Counting the Cost of Daptomycin Versus Vancomycin in Hospitalized Patients: A Cost Minimization Analysis.

Daptomycin use for gram-positive infections has increased. This cost minimization analysis aimed to determine cost and/or time savings of daptomycin over vancomycin. The estimated hospital cost savings was US$166.41 per patient, and pharmacist time saved of almost 20 minutes per patient. Daptomycin has the potential to save both time and money.

Widespread alternative splicing dysregulation occurs presymptomatically in CAG expansion spinocerebellar ataxias.

The spinocerebellar ataxias (SCAs) are a group of dominantly inherited neurodegenerative diseases, several of which are caused by CAG expansion mutations (SCAs 1, 2, 3, 6, 7 and 12) and more broadly belong to the large family of over 40 microsatellite expansion diseases. While dysregulation of alternative splicing is a well defined driver of disease pathogenesis across several microsatellite diseases, the contribution of alternative splicing in CAG expansion SCAs is poorly understood. Furthermore, despite extensive studies on differential gene expression, there remains a gap in our understanding of presymptomatic transcriptomic drivers of disease. We sought to address these knowledge gaps through a comprehensive study of 29 publicly available RNA-sequencing datasets. We identified that dysregulation of alternative splicing is widespread across CAG expansion mouse models of SCAs 1, 3 and 7. These changes were detected presymptomatically, persisted throughout disease progression, were repeat length-dependent, and were present in brain regions implicated in SCA pathogenesis including the cerebellum, pons and medulla. Across disease progression, changes in alternative splicing occurred in genes that function in pathways and processes known to be impaired in SCAs, such as ion channels, synaptic signalling, transcriptional regulation and the cytoskeleton. We validated several key alternative splicing events with known functional consequences, including Trpc3 exon 9 and Kcnma1 exon 23b, in the Atxn1154Q/2Q mouse model. Finally, we demonstrated that alternative splicing dysregulation is responsive to therapeutic intervention in CAG expansion SCAs with Atxn1 targeting antisense oligonucleotide rescuing key splicing events. Taken together, these data demonstrate that widespread presymptomatic dysregulation of alternative splicing in CAG expansion SCAs may contribute to disease onset, early neuronal dysfunction and may represent novel biomarkers across this devastating group of neurodegenerative disorders.

Spectral features of non-nutritive suck dynamics in extremely preterm infants.

Background: Non-nutritive suck (NNS) is used to promote ororhythmic patterning and assess oral feeding readiness in preterm infants in the neonatal intensive care unit (NICU). While time domain measures of NNS are available in real time at cribside, our understanding of suck pattern generation in the frequency domain is limited. The aim of this study is to model the development of NNS in the frequency domain using Fourier and machine learning (ML) techniques in extremely preterm infants (EPIs). Methods: A total of 117 EPIs were randomized to a pulsed or sham orocutaneous intervention during tube feedings 3 times/day for 4 weeks, beginning at 30 weeks post-menstrual age (PMA). Infants were assessed 3 times/week for NNS dynamics until they attained 100% oral feeding or NICU discharge. Digitized NNS signals were processed in the frequency domain using two transforms, including the Welch power spectral density (PSD) method, and the Yule-Walker PSD method. Data analysis proceeded in two stages. Stage 1: ML longitudinal cluster analysis was conducted to identify groups (classes) of infants, each showing a unique pattern of change in Welch and Yule-Walker calculations during the interventions. Stage 2: linear mixed modeling (LMM) was performed for the Welch and Yule-Walker dependent variables to examine the effects of gestationally-aged (GA), PMA, sex (male, female), patient type [respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD)], treatment (NTrainer, Sham), intervention phase [1, 2, 3], cluster class, and phase-by-class interaction. Results: ML of Welch PSD method and Yule-Walker PSD method measures revealed three membership classes of NNS growth patterns. The dependent measures peak_Hz, PSD amplitude, and area under the curve (AUC) are highly dependent on PMA, but show little relation to respiratory status (RDS, BPD) or somatosensory intervention. Thus, neural regulation of NNS in the frequency domain is significantly different for each identified cluster (classes A, B, C) during this developmental period. Conclusions: Efforts to increase our knowledge of the evolution of the suck central pattern generator (sCPG) in preterm infants, including NNS rhythmogenesis will help us better understand the observed phenotypes of NNS production in both the frequency and time domains. Knowledge of those features of the NNS which are relatively invariant vs. other features which are modifiable by experience will likewise inform more effective treatment strategies in this fragile population.

Individual transcriptomic response to strength training for patients with myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1), the most common form of adult-onset muscular dystrophy, is caused by a CTG expansion resulting in significant transcriptomic dysregulation that leads to muscle weakness and wasting. While strength training is clinically beneficial in DM1, molecular effects had not been studied. To determine whether training rescued transcriptomic defects, RNA-Seq was performed on vastus lateralis samples from 9 male patients with DM1 before and after a 12-week strength-training program and 6 male controls who did not undergo training. Differential gene expression and alternative splicing analysis were correlated with the one-repetition maximum strength evaluation method (leg extension, leg press, hip abduction, and squat). While training program-induced improvements in splicing were similar among most individuals, rescued splicing events varied considerably between individuals. Gene expression improvements were highly varied between individuals, and the percentage of differentially expressed genes rescued after training were strongly correlated with strength improvements. Evaluating transcriptome changes individually revealed responses to the training not evident from grouped analysis, likely due to disease heterogeneity and individual exercise response differences. Our analyses indicate that transcriptomic changes are associated with clinical outcomes in patients with DM1 undergoing training and that these changes are often specific to the individual and should be analyzed accordingly.

Alternative Splicing Outcomes Across an RNA-Binding Protein Concentration Gradient.

Alternative splicing (AS) is a dynamic RNA processing step that produces multiple RNA isoforms from a single pre-mRNA transcript and contributes to the complexity of the cellular transcriptome and proteome. This process is regulated through a network of cis-regulatory sequence elements and trans-acting factors, most-notably RNA binding proteins (RBPs). The muscleblind-like (MBNL) and RNA binding fox-1 homolog (RBFOX) are two well characterized families of RBPs that regulate fetal to adult AS transitions critical for proper muscle, heart, and central nervous system development. To better understand how the concentration of these RBPs influences AS transcriptome wide, we engineered a MBNL1 and RBFOX1 inducible HEK-293 cell line. Modest induction of exogenous RBFOX1 in this cell line modulated MBNL1-dependent AS outcomes in 3 skipped exon events, despite significant levels of endogenous RBFOX1 and RBFOX2. Due to background RBFOX levels, we conducted a focused analysis of dose-dependent MBNL1 skipped exon AS outcomes and generated transcriptome wide dose-response curves. Analysis of this data demonstrates that MBNL1-regulated exclusion events may require higher concentrations of MBNL1 protein to properly regulate AS outcomes compared to inclusion events and that multiple arrangements of YGCY motifs can produce similar splicing outcomes. These results suggest that rather than a simple relationship between the organization of RBP binding sites and a specific splicing outcome, that complex interaction networks govern both AS inclusion and exclusion events across a RBP gradient.

Quercetin selectively reduces expanded repeat RNA levels in models of myotonic dystrophy.

Myotonic dystrophy is a multisystemic neuromuscular disease caused by either a CTG repeat expansion in DMPK (DM1) or a CCTG repeat expansion in CNBP (DM2). Transcription of the expanded alleles produces toxic gain-of-function RNA that sequester the MBNL family of alternative splicing regulators into ribonuclear foci, leading to pathogenic mis-splicing. There are currently no approved treatments that target the root cause of disease which is the production of the toxic expansion RNA molecules. In this study, using our previously established HeLa DM1 repeat selective screening platform, we identified the natural product quercetin as a selective modulator of toxic RNA levels. Quercetin treatment selectively reduced toxic RNA levels and rescued MBNL dependent mis-splicing in DM1 and DM2 patient derived cell lines and in the HSALR transgenic DM1 mouse model where rescue of myotonia was also observed. Based on our data and its safety profile for use in humans, we have identified quercetin as a priority disease-targeting therapeutic lead for clinical evaluation for the treatment of DM1 and DM2.

Ventilation Strategies During Extracorporeal Membrane Oxygenation for Neonatal Respiratory Failure: Current Approaches Among Level IV Neonatal ICUs.

To describe ventilation strategies used during extracorporeal membrane oxygenation (ECMO) for neonatal respiratory failure among level IV neonatal ICUs (NICUs). DESIGN: Cross-sectional electronic survey. SETTING: Email-based Research Electronic Data Capture survey. PATIENTS: Neonates undergoing ECMO for respiratory failure at level IV NICUs. INTERVENTIONS: A 40-question survey was sent to site sponsors of regional referral neonatal ECMO centers participating in the Children's Hospitals Neonatal Consortium. Reminder emails were sent at 2- and 4-week intervals. MEASUREMENTS AND MAIN RESULTS: Twenty ECMO centers responded to the survey. Most primarily use venoarterial ECMO (65%); this percentage is higher (90%) for congenital diaphragmatic hernia. Sixty-five percent reported following protocol-based guidelines, with neonatologists primarily responsible for ventilator management (80%). The primary mode of ventilation was pressure control (90%), with synchronized intermittent mechanical ventilation (SIMV) comprising 80%. Common settings included peak inspiratory pressure (PIP) of 16-20 cm H2O (55%), positive end-expiratory pressure (PEEP) of 9-10 cm H2O (40%), I-time 0.5 seconds (55%), rate of 10-15 (60%), and Fio2 22-30% (65%). A minority of sites use high-frequency ventilation (HFV) as the primary mode (5%). During ECMO, 55% of sites target some degree of lung aeration to avoid complete atelectasis. Fifty-five percent discontinue inhaled nitric oxide (iNO) during ECMO, while 60% use iNO when trialing off ECMO. Nonventilator practices to facilitate decannulation include bronchoscopy (50%), exogenous surfactant (25%), and noninhaled pulmonary vasodilators (50%). Common ventilator thresholds for decannulation include PEEP of 6-7 (45%), PIP of 21-25 (55%), and tidal volume 5-5.9 mL/kg (50%). CONCLUSIONS: The majority of level IV NICUs follow internal protocols for ventilator management during neonatal respiratory ECMO, and neonatologists primarily direct management in the NICU. While most centers use pressure-controlled SIMV, there is considerable variability in the range of settings used, with few centers using HFV primarily. Future studies should focus on identifying respiratory management practices that improve outcomes for neonatal ECMO patients.

Assessment of microplastics in Irish river sediment.

Microplastics (MPs) are environmental pollutants of growing concern, and awareness of MPs pollution in marine and freshwater environments has increased in recent years. However, knowledge of MPs contamination in riverine sediments in Ireland is limited. To address this, we collected and analysed sediment samples from 16 selected sites along the River Barrow. Microplastics were extracted through a density separation method, after which their size, colour, and shape were analysed under a stereo microscope (Optica SZM-2). Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to identify polymer types. A total of 690 MPs were recovered from the 16 sites, with fibres as the dominant MP type. The highest concentration of MPs was 155 MP fibres kg-1 wet sediment found in samples collected from Graiguenamanagh, Co. Kilkenny (GK). The majority of the recovered MPs were polyethylene (PE), polypropylene (PP), nylon, and cellulose acetate (CA) fibres. Overall, this study highlighted the presence of MPs in Irish river sediments and provided a baseline for future studies on MPs pollution. Further research is needed to better understand sources, distribution, and effects of MPs in freshwater ecosystems.

Chronic lung disease in full-term infants: Characteristics and neonatal intensive care outcomes in infants referred to children's hospitals.

OBJECTIVE: To describe characteristics, outcomes, and risk factors for death or tracheostomy with home mechanical ventilation in full-term infants with chronic lung disease (CLD) admitted to regional neonatal intensive care units. STUDY DESIGN: This was a multicenter, retrospective cohort study of infants born ≥37 weeks of gestation in the Children's Hospitals Neonatal Consortium. RESULTS: Out of 67,367 full-term infants admitted in 2010-2016, 4886 (7%) had CLD based on receiving respiratory support at either 28 days of life or discharge. 3286 (67%) were still hospitalized at 28 days receiving respiratory support, with higher mortality risk than those without CLD (10% vs. 2%, p < 0.001). A higher proportion received tracheostomy (13% vs. 0.3% vs. 0.4%, p < 0.001) and gastrostomy (30% vs. 1.7% vs. 3.7%, p < 0.001) compared to infants with CLD discharged home before 28 days and infants without CLD, respectively. The diagnoses and surgical procedures differed significantly between the two CLD subgroups. Small for gestational age, congenital pulmonary, airway, and cardiac anomalies and bloodstream infections were more common among infants with CLD who died or required tracheostomy with home ventilation (p < 0.001). Invasive ventilation at 28 days was independently associated with death or tracheostomy and home mechanical ventilation (odds ratio 7.6, 95% confidence interval 5.9-9.6, p < 0.0001). CONCLUSION: Full-term infants with CLD are at increased risk for morbidity and mortality. We propose a severity-based classification for CLD in full-term infants. Future work to validate this classification and its association with early childhood outcomes is necessary.

Molecular characterization of myotonic dystrophy fibroblast cell lines for use in small molecule screening.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are common forms of adult onset muscular dystrophy. Pathogenesis in both diseases is largely driven by production of toxic-expanded repeat RNAs that sequester MBNL RNA-binding proteins, causing mis-splicing. Given this shared pathogenesis, we hypothesized that diamidines, small molecules that rescue mis-splicing in DM1 models, could also rescue mis-splicing in DM2 models. While several DM1 cell models exist, few are available for DM2 limiting research and therapeutic development. Here, we characterize DM1 and DM2 patient-derived fibroblasts for use in small molecule screens and therapeutic studies. We identify mis-splicing events unique to DM2 fibroblasts and common events shared with DM1 fibroblasts. We show that diamidines can partially rescue molecular phenotypes in both DM1 and DM2 fibroblasts. This study demonstrates the potential of fibroblasts as models for DM1 and DM2, which will help meet an important need for well-characterized DM2 cell models.

Use of Telehealth Across Pediatric Subspecialties Before and During the COVID-19 Pandemic.

Importance: The identification of variation in health care is important for quality improvement. Little is known about how different pediatric subspecialties are using telehealth and what is driving variation. Objective: To characterize trends in telehealth use before and during the COVID-19 pandemic across pediatric subspecialties and the association of delivery change with no-show rates and access disparities. Design, Setting, and Participants: In this cohort study, 8 large pediatric medical groups in California collaborated to share aggregate data on telehealth use for 11 pediatric subspecialties from January 1, 2019, to December 31, 2021. Main Outcomes and Measures: Monthly in-person and telehealth visits for 11 subspecialties, characteristics of patients participating in in-person and telehealth visits, and no-show rates. Monthly use rates per 1000 unique patients were calculated. To assess changes in no-show rates, a series of linear regression models that included fixed effects for medical groups and calendar month were used. The demographic characteristics of patients served in person during the prepandemic period were compared with those of patients who received in-person and telehealth care during the pandemic period. Results: In 2019, participating medical groups conducted 1.8 million visits with 549 306 unique patients younger than 18 years (228 120 [41.5%] White and 277 167 [50.5%] not Hispanic). A total of 72 928 patients (13.3%) preferred a language other than English, and 250 329 (45.6%) had Medicaid. In specialties with lower telehealth use (cardiology, orthopedics, urology, nephrology, and dermatology), telehealth visits ranged from 6% to 29% of total visits from May 1, 2020, to April 30, 2021. In specialties with higher telehealth use (genetics, behavioral health, pulmonology, endocrinology, gastroenterology, and neurology), telehealth constituted 38.8% to 73.0% of total visits. From the prepandemic to the pandemic periods, no-show rates slightly increased for lower-telehealth-use subspecialties (9.2% to 9.4%) and higher-telehealth-use subspecialties (13.0% to 15.3%), but adjusted differences (comparing lower-use and higher-use subspecialties) in changes were not statistically significant (difference, 2.5 percentage points; 95% CI, -1.2 to 6.3 percentage points; P = .15). Patients who preferred a language other than English constituted 6140 in-person visits (22.2%) vs 2707 telehealth visits (11.4%) in neurology (P < .001). Conclusions and Relevance: There was high variability in adoption of telehealth across subspecialties and in patterns of use over time. The documentation of variation in telehealth adoption can inform evolving telehealth policy for pediatric patients, including the appropriateness of telehealth for different patient needs and areas where additional tools are needed to promote appropriate use.

Cardiac Interventions for Patients With Trisomy 13 and Trisomy 18: Experience, Ethical Issues, Communication, and the Case for Individualized Family-Centered Care.

This report is informed by the themes of the session Trisomy 13/18, Exploring the Changing Landscape of Interventions at NeoHeart 2020-The Fifth International Conference of the Neonatal Heart Society. The faculty reviewed the present evidence in the management of patients and the support of families in the setting of trisomy 13 and trisomy 18 with congenital heart disease. Until recently medical professionals were taught that T13 and 18 were "lethal conditions" that were "incompatible with life" for which measures to prolong life are therefore ethically questionable and likely futile. While the medical literature painted one picture, family support groups shared stories of the long-term survival of children who displayed happiness and brought joy along with challenges to families. Data generated from such care shows that surgery can, in some cases, prolong survival and increase the likelihood of time at home. The authors caution against a change from never performing heart surgery to always-we suggest that the pendulum of intervention find a balanced position where all therapies including comfort care and surgery can be reviewed. Families and clinicians should typically be supported and empowered to define the best care for their children and patients. Key concepts in communication and case vignettes are reviewed including the importance of supportive relationships and the fact that palliative care may serve as an additional layer of support for decision-making and quality of life interventions. While cardiac surgery may be beneficial in some cases, surgery should not be the primary focus of initial family education and support.

Effect of Whole-Genome Sequencing on the Clinical Management of Acutely Ill Infants With Suspected Genetic Disease: A Randomized Clinical Trial.

Importance: Whole-genome sequencing (WGS) shows promise as a first-line genetic test for acutely ill infants, but widespread adoption and implementation requires evidence of an effect on clinical management. Objective: To determine the effect of WGS on clinical management in a racially and ethnically diverse and geographically distributed population of acutely ill infants in the US. Design, Setting, and Participants: This randomized, time-delayed clinical trial enrolled participants from September 11, 2017, to April 30, 2019, with an observation period extending to July 2, 2019. The study was conducted at 5 US academic medical centers and affiliated children's hospitals. Participants included infants aged between 0 and 120 days who were admitted to an intensive care unit with a suspected genetic disease. Data were analyzed from January 14 to August 20, 2020. Interventions: Patients were randomized to receive clinical WGS results 15 days (early) or 60 days (delayed) after enrollment, with the observation period extending to 90 days. Usual care was continued throughout the study. Main Outcomes and Measures: The main outcome was the difference in the proportion of infants in the early and delayed groups who received a change of management (COM) 60 days after enrollment. Additional outcome measures included WGS diagnostic efficacy, within-group COM at 90 days, length of hospital stay, and mortality. Results: A total of 354 infants were randomized to the early (n = 176) or delayed (n = 178) arms. The mean participant age was 15 days (IQR, 7-32 days); 201 participants (56.8%) were boys; 19 (5.4%) were Asian; 47 (13.3%) were Black; 250 (70.6%) were White; and 38 (10.7%) were of other race. At 60 days, twice as many infants in the early group vs the delayed group received a COM (34 of 161 [21.1%; 95% CI, 15.1%-28.2%] vs 17 of 165 [10.3%; 95% CI, 6.1%-16.0%]; P = .009; odds ratio, 2.3; 95% CI, 1.22-4.32) and a molecular diagnosis (55 of 176 [31.0%; 95% CI, 24.5%-38.7%] vs 27 of 178 [15.0%; 95% CI, 10.2%-21.3%]; P < .001). At 90 days, the delayed group showed a doubling of COM (to 45 of 161 [28.0%; 95% CI, 21.2%-35.6%]) and diagnostic efficacy (to 56 of 178 [31.0%; 95% CI, 24.7%-38.8%]). The most frequent COMs across the observation window were subspecialty referrals (39 of 354; 11%), surgery or other invasive procedures (17 of 354; 4%), condition-specific medications (9 of 354; 2%), or other supportive alterations in medication (12 of 354; 3%). No differences in length of stay or survival were observed. Conclusions and Relevance: In this randomized clinical trial, for acutely ill infants in an intensive care unit, introduction of WGS was associated with a significant increase in focused clinical management compared with usual care. Access to first-line WGS may reduce health care disparities by enabling diagnostic equity. These data support WGS adoption and implementation in this population. Trail Registration: ClinicalTrials.gov Identifier: NCT03290469.

Therapeutic strategies for C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia.

PURPOSE OF REVIEW: An intronic G4C2 expansion mutation in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). Although there are currently no treatments for this insidious, fatal disease, intense research has led to promising therapeutic strategies, which will be discussed here. RECENT FINDINGS: Therapeutic strategies for C9-ALS/FTD have primarily focused on reducing the toxic effects of mutant expansion RNAs or the dipeptide repeat proteins (DPRs). The pathogenic effects of G4C2 expansion transcripts have been targeted using approaches aimed at promoting their degradation, inhibiting nuclear export or silencing transcription. Other promising strategies include immunotherapy to reduce the DPRs themselves, reducing RAN translation, removing the repeats using DNA or RNA editing and manipulation of downstream disease-altered stress granule pathways. Finally, understanding the molecular triggers that lead to pheno-conversion may lead to opportunities that can delay symptomatic disease onset. SUMMARY: A large body of evidence implicates RAN-translated DPRs as a main driver of C9-ALS/FTD. Promising therapeutic strategies for these devastating diseases are being rapidly developed with several approaches already in or approaching clinical trials.

Project Baby Bear: Rapid precision care incorporating rWGS in 5 California children's hospitals demonstrates improved clinical outcomes and reduced costs of care.

Genetic disorders are a leading contributor to mortality in neonatal and pediatric intensive care units (ICUs). Rapid whole-genome sequencing (rWGS)-based rapid precision medicine (RPM) is an intervention that has demonstrated improved clinical outcomes and reduced costs of care. However, the feasibility of broad clinical deployment has not been established. The objective of this study was to implement RPM based on rWGS and evaluate the clinical and economic impact of this implementation as a first line diagnostic test in the California Medicaid (Medi-Cal) program. Project Baby Bear was a payor funded, prospective, real-world quality improvement project in the regional ICUs of five tertiary care children's hospitals. Participation was limited to acutely ill Medi-Cal beneficiaries who were admitted November 2018 to May 2020, were <1 year old and within one week of hospitalization, or had just developed an abnormal response to therapy. The whole cohort received RPM. There were two prespecified primary outcomes-changes in medical care reported by physicians and changes in the cost of care. The majority of infants were from underserved populations. Of 184 infants enrolled, 74 (40%) received a diagnosis by rWGS that explained their admission in a median time of 3 days. In 58 (32%) affected individuals, rWGS led to changes in medical care. Testing and precision medicine cost $1.7 million and led to $2.2-2.9 million cost savings. rWGS-based RPM had clinical utility and reduced net health care expenditures for infants in regional ICUs. rWGS should be considered early in ICU admission when the underlying etiology is unclear.

Venovenous versus venoarterial extracorporeal membrane oxygenation among infants with hypoxic-ischemic encephalopathy: is there a difference in outcome?

OBJECTIVE: Our hypothesis was that among infants with hypoxic-ischemic encephalopathy (HIE), venoarterial (VA), compared to venovenous (VV), extracorporeal membrane oxygenation (ECMO) is associated with an increased risk of mortality or intracranial hemorrhage (ICH). DESIGN/METHODS: Retrospective cohort analysis of infants in the Children's Hospitals Neonatal Database from 2010 to 2016 with moderate or severe HIE, gestational age ≥36 weeks, and ECMO initiation <7 days of age. The primary outcome was mortality or ICH. RESULTS: Severe HIE was more common in the VA ECMO group (n = 57), compared to the VV ECMO group (n = 53) (47.4% vs. 26.4%, P = 0.02). VA ECMO was associated with a significantly higher risk of death or ICH [57.9% vs. 34.0%, aOR 2.39 (1.08-5.28)] and mortality [31.6% vs. 11.3%, aOR 3.06 (1.08-8.68)], after adjusting for HIE severity. CONCLUSIONS: In HIE, VA ECMO was associated with a higher incidence of mortality or ICH. VV ECMO may be beneficial in this population.

The alternative initiation factor eIF2A plays key role in RAN translation of myotonic dystrophy type 2 CCUG•CAGG repeats.

Repeat-associated non-ATG (RAN) proteins have been reported in 11 microsatellite expansion disorders but the factors that allow RAN translation to occur and the effects of different repeat motifs and alternative AUG-like initiation codons are unclear. We studied the mechanisms of RAN translation across myotonic dystrophy type 2 (DM2) expansion transcripts with (CCUG) or without (CAGG) efficient alternative AUG-like codons. To better understand how DM2 LPAC and QAGR RAN proteins are expressed, we generated a series of CRISPR/Cas9-edited HEK293T cell lines. We show that LPAC and QAGR RAN protein levels are reduced in protein kinase R (PKR)-/- and PKR-like endoplasmic reticulum kinase (PERK)-/- cells, with more substantial reductions of CAGG-encoded QAGR in PKR-/- cells. Experiments using mutant eIF2α-S51A HEK293T cells show that p-eIF2α is required for QAGR production. In contrast, LPAC levels were only partially reduced in these cells, suggesting that both non-AUG and close-cognate initiation occur across CCUG RNAs. Overexpression of the alternative initiation factor eIF2A increases LPAC and QAGR protein levels but, notably, has a much larger effect on QAGR expressed from CAGG-expansion RNAs that lack efficient close-cognate codons. The effects of eIF2A on increasing LPAC are consistent with previous reports that eIF2A affects CUG-initiation translation. The observation that eIF2A also increases QAGR proteins is novel because CAGG expansion transcripts do not contain CUG or similarly efficient close-cognate AUG-like codons. For QAGR but not LPAC, the eIF2A-dependent increases are not seen when p-eIF2α is blocked. These data highlight the differential regulation of DM2 RAN proteins and eIF2A as a potential therapeutic target for DM2 and other RAN diseases.

Management of Congenital Diaphragmatic Hernia Treated With Extracorporeal Life Support: Interim Guidelines Consensus Statement From the Extracorporeal Life Support Organization.

The management of infants with congenital diaphragmatic hernia (CDH) receiving extracorporeal life support (ECLS) is complex. Significant variability in both practice and prevalence of ECLS use exists among centers, given the lack of evidence to guide management decisions. The purpose of this report is to review existing evidence and develop management recommendations for CDH patients treated with ECLS. This article was developed by the Extracorporeal Life Support Organization CDH interest group in cooperation with members of the CDH Study Group and the Children's Hospitals Neonatal Consortium.

Single cell transcriptomics reveals opioid usage evokes widespread suppression of antiviral gene program.

Chronic opioid usage not only causes addiction behavior through the central nervous system, but also modulates the peripheral immune system. However, how opioid impacts the immune system is still barely characterized systematically. In order to understand the immune modulatory effect of opioids in an unbiased way, here we perform single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells from opioid-dependent individuals and controls to show that chronic opioid usage evokes widespread suppression of antiviral gene program in naive monocytes, as well as in multiple immune cell types upon stimulation with the pathogen component lipopolysaccharide. Furthermore, scRNA-seq reveals the same phenomenon after a short in vitro morphine treatment. These findings indicate that both acute and chronic opioid exposure may be harmful to our immune system by suppressing the antiviral gene program. Our results suggest that further characterization of the immune modulatory effects of opioid is critical to ensure the safety of clinical opioids.

An empirical investigation into the effectiveness of genetic genealogy to identify individuals in the UK.

The use of genetic genealogy techniques to identify Joseph James DeAngelo as the prime suspect in the Golden State Killer case in 2018 has opened up a new approach to investigation of cold cases. Since that breakthrough, genetic genealogy methods have been reported to be applied to around 100 cases. To date, all of these reports relate to investigations in the US, where the high uptake of "direct-to-consumer" (DTC) genetic testing by individuals conducting private ancestral research has provided the necessary publicly available data for successful forensic investigations. We have conducted a study to assess the likely effectiveness of genetic genealogy techniques if applied to investigations in the UK. Ten volunteers provided their own SNP array data, downloaded from a DTC provider of their choice. These data sets were anonymised and uploaded to the GEDmatch Genesis genealogy website, mimicking data sets from unsourced crime samples or unidentified human remains. A team of experienced genealogists then attempted to identify the donors of the anonymised data sets by working with matches on the database and identifying points where the matches' trees intersect to determine their shared family lineages which were further investigated using traditional resources (such as birth, marriage, death and census records, social media and online family trees). Through these methods, four of the ten donors were identified, at least to the level of one of a set of siblings. This confirms that, despite the over-representation of US citizens on publicly accessible genealogy databases, there is still potential for effective use in investigations outside the US where legislation permits. One of our four identified individuals was of Indian heritage (via St Vincent and the Grenadines) highlighting that in the right circumstances individuals of non-European origin can be identified.