Liver disease and transplantation in telomere biology disorders: An international multicenter cohort.

BACKGROUND: Patients with telomere biology disorders (TBD) develop hepatic disease, including hepatitis, cirrhosis, and hepatopulmonary syndrome. No specific treatment exists for TBD-related liver disease, and the role of liver transplantation (LT) remains controversial. Our study objectives were to describe the clinical characteristics, management, and outcomes in patients with TBD-related liver disease, and their LT outcomes. METHODS: Data from 83 patients with TBD-associated liver disease were obtained from 17 participating centers in the Clinical Care Consortium of Telomere-Associated Ailments and by self-report for our retrospective, multicenter, international cohort study. RESULTS: Group A ("Advanced") included 40 patients with advanced liver disease. Of these, 20 underwent LT (Group AT). Group M ("Mild") included 43 patients not warranting LT evaluation, none of whom were felt to be medically unfit for liver transplantation. Supplemental oxygen requirement, pulmonary arteriovenous malformation, hepatopulmonary syndrome, and higher bilirubin and international normalized ratio values were associated with Group A. Other demographics, clinical manifestations, and laboratory findings were similar between groups. Six group A patients were declined for LT; 3 died on the waitlist. Median follow-up post-LT was 2.9 years (range 0.6-13.2 y). One-year survival post-LT was 73%. Median survival post-LT has not been reached. Group AT patients had improved survival by age compared to all nontransplant patients (log-rank test p = 0.02). Of 14 patients with pretransplant hypoxemia, 8 (57%) had improved oxygenation after transplant. CONCLUSIONS: LT recipients with TBD do not exhibit excessive posttransplant mortality, and LT improved respiratory status in 57%. A TBD diagnosis should not exclude LT consideration.

Loss of an extensive ciliary connectome induces proteostasis and cell fate switching in a severe motile ciliopathy.

Motile cilia have essential cellular functions in development, reproduction, and homeostasis. Genetic causes for motile ciliopathies have been identified, but the consequences on cellular functions beyond impaired motility remain unknown. Variants in CCDC39 and CCDC40 cause severe disease not explained by loss of motility. Using human cells with pathological variants in these genes, Chlamydomonas genetics, cryo-electron microscopy, single cell RNA transcriptomics, and proteomics, we identified perturbations in multiple cilia-independent pathways. Absence of the axonemal CCDC39/CCDC40 heterodimer results in loss of a connectome of over 90 proteins. The undocked connectome activates cell quality control pathways, switches multiciliated cell fate, impairs microtubule architecture, and creates a defective periciliary barrier. Both cilia-dependent and independent defects are likely responsible for the disease severity. Our findings provide a foundation for reconsidering the broad cellular impact of pathologic variants in ciliopathies and suggest new directions for therapies.

Evaluating caregiver-clinician communication for tracheostomy placement in the neonatal intensive care unit: a qualitative inquiry.

OBJECTIVE: Identify stakeholders' tracheostomy decision-making information priorities in the Neonatal Intensive Care Unit (NICU). STUDY DESIGN: English-speaking caregivers and clinicians who participated in NICU tracheostomy discussions between January 2017 and December 2021 were eligible. They reviewed a pediatric tracheostomy communication guide prior to meeting. Interviews focused on tracheostomy decision-making experiences, communication preferences, and guide perceptions. Interviews were recorded, transcribed, and analyzed using iterative inductive/deductive coding to inform thematic analysis. RESULTS: Ten caregivers and nine clinicians were interviewed. Caregivers were surprised by the severity of their child's diagnosis and the intensive home care required, but proceeded with tracheostomy because it was the only chance for survival. All recommended that tracheostomy information be introduced early and in phases. Inadequate communication limited caregivers' understanding of post-surgical care and discharge requirements. All felt a guide could standardize communication. CONCLUSIONS: Caregivers seek detailed information regarding expectations after tracheostomy placement in the NICU and at home.

Evaluating Caregiver-Clinician Communication for Tracheostomy Placement in the Neonatal Intensive Care Unit: A Qualitative Inquiry.

Objective: Identify stakeholders' tracheostomy decision-making information priorities in the Neonatal Intensive Care Unit (NICU). Study Design: English-speaking caregivers and clinicians who participated in NICU tracheostomy discussions between January 2017 and December 2021 were eligible. They reviewed a pediatric tracheostomy communication guide prior to meeting. Interviews focused on tracheostomy decision-making experiences, communication preferences, and guide perceptions. Interviews were recorded, transcribed, and analyzed using iterative inductive/deductive coding to inform thematic analysis. Results: Ten caregivers and nine clinicians were interviewed. Caregivers were surprised by the severity of their child's diagnosis and the intensive home care required, but proceeded with tracheostomy because it was the only chance for survival. All recommended that tracheostomy information be introduced early and in phases. Inadequate communication limited caregivers' understanding of post-surgical care and discharge requirements. All felt a guide could standardize communication. Conclusions: Caregivers seek detailed information regarding expectations after tracheostomy placement in the NICU and at home.

The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes.

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift-null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partially preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. Transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. These findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies.

A novel high-prevalence antigen in the Lutheran system, LUGA (LU24), and an updated, full-length 3D BCAM model.

BACKGROUND: The basal cell adhesion molecule (BCAM) carries the antigens of the Lutheran (LU, ISBT005) system. We report a novel Lutheran antigen and propose an updated, full-length 3D model of BCAM. STUDY DESIGN AND METHODS: Red blood cell testing, antibody identification, and BCAM genomic DNA sequencing were done by standard methods. Multi-template homology modeling of BCAM used structural templates selected for coverage, highest sequence identity, and protein domain family. All variants causing the loss or gain of a Lutheran antigen were analyzed for residue accessibility and intraprotein interactions. RESULTS: An antibody to a high-prevalence antigen in the plasma of a pregnant woman was determined to be directed at a novel Lutheran antigen. Sequencing of BCAM found three homozygous changes: c.212G > A (p.Arg71His) and two silent, c.711C > T and c.714C > T. The model was built from the first two immunoglobulin crystallized domains of BCAM (D1, D2), three other templates (for D3, D4 and D5 with a higher sequence identity with the target than those used for the model proposed by Burton and Brady in 2008, and for the transmembrane region) and RaptorX (for the intracellular domain). All residues associated with a Lutheran antigen were found to be exposed in wild-type or variant proteins, except p.447 associated with loss of Lu13 expression. CONCLUSION: The c.212G > A change results in the loss of LUGA (LU24) antigen. Whole genome sequencing continues to reveal polymorphisms with uncertain immunogenicity. This model and demonstration that nearly all residues associated with the expression of a Lutheran antigen are exposed will help evaluate the significance of new polymorphisms.

Markers for severe disease and long-term sequelae in pediatric patients with severe acute respiratory syndrome coronavirus 2 infection.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has resulted in substantial global morbidity and mortality since late 2019. Children can be infected but the disease predominantly affects adults, and research into the acute and chronic sequelae mostly pertains to this population. This study determines the clinical and demographic parameters associated with severe acute disease and chronic complications from COVID-19 in the pediatric population. METHODS: A retrospective chart review was undertaken of all patients between birth and 21 years of age who were positive for SARS-CoV-2 by polymerase chain reaction (PCR) and were admitted to two tertiary care hospitals between March 1, 2020, and January 21, 2021. Markers for severe disease were defined as supplemental oxygen requirement, positive pressure ventilation, and acute chest radiograph abnormality at presentation. Chronic disease was defined as symptoms persisting >4 weeks. RESULTS: Review of 101 patients with positive SARS-CoV2 testing found 67 presentations consistent with acute symptomatic infection. Age distribution was bimodal, with predominance in infancy and adolescence. Most (75%) had an extrapulmonary comorbidity, and fewer patients (33%) had pre-existing lung disease. A history of pulmonary comorbidity and obesity was significantly associated with markers for severe disease. Long-term chronic complications were associated with history of underlying lung disease and acute severe COVID-19. CONCLUSIONS: Demographic and clinical markers were associated with severe COVID-19 in children. Moreover, both the presence of pulmonary comorbidity and severe acute COVID-19 are associated with long-term sequelae.

Emerging Genotype-Phenotype Relationships in Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare inherited condition affecting motile cilia and leading to organ laterality defects, recurrent sino-pulmonary infections, bronchiectasis, and severe lung disease. Research over the past twenty years has revealed variability in clinical presentations, ranging from mild to more severe phenotypes. Genotype and phenotype relationships have emerged. The increasing availability of genetic panels for PCD continue to redefine these genotype-phenotype relationships and reveal milder forms of disease that had previously gone unrecognized.

Implementation of a screening tool for primary ciliary dyskinesia (PCD) in a pediatric otolaryngology clinic.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare genetic disease arising from motile ciliary dysfunction and associated with recurrent and chronic upper and lower respiratory tract infections. Pediatric otolaryngologists may see these patients prior to the development of lung disease. Features of PCD may overlap with other suppurative respiratory diseases, creating diagnostic challenges. A simple screening tool would be beneficial to identify potential patients who have chronic upper respiratory tract disease requiring further specialist evaluation. OBJECTIVE: To test a simple screening tool consisting of four questions to detect PCD in children with chronic otitis media and chronic rhinosinusitis seen in a tertiary otolaryngology clinic. METHODS: A prospective, single site, observational study in a tertiary care pediatric otolaryngology clinic. Children aged 3-17 years diagnosed with chronic otitis media or rhinosinusitis with onset at less than 2 years of age were recruited. All study subjects had at least one of four key clinical features for PCD as determined by answers to screening questions, while control subjects had none. All participants completed a medical history questionnaire and nasal nitric oxide measurements. Those with reduced nasal nitric oxide levels were referred to our PCD center for further evaluation. RESULTS: A total of 153 patients were screened and 62 subjects were enrolled. Of those, 35 were enrolled as study subjects and 27 as matched controls. Study subjects had mean age of 7.5 years (3.2-16.5) with pre-screening diagnosis of chronic otitis media (n = 29) or chronic rhinosinusitis (n = 6). Control subjects (n = 27) had mean age 7.2 years (3.0-16.3) with pre-screening diagnosis of chronic otitis media (n = 25), and chronic rhinosinusitis (n = 2). There were no differences in subject demographics or mean nasal nitric oxide values between the two groups (179.8 vs 210.8 nl/min). Ten individuals had low nasal nitric oxide values, 7 of which were normal on repeat testing. Three subjects failed to return for follow up evaluations. Four referrals were made for further evaluation on the basis of clinical symptoms and nasal nitric oxide results. While no new cases of PCD were detected, a subject and his sibling with recurrent sinopulmonary infections were referred for immunologic evaluation. CONCLUSION: The use of standardized screening questions can be used in an otolaryngology clinic to identify patients who require further evaluation for PCD or primary immunodeficiency.

Effects of a PICU Status Asthmaticus De-Escalation Pathway on Length of Stay and Albuterol Use.

OBJECTIVES: Evaluate the effects of an asthma de-escalation clinical pathway on selected outcomes for patients admitted to a PICU with status asthmaticus. DESIGN: Time series quality improvement trial. SETTING: PICU in a tertiary care children's hospital. PATIENTS: Children age 2-18 years old with a known diagnosis of asthma presenting with status asthmaticus. INTERVENTION: One-hundred five admissions to a PICU for status asthmaticus were treated according to a new de-escalation pathway between August 15, 2015, and August 30, 2016. This group was compared with a prepathway group of 141. MEASUREMENTS AND MAIN RESULTS: Primary outcome was variability in PICU length of stay. Secondary outcomes were median PICU length of stay, median hospital length of stay, and median duration a patient received continuous nebulized albuterol. The effectiveness of the intervention was tracked using control charts. The postpathway group demonstrated decreased variability of PICU length of stay and time receiving continuous albuterol. Statistically significant decreases were seen in median PICU length of stay (16 vs 13 hr; p = 0.0009), median duration a child spent receiving continuous nebulized albuterol (10.8 vs 7.3 hr; p = 0.0008), and median hospital length of stay (37 vs 31 hr; p = 0.02). Total number of asthma assessments completed by respiratory therapists increased from 741 to 1,087. CONCLUSIONS: Implementation of a PICU asthma de-escalation pathway demonstrated statistical decrease in the reported measures for children with status asthmaticus. Although the clinical significance of these changes may be debatable, the results demonstrate that efforts to standardize asthma care in the PICU setting is an area in need of further study.

Utilization of sequence variants as biomarkers to analyze population dynamics in cloned cell lines.

The inherent nature of cloned CHO cell lines includes the presence of genetic and phenotypic drift that leads to heterogeneous populations. The genetic heterogeneity exhibited by these cells can be exploited to understand the population dynamics of cloned cell lines. Understanding the interplay between heterogeneity, cell culture conditions, and population dynamics will allow for critical assessment of overarching cell line development methods and strategies in terms of population and monoclonality. Sequence variants (SVs) are protein isoforms of the gene-of-interest that contain unintended amino acid substitutions, extensions, or truncations that may contribute to heterogeneity. In this case, SVs are unique sequences in the genome of the integrated transgene that can be used as biomarkers to understand the heterogeneity of a monoclonal cell line and how production process conditions can impact population dynamics. In this study, orthogonal genetic and analytical methods were used to examine the variability of SV levels in four different SV-containing cell lines under varied culture conditions and generational ages. Culture conditions tested had little to no impact on SV levels. However, generational age studies showed two distinct trends: stability of SV levels out to approximately 100 generations in cell lines with higher level SVs (>10%) and a progressive decrease of SV levels as the cells age to approximately 100 generations in cell lines with lower level SVs (<10%). The results suggest that the four SV-containing cell lines fall into two distinct population models; SVs present in the whole population of cells and SVs present in only a sub-population of cells. The data presented here are one of the first studies to not only analyze and compare SV levels in both genetic and protein material but also to utilize SVs as biomarkers to probe distinct populations of cloned cell lines. Biotechnol. Bioeng. 2017;114: 1744-1752. © 2017 Wiley Periodicals, Inc.

Raman Analysis of Dilute Aqueous Samples by Localized Evaporation of Submicroliter Droplets on the Tips of Superhydrophobic Copper Wires.

Raman analysis of dilute aqueous solutions is normally prevented by their low signal levels. A very general method to increase the concentration to detectable levels is to evaporate droplets of the sample to dryness, creating solid deposits which are then Raman probed. Here, superhydrophobic (SHP) wires with hydrophilic tips have been used as supports for drying droplets, which have the advantage that the residue is automatically deposited at the tip. The SHP wires were readily prepared in minutes using electroless galvanic deposition of Ag onto copper wires followed by modification with a polyfluorothiol (3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro-1-decanethiol, HDFT). Cutting the coated wires with a scalpel revealed hydrophilic tips which could support droplets whose maximum size was determined by the wire diameter. Typically, 230 µm wires were used to support 0.6 µL droplets. Evaporation of dilute melamine droplets gave solid deposits which could be observed by scanning electron microscopy (SEM) and Raman spectroscopy. The limit of detection for melamine using a two stage evaporation procedure was 1 × 10(-6) mol dm(-3). The physical appearance of dried droplets of sucrose and glucose showed that the samples retained significant amounts of water, even under high vacuum. Nonetheless, the Raman detection limits of sucrose and glucose were 5 × 10(-4) and 2.5 × 10(-3) mol dm(-3), respectively, which is similar to the sensitivity reported for surface-enhanced Raman spectroscopy (SERS) detection of glucose. It was also possible to quantify the two sugars in mixtures at concentrations which were similar to those found in human blood through multivariate analysis.

The role of the laparoendoscopic single site totally extraperitoneal approach to inguinal hernia repairs: a review and meta-analysis of the literature.

BACKGROUND: Laparoendoscopic single site (LESS) surgery may have perceived benefits of reduced visible scarring compared to conventional laparoscopic (LAP) totally extraperitoneal (TEP) hernia repairs. We reviewed the literature to compare LESS TEP inguinal hernia repairs with LAP TEP repairs. METHODS: We searched electronic databases for research published between January 2008 and January 2012. RESULTS: A total of 13 studies reported on 325 patients. The duration of surgery was 40-98 minutes for unilateral hernia and 41-121 minutes for bilateral repairs. Three studies involving 287 patients compared LESS TEP (n = 128) with LAP TEP (n = 159). There were no significant differences in operative duration for unilateral hernias (p = 0.63) or bilateral repairs (p = 0.29), and there were no significant differences in hospital stay (p > 0.99), intraoperative complications (p = 0.82) or early recurrence rates (p = 0.82). There was a trend toward earlier return to activity in the LESS TEP group (p = 0.07). CONCLUSION: Laparoendoscopic single site surgery TEP hernia repair is a relatively new technique and appears to be safe and effective. Advantages, such as less visible scarring, mean patients may opt for LESS TEP over LAP TEP. Further studies with clear definitions of outcome measures and robust follow-up to assess patient satisfaction, return to normal daily activities and recurrence are needed to strengthen the evidence.

CONTEXTE: La chirurgie laparoendoscopique à orifice unique (LESS) a comme avantage perçu une réduction des cicatrices apparentes comparativement aux réparations laparoscopiques (LAP) classiques totalement extrapéritonéales (TEP) des hernies. Nous avons passé en revue la littérature afin de comparer les réparations des hernies inguinales par chirurgie LESS TEP et par LAP TEP. MÉTHODES: Nous avons interrogé les bases de données électroniques pour y recenser la recherche publiée entre janvier 2008 et janvier 2012. RÉSULTATS: En tout, 13 études ont porté sur 325 patients. La durée de la chirurgie a été de 40 à 98 minutes pour les réparations de hernies unilatérales et de 41 à 121 minutes pour les réparations de hernies bilatérales. Trois études regroupant 287 patients ont comparé la technique LESS TEP (n = 128) à la technique LAP TEP (n = 159). On n'a observé aucune différence significative quant à la durée de la chirurgie des réparations de hernies unilatérales (p = 0,63) ou bilatérales (p = 0,29) et aucune différence significative de durée des séjours hospitaliers (p > 0,99), de complications peropératoires (p = 0,82) ou de taux de récurrences précoces (p = 0,82). On a noté une tendance à un retour plus rapide aux activités dans le groupe soumis à la technique LESS TEP (p = 0,07). CONCLUSION: La réparation de hernie par chirurgie TEP laparoendoscopique à un seul orifice est une technique relativement nouvelle et semble sécuritaire et efficace. Ses avantages, par exemple des cicatrices moins apparentes, pourraient pousser les patients à opter pour la technique LESS TEP plutôt que LAP TEP. Il faudra procéder à d'autres études fondées sur des définitions paramétriques claires et comportant un suivi robuste pour évaluer la satisfaction des patients, la reprise des activités quotidiennes normales et les taux de récurrences afin de consolider les preuves.