Use.

INTRODUCTION: Bariatric surgery improves many obesity-related comorbidities, yet the literature remains inconclusive on the impact of bariatric surgery on asthma. Our primary objective was to identify the long-term impact of bariatric surgery on asthma severity and medication use. METHODS: A retrospective review was completed of all patients with a diagnosis of asthma who underwent bariatric surgery over 10 years at a single institution. Primary outcomes were the number of asthma medications prescribed at five time points (preoperative, postoperative < 18 months, 19-36 months, 37-60 months, 60 + months) after bariatric surgery. Secondary outcomes were spirometry results and BMI. RESULTS: There were 260 patients with 84.6% female predominance. There were 168 sleeve gastrectomy patients and 92 Roux-en-Y gastric bypass patients. Mean age was 47.6 ± 10.7 years, mean BMI was 46.0 ± 6.8 kg/m2, and 54.2% were previous tobacco users. The total number of patients on two or more asthma medications decreased from 46% preoperatively to 41% at 18 months, to 36% at 36 months, and to 32% at 60 months after surgery. The total number of patients free from asthma medication increased from 25% preoperatively to 33% at 60 months postoperatively. Asthma medication use decreased in both surgery groups, and neither operation demonstrated superiority. No significant improvement nor differences were found between groups at any time point regarding FEV1/FVC ratio spirometry measures. CONCLUSION: Bariatric surgery reduces the use of medications taken for management of asthma. The amount of asthma medication usage decreases with time and is sustained at 60 months after bariatric surgery.

Are Internal Fragments Observable in Electron Based Top-Down Mass Spectrometry?

Protein tandem mass spectrometry (MS/MS) often generates sequence-informative fragments from backbone bond cleavages near the termini. This lack of fragmentation in the protein interior is particularly apparent in native top-down mass spectrometry (MS). Improved sequence coverage, critical for reliable annotation of posttranslational modifications and sequence variants, may be obtained from internal fragments generated by multiple backbone cleavage events. However, internal fragment assignments can be error prone due to isomeric/isobaric fragments from different parts of a protein sequence. Also, internal fragment generation propensity depends on the chosen MS/MS activation strategy. Here, we examine internal fragment formation in electron capture dissociation (ECD) and electron transfer dissociation (ETD) following native and denaturing MS, as well as LC/MS of several proteins. Experiments were undertaken on multiple instruments, including quadrupole time-of-flight, Orbitrap, and high-field Fourier-transform ion cyclotron resonance (FT-ICR) across four laboratories. ECD was performed at both ultrahigh vacuum and at similar pressure to ETD conditions. Two complementary software packages were used for data analysis. When feasible, ETD-higher energy collision dissociation MS3 was performed to validate/refute potential internal fragment assignments, including differentiating MS3 fragmentation behavior of radical versus even-electron primary fragments. We show that, under typical operating conditions, internal fragments cannot be confidently assigned in ECD or ETD. On the other hand, such fragments, along with some b-type terminal fragments (not typically observed in ECD/ETD spectra) appear at atypical ECD operating conditions, suggesting they originate from a separate ion-electron activation process. Furthermore, atypical fragment ion types, e.g., x ions, are observed at such conditions as well as upon EThcD, presumably due to vibrational activation of radical z-type ions.

CIUSuite 3: Next-Generation CCS Calibration and Automated Data Analysis Tools for Gas-Phase Protein Unfolding Data.

Ion mobility-mass spectrometry (IM-MS) has become a technology deployed across a wide range of structural biology applications despite the challenges in characterizing closely related protein structures. Collision-induced unfolding (CIU) has emerged as a valuable technique for distinguishing closely related, iso-cross-sectional protein and protein complex ions through their distinct unfolding pathways in the gas phase. With the speed and sensitivity of CIU analyses, there has been a rapid growth of CIU-based assays, especially regarding biomolecular targets that remain challenging to assess and characterize with other structural biology tools. With information-rich CIU data, many software tools have been developed to automate laborious data analysis. However, with the recent development of new IM-MS technologies, such as cyclic IM-MS, CIU continues to evolve, necessitating improved data analysis tools to keep pace with new technologies and facilitating the automation of various data processing tasks. Here, we present CIUSuite 3, a software package that contains updated algorithms that support various IM-MS platforms and supports the automation of various data analysis tasks such as peak detection, multidimensional classification, and collision cross section (CCS) calibration. CIUSuite 3 uses local maxima searches along with peak width and prominence filters to detect peaks to automate CIU data extraction. To support both the primary CIU (CIU1) and secondary CIU (CIU2) experiments enabled by cyclic IM-MS, two-dimensional data preprocessing is deployed, which allows multidimensional classification. Our data suggest that additional dimensions in classification improve the overall accuracy of class assignments. CIUSuite 3 also supports CCS calibration for both traveling wave and drift tube IM-MS, and we demonstrate the accuracy of a new single-field CCS calibration method designed for drift tube IM-MS leveraging calibrant CIU data. Overall, CIUSuite 3 is positioned to support current and next-generation IM-MS and CIU assay development deployed in an automated format.

Time-Resolved Ion Mobility-Mass Spectrometry Reveals Structural Transitions in the Disassembly of Modular Polyketide Syntheses.

The type 1 polyketide synthase (PKS) assembly line uses its modular structure to produce polyketide natural products that form the basis of many pharmaceuticals. Currently, several cryoelectron microscopy (cryo-EM) structures of a multidomain PKS module have been constructed, but much remains to be learned. Here we utilize ion-mobility mass spectrometry (IM-MS) to record size and shape information and detect different conformational states of a 207 kDa didomain dimer comprised of ketosynthase (KS) and acyl transferase (AT), excised from full-length module. Furthermore, gas-phase stability differences between these different conformations are captured by collision induced unfolding (CIU) technology. Additionally, through tracking these forms as a function of time, we elucidate a detailed disassembly pathway for KS-AT dimers for the first time.

The LTAR Grazing Land Common Experiment at the Jornada Experimental Range: Old genetics, new precision technologies, and adaptive value chains.

Rangelands and the supply chains connected to them are central to the agrifood systems of the Southwestern United States. Local ranchers are simultaneously arid lands managers, herd managers, and marketing managers. To stay in business, they must adapt to unpredictable forage resources and markets while conserving soils and vegetation resources for the long term. As climate warming and drying exacerbate the complexity and difficulty of day-to-day production, producers and policymakers are seeking alternatives to "business as usual." The Long-Term Agroecosystem Research (LTAR)-Jornada team has developed a package of strategies to help producers adapt to the local and inter-regional challenges. The package includes heritage cattle, precision ranching systems, and adaptive value chains. Five ranches across the Southwest have adopted different combinations and are partnering with LTAR and each other to measure their benefits and drawbacks in real-world conditions. Opportunities for controlled experimentation differ among the ranches, so we use LTAR's indicator system to assess and compare results. Even as we invest in co-producing knowledge about these three strategies, we recognize that progressive aridification and urbanization of Southwestern rangelands create challenges for which a single "silver bullet" of agricultural innovation is unlikely to provide durable solutions. We are learning from our customers and stakeholders about ways to adjust the development of new options.

Gas-Phase Unfolding Reveals Stability Shifts Associated with Substrate Binding in Modular Polyketide Synthases.

Native mass spectrometry (MS), ion mobility (IM), and collision-induced unfolding (CIU) have all been widely used to study the binding of small molecules to proteins and their complexes. Despite many successes in detecting subtle gas-phase stability differences in smaller systems dominated by single-domain subunits, studies targeting complexes comprised of large, multidomain subunits still face many challenges. For example, polyketide synthases (PKSs) are multiprotein enzymes that use their modular architecture to produce polyketide natural products and form the basis for nearly one-third of pharmaceuticals. Here, we describe the development of CIU methods capable of extracting information from these multiprotein complexes and demonstrate the current limits of quantitative CIU technology by probing the stabilities ∼280 kDa PKS dimer protein complexes. Our approach detects the evidence of the stability shifts associated with substrate binding that accounts for <0.1% of the mass for the intact assembly.

A Phase 1 Assessment of the Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of (2R,6R)-Hydroxynorketamine in Healthy Volunteers.

(R,S)-Ketamine (ketamine) is a dissociative anesthetic that also possesses analgesic and antidepressant activity. Undesirable dissociative side effects and misuse potential limit expanded use of ketamine in several mental health disorders despite promising clinical activity and intensifying medical need. (2R,6R)-Hydroxynorketamine (RR-HNK) is a metabolite of ketamine that lacks anesthetic and dissociative activity but maintains antidepressant and analgesic activity in multiple preclinical models. To enable future assessments in selected human indications, we report the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of RR-HNK in a Phase 1 study in healthy volunteers (NCT04711005). A six-level single-ascending dose (SAD) (0.1-4 mg/kg) and a two-level multiple ascending dose (MAD) (1 and 2 mg/kg) study was performed using a 40-minute IV administration emulating the common practice for ketamine administration for depression. Safety assessments showed RR-HNK possessed a minimal adverse event profile and no serious adverse events at all doses examined. Evaluations of dissociation and sedation demonstrated that RR-HNK did not possess anesthetic or dissociative characteristics in the doses examined. RR-HNK PK parameters were measured in both the SAD and MAD studies and exhibited dose-proportional increases in exposure. Quantitative electroencephalography (EEG) measurements collected as a PD parameter based on preclinical findings and ketamine's established effect on gamma-power oscillations demonstrated increases of gamma power in some participants at the lower/mid-range doses examined. Cerebrospinal fluid examination confirmed RR-HNK exposure within the central nervous system (CNS). Collectively, these data demonstrate RR-HNK is well tolerated with an acceptable PK profile and promising PD outcomes to support the progression into Phase 2.

Let-7 restrains an oncogenic epigenetic circuit in AT2 cells to prevent ectopic formation of fibrogenic transitional cell intermediates and pulmonary fibrosis.

Analysis of lung alveolar type 2 (AT2) progenitor stem cells has highlighted fundamental mechanisms that direct their differentiation into alveolar type 1 cells (AT1s) in lung repair and disease. However, microRNA (miRNA) mediated post-transcriptional mechanisms which govern this nexus remain understudied. We show here that the let-7 miRNA family serves a homeostatic role in governance of AT2 quiescence, specifically by preventing the uncontrolled accumulation of AT2 transitional cells and by promoting AT1 differentiation to safeguard the lung from spontaneous alveolar destruction and fibrosis. Using mice and organoid models with genetic ablation of let-7a1/let-7f1/let-7d cluster (let-7afd) in AT2 cells, we demonstrate prevents AT1 differentiation and results in aberrant accumulation of AT2 transitional cells in progressive pulmonary fibrosis. Integration of enhanced AGO2 UV-crosslinking and immunoprecipitation sequencing (AGO2-eCLIP) with RNA-sequencing from AT2 cells uncovered the induction of direct targets of let-7 in an oncogene feed-forward regulatory network including BACH1/EZH2 which drives an aberrant fibrotic cascade. Additional analyses by CUT&RUN-sequencing revealed loss of let-7afd hampers AT1 differentiation by eliciting aberrant histone EZH2 methylation which prevents the exit of AT2 transitional cells into terminal AT1s. This study identifies let-7 as a key gatekeeper of post-transcriptional and epigenetic chromatin signals to prevent AT2-driven pulmonary fibrosis.

Noninvasive Ventilation for Moderate to Severe Acute Respiratory Distress Syndrome Due to COVID-19.

BACKGROUND: Use of noninvasive ventilation in patients with acute respiratory distress syndrome (ARDS) is debated. The COVID-19 pandemic posed challenges due to high patient volumes and worldwide resource strain. OBJECTIVES: To determine associations between use of noninvasive ventilation in adult patients with moderate to severe ARDS due to SARS-CoV-2 pneumonia, progression to intubation, and hospital mortality. METHODS: This retrospective cohort study included patients in an institutional COVID-19 registry. Adult patients were included if they were admitted for COVID-19 between March 1, 2020, and March 31, 2022, and developed moderate to severe ARDS. Primary outcomes were progression to intubation and hospital mortality in patients who received noninvasive ventilation or mechanical ventilation. A secondary outcome was successful treatment with noninvasive ventilation without intubation. RESULTS: Of 823 patients who met inclusion criteria, 454 (55.2%) did not receive noninvasive ventilation and 369 (44.8%) received noninvasive ventilation. Patients receiving noninvasive ventilation were more likely to require mechanical ventilation than were patients not receiving noninvasive ventilation. Among patients requiring endotracheal intubation, those receiving noninvasive ventilation had a higher likelihood of mortality. Patients receiving noninvasive ventilation had lower severity-adjusted odds of survival to discharge without intubation than did patients not receiving noninvasive ventilation. CONCLUSION: Patients with moderate to severe ARDS due to SARS-CoV-2 pneumonia treated with noninvasive ventilation had increased likelihood of progression to endotracheal intubation and hospital mortality.

Molecular insights into the interaction between a disordered protein and a folded RNA.

Intrinsically disordered protein regions (IDRs) are well-established as contributors to intermolecular interactions and the formation of biomolecular condensates. In particular, RNA-binding proteins (RBPs) often harbor IDRs in addition to folded RNA-binding domains that contribute to RBP function. To understand the dynamic interactions of an IDR-RNA complex, we characterized the RNA-binding features of a small (68 residues), positively charged IDR-containing protein, SERF. At high concentrations, SERF and RNA undergo charge-driven associative phase separation to form a protein- and RNA-rich dense phase. A key advantage of this model system is that this threshold for demixing is sufficiently high that we could use solution-state biophysical methods to interrogate the stoichiometric complexes of SERF with RNA in the one-phase regime. Herein, we describe our comprehensive characterization of SERF alone and in complex with a small fragment of the HIV-1 TAR RNA (TAR) with complementary biophysical methods and molecular simulations. We find that this binding event is not accompanied by the acquisition of structure by either molecule; however, we see evidence for a modest global compaction of the SERF ensemble when bound to RNA. This behavior likely reflects attenuated charge repulsion within SERF via binding to the polyanionic RNA and provides a rationale for the higher-order assembly of SERF in the context of RNA. We envision that the SERF-RNA system will lower the barrier to accessing the details that support IDR-RNA interactions and likewise deepen our understanding of the role of IDR-RNA contacts in complex formation and liquid-liquid phase separation.

Liquid-phase separations coupled with ion mobility-mass spectrometry for next-generation biopharmaceutical analysis.

INTRODUCTION: The pharmaceutical industry continues to expand its search for innovative biotherapeutics. The comprehensive characterization of such therapeutics requires many analytical techniques to fully evaluate critical quality attributes, making analysis a bottleneck in discovery and development timelines. While thorough characterization is crucial for ensuring the safety and efficacy of biotherapeutics, there is a need to further streamline analytical characterization and expedite the overall timeline from discovery to market. AREAS COVERED: This review focuses on recent developments in liquid-phase separations coupled with ion mobility-mass spectrometry (IM-MS) for the development and characterization of biotherapeutics. We cover uses of IM-MS to improve the characterization of monoclonal antibodies, antibody-drug conjugates, host cell proteins, glycans, and nucleic acids. This discussion is based on an extensive literature search using Web of Science, Google Scholar, and SciFinder. EXPERT OPINION: IM-MS has the potential to enhance the depth and efficiency of biotherapeutic characterization by providing additional insights into conformational changes, post-translational modifications, and impurity profiles. The rapid timescale of IM-MS positions it well to enhance the information content of existing assays through its facile integration with standard liquid-phase separation techniques that are commonly used for biopharmaceutical analysis.