ALKBH5 regulates somatic cell reprogramming in a phase-specific manner.

Establishment of the pluripotency regulatory network in somatic cells by introducing four transcription factors [octamer binding transcription factor 4 (OCT4; also known as POU5F1), sex determining region Y (SRY)-box 2 (SOX2), Kruppel-like factor 4 (KLF4) and cellular myelocytomatosis (c-MYC)] provides a promising tool for cell-based therapies in regenerative medicine. Nevertheless, the mechanisms at play when generating induced pluripotent stem cells from somatic cells are only partly understood. Here, we show that the RNA-specific N6-methyladenosine (m6A) demethylase ALKBH5 regulates somatic cell reprogramming in a stage-specific manner through its catalytic activity. Knockdown or knockout of Alkbh5 in the early reprogramming phase impairs reprogramming efficiency by reducing the proliferation rate through arresting the cells at G2/M phase and decreasing the upregulation of epithelial markers. On the other hand, ALKBH5 overexpression at the early reprogramming phase has no significant impact on reprogramming efficiency, whereas overexpression at the late phase enhances reprogramming by stabilizing Nanog transcripts, resulting in upregulated Nanog expression. Our study provides mechanistic insight into the crucial dynamic role of ALKBH5, mediated through its catalytic activity, in regulating somatic cell reprogramming at the post-transcriptional level. This article has an associated First Person interview with the first author of the paper.

Surgical management of rare tumors (Part 1).

With an annual cumulative occurrence of approximately 15,000 in North America, all childhood cancers are rare. Very rare cancers as defined by both the European Cooperative Study Group for Rare Pediatric Cancers and the Children's Oncology Group fall into two principal categories: those so uncommon (fewer than 2 cases/million) that their study is challenging even through cooperative group efforts (e.g., pleuropulmonary blastoma and desmoplastic small round cell tumor) and those that are far more common in adults and therefore rarely studied in children (e.g., thyroid, melanoma, and gastrointestinal stromal tumor). Treatment strategies for these latter tumors are typically based on adult guidelines, although the pediatric variants of these tumors may harbor different genetic signatures and demonstrate different behavior. If melanoma and differentiated thyroid cancer are excluded, other rare cancer types account for only 2% of the cancers in children aged 0 to 14. This article highlights several of the most common rare tumor types.

Optimizing pediatric tonsillectomy outcomes with an opioid sparing anesthesia protocol: Learning and continuously improving with real-world data.

INTRODUCTION: This quality improvement initiative is a continued pursuit to optimize outcomes by iteratively improving our opioid sparing anesthesia protocol for tonsillectomy with or without adenoidectomy at our pediatric ambulatory surgical center through data driven Plan-Do-Study-Act cycles. METHODS: From 1/2015 through 12/2023, our standardized tonsillectomy protocol underwent nine procedure-specific perioperative Plan-Do-Study-Act cycles, three procedure-specific postoperative prescription Plan-Do-Study-Act cycles, and four general ambulatory surgical center enhanced recovery Plan-Do-Study-Act cycles. We analyzed data from the medical record using statistical process control charts. The primary outcome measure was the percent of patients requiring intravenous opioid in the post anesthesia care unit. Secondary outcomes included maximum post anesthesia care unit pain score, the percent of patients requiring treatment for nausea and/or vomiting in the post anesthesia care unit, and the number of postoperative opioid prescription dosages. Balancing measures were average post anesthesia care unit length of stay, percent of patients with prolonged Post Anesthesia Care Unit length of stay (>120 min), and 30-day reoperation rate. RESULTS: A total of 5654 tonsillectomy with or without adenoidectomy cases were performed at our ambulatory surgical center from 2015 to 2023. The incidence of intravenous opioid administered in the post anesthesia care unit initially rose with opioid free anesthesia launch, but subsequently decreased below the target of 10%. Maximum post anesthesia care unit pain scores rose from mean 3.6 to 4.5, but subsequently returned to the baseline of 3.5, while the incidence of postoperative nausea and/or vomiting improved. The average post anesthesia care unit length of stay increased by 10 min with opioid free anesthesia; however, prolonged post anesthesia care unit stay and 30-day reoperation rates were unchanged. CONCLUSIONS: The continued refinement of our opioid sparing anesthesia protocol has led to reduced perioperative and home opioid use, stable maximum post anesthesia care unit pain scores, and improved postoperative nausea and vomiting rates, with only a slight increase in mean post anesthesia care unit length of stay.

Intestinal-epithelial LSD1 controls goblet cell maturation and effector responses required for gut immunity to bacterial and helminth infection.

Infectious and inflammatory diseases in the intestine remain a serious threat for patients world-wide. Reprogramming of the intestinal epithelium towards a protective effector state is important to manage inflammation and immunity and can be therapeutically targeted. The role of epigenetic regulatory enzymes within these processes is not yet defined. Here, we use a mouse model that has an intestinal-epithelial specific deletion of the histone demethylase Lsd1 (cKO mice), which maintains the epithelium in a fixed reparative state. Challenge of cKO mice with bacteria-induced colitis or a helminth infection model both resulted in increased pathogenesis. Mechanistically, we discovered that LSD1 is important for goblet cell maturation and goblet-cell effector molecules such as RELMß. We propose that this may be in part mediated by directly controlling genes that facilitate cytoskeletal organization, which is important in goblet cell biology. This study therefore identifies intestinal-epithelial epigenetic regulation by LSD1 as a critical element in host protection from infection.

Surgery for Velopharyngeal Insufficiency Following Cleft Palate Repair: An Audit of Contemporary Practice and Proposed Schema of Techniques and Variations.

OBJECTIVE: Surgical treatment of velopharyngeal insufficiency (VPI) includes a wide array of procedures. The purpose of this study was to develop a classification for VPI procedures and to describe variations in how they are performed.Design/participants/setting/outcomes: We completed an in-depth review of the literature to develop a preliminary schema that encompassed existing VPI procedures. Forty-one cleft surgeons from twelve hospitals across the USA and Canada reviewed the schema and either confirmed that it encompassed all VPI procedures they performed or requested additions. Two surgeons then observed the conduct of the procedures by surgeons at each hospital. Standardized reports were completed with each visit to further explore the literature, refine the schema, and delineate the common and unique aspects of each surgeon's technique. RESULTS: Procedures were divided into three groups: palate-based surgery; pharynx-based surgery; and augmentation. Palate-based operations included straight line mucosal incision with intravelar veloplasty, double-opposing Z-plasty, and palate lengthening with buccal myomucosal flaps. Many surgeons blended maneuvers from these three techniques, so a more descriptive schema was developed classifying the maneuvers employed on the oral mucosa, nasal mucosa, and muscle. Pharynx-based surgery included pharyngeal flap and sphincter pharyngoplasty, with variations in design for each. Augmentation procedures included palate and posterior wall augmentation. CONCLUSIONS: A comprehensive schema for VPI procedures was developed incorporating intentional adaptations in technique. There was substantial variation amongst surgeons in how each procedure was performed. The schema may enable more specific evaluations of surgical outcomes and exploration of the mechanisms through which these procedures improve speech.

Alpelisib for the treatment of PIK3CA-related head and neck lymphatic malformations and overgrowth.

PURPOSE: PIK3CA-related overgrowth spectrum (PROS) conditions of the head and neck are treatment challenges. Traditionally, these conditions require multiple invasive interventions, with incomplete malformation removal, disfigurement, and possible dysfunction. Use of the PI3K inhibitor alpelisib, previously shown to be effective in PROS, has not been reported in PIK3CA-associated head and neck lymphatic malformations (HNLMs) or facial infiltrating lipomatosis (FIL). We describe prospective treatment of 5 children with PIK3CA-associated HNLMs or head and neck FIL with alpelisib monotherapy. METHODS: A total of 5 children with PIK3CA-associated HNLMs (n = 4) or FIL (n = 1) received alpelisib monotherapy (aged 2-12 years). Treatment response was determined by parental report, clinical evaluation, diary/questionnaire, and standardized clinical photography, measuring facial volume through 3-dimensional photos and magnetic resonance imaging. RESULTS: All participants had reduction in the size of lesion, and all had improvement or resolution of malformation inflammation/pain/bleeding. Common invasive therapy was avoided (ie, tracheotomy). After 6 or more months of alpelisib therapy, facial volume was reduced (range 1%-20%) and magnetic resonance imaging anomaly volume (range 0%-23%) were reduced, and there was improvement in swallowing, upper airway patency, and speech clarity. CONCLUSION: Individuals with head and neck PROS treated with alpelisib had decreased malformation size and locoregional overgrowth, improved function and symptoms, and fewer invasive procedures.

The use of public transport and contraction of SARS-CoV-2 in a large prospective cohort in Norway.

BACKGROUND: For many people public transport is the only mode of travel, and it can be challenging to keep the necessary distances in such a restricted space. The exact role of public transportation and risk of SARS-CoV-2 transmission is not known. METHODS: Participants (n = 121,374) were untested adult Norwegian residents recruited through social media who in the spring of 2020 completed a baseline questionnaire on demographics and the use of public transport. Incident cases (n = 1069) had a positive SARS-CoV-2 polymerase chain reaction test registered at the Norwegian Messaging System for Infectious Diseases by January 27, 2021. We investigated the association between the use of public transport and SARS-CoV-2 using logistic regression. Odds ratios (ORs) with 95% confidence intervals (CIs) adjusted for age, calendar time, gender, municipality, smoking, income level, fitness and underlying medical conditions were estimated. Frequency of the use of public transport was reported for 2 week-periods. RESULTS: Before lockdown, those who tested positive on SARS-CoV-2 were more likely to have used public transport 1-3 times (OR = 1.28, CI 1.09-1.51), 4-10 times (OR = 1.49, CI 1.26-1.77) and ≥ 11 times (OR = 1.50, CI 1.27-1.78, p for trend < 0.0001) than those who had not tested positive. CONCLUSION: The use of public transport was positively associated with contracting SARS-CoV-2 both before and after lockdown.

Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition.

Maternal-to-zygotic transition (MZT) is essential for the formation of a new individual, but is still poorly understood despite recent progress in analysis of gene expression and DNA methylation in early embryogenesis. Dynamic histone modifications may have important roles in MZT, but direct measurements of chromatin states have been hindered by technical difficulties in profiling histone modifications from small quantities of cells. Recent improvements allow for 500 cell-equivalents of chromatin per reaction, but require 10,000 cells for initial steps or require a highly specialized microfluidics device that is not readily available. We developed a micro-scale chromatin immunoprecipitation and sequencing (µChIP-seq) method, which we used to profile genome-wide histone H3 lysine methylation (H3K4me3) and acetylation (H3K27ac) in mouse immature and metaphase II oocytes and in 2-cell and 8-cell embryos. Notably, we show that ~22% of the oocyte genome is associated with broad H3K4me3 domains that are anti-correlated with DNA methylation. The H3K4me3 signal becomes confined to transcriptional-start-site regions in 2-cell embryos, concomitant with the onset of major zygotic genome activation. Active removal of broad H3K4me3 domains by the lysine demethylases KDM5A and KDM5B is required for normal zygotic genome activation and is essential for early embryo development. Our results provide insight into the onset of the developmental program in mouse embryos and demonstrate a role for broad H3K4me3 domains in MZT.

Cleft palate morphology, genetic etiology, and risk of mortality in infants with Robin sequence.

Robin sequence (RS) has many genetic and nongenetic causes, including isolated Robin sequence (iRS), Stickler syndrome (SS), and other syndromes (SyndRS). The purpose of this study was to determine if the presence and type of cleft palate varies between etiologic groups. A secondary endpoint was to determine the relationship of etiologic group, cleft type, and mortality. Retrospective chart review of patients with RS at two high-volume craniofacial centers. 295 patients with RS identified. CP was identified in 97% with iRS, 95% with SS, and 70% of those with SyndRS (p < .0001). U-shaped CP was seen in 86% of iRS, 82% with SS, but only 27% with SyndRS (p < .0001). At one institution, 12 children (6%) with RS died, all from the SyndRS group (p < .0001). All died due to medical comorbidities related to their syndrome. Only 25% of children who died had a U-shaped CP. The most common palatal morphology among those who died was an intact palate. U-shaped CP was most strongly associated with iRS and SS, and with a lower risk of mortality. RS with submucous CP, cleft lip and palate or intact palate was strongly suggestive of an underlying genetic syndrome and higher risk of mortality.

ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.

N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m(6)A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m(6)A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m(6)A modification has fundamental and broad functions in mammalian cells.

Activating variants in PDGFRB result in a spectrum of disorders responsive to imatinib monotherapy.

More than 50 individuals with activating variants in the receptor tyrosine kinase PDGFRB have been reported, separated based on clinical features into solitary myofibromas, infantile myofibromatosis, Penttinen syndrome with premature aging and osteopenia, Kosaki overgrowth syndrome, and fusiform aneurysms. Despite their descriptions as distinct clinical entities, review of previous reports demonstrates substantial phenotypic overlap. We present a case series of 12 patients with activating variants in PDGFRB and review of the literature. We describe five patients with PDGFRB activating variants whose clinical features overlap multiple diagnostic entities. Seven additional patients from a large family had variable expressivity and late-onset disease, including adult onset features and two individuals with sudden death. Three patients were treated with imatinib and had robust and rapid response, including the first two reported infants with multicentric myofibromas treated with imatinib monotherapy and one with a recurrent p.Val665Ala (Penttinen) variant. Along with previously reported individuals, our cohort suggests infants and young children had few abnormal features, while older individuals had multiple additional features, several of which appeared to worsen with advancing age. Our analysis supports a diagnostic entity of a spectrum disorders due to activating variants in PDGFRB. Differences in reported phenotypes can be dramatic and correlate with advancing age, genotype, and to mosaicism in some individuals.

Reversible RNA modifications in meiosis and pluripotency.

Post-transcriptional RNA modifications were discovered several decades ago, but the reversible nature of RNA modifications has only recently been discovered. Owing to technological advances, knowledge of epitranscriptomic marks and their writers, readers and erasers has recently advanced tremendously. Here we focus on the roles of the dynamic methylation and demethylation of internal adenosines in mRNA in germ cells and pluripotent stem cells.

Mycolicibacterium nivoides sp. nov isolated from a peat bog.

A fast-growing, non-chromogenic, acid-fast-staining bacterium (DL90T) was isolated from a peat bog in northern Minnesota. On the basis of 16S rRNA gene sequence similarity (99.8 % identity with Mycolicibacterium septicum and 98 % with Mycolicibacterium peregrinum) and chemotaxonomic data (fatty acid content), strain DL90T represents a member of the genus Mycolicibacterium. Physiological tests (growth curves, biofilm formation, antibiotic sensitivity, colony morphologies and heat tolerance) and biochemical analysis (arylsulfatase activity and fatty acid profiles) distinguish DL90T from its closest relative M. septicum. Phylogenomic reconstruction of the 'Fortuitium-Vaccae' clade, digital DNA-DNA hybridization (DDH) values of 61 %, and average nucleotide identity (ANI) values of approximately 95 % indicate that DL90T is likely to be diverged from M. septicum. Thus, we propose that DL90T represents a novel species, given the name Mycolicibacterium nivoides with the type strain being isolate DL90T (=JCM 32796T=NCCB 100660T).

The development of an opioid sparing anesthesia protocol for pediatric ambulatory tonsillectomy and adenotonsillectomy surgery-A quality improvement project.

Pain management following pediatric tonsillectomy and adenotonsillectomy surgery is challenging and traditionally involves perioperative opioids. However, the recent national opioid shortage compelled anesthesiologists at Bellevue Surgery Center to identify an alternative perioperative analgesic regimen that minimizes opioids yet provides effective pain relief. We assembled an interdisciplinary quality improvement team to trial a series of analgesic protocols using the Plan-Do-Study-Act cycle. Initially, we replaced intraoperative morphine and acetaminophen (M/A protocol) with intraoperative dexmedetomidine and preoperative ibuprofen (D/I protocol). However, when results were not favorable, we rapidly transitioned to intraoperative ketorolac and dexmedetomidine (D/K protocol). The following measures were evaluated using statistical process control chart methodology and interpreted using Shewhart's theory of variation: maximum pain score in the postanesthesia care unit, postoperative morphine rescue rate, postanesthesia care unit length of stay, total anesthesia time, postoperative nausea and vomiting rescue rate, and reoperation rate within 30 days of surgery. There were 333 patients in the M/A protocol, 211 patients in the D/I protocol, and 196 patients in the D/K protocol. With the D/I protocol, there were small increases in maximum pain score and postanesthesia care unit length of stay, but no difference in morphine rescue rate or total anesthesia time compared to the M/A protocol. With the D/K protocol, postoperative pain control and postanesthesia care unit length of stay were similar compared to the M/A protocol. Both the D/I and D/K protocols had reduced nausea and vomiting rescue rates. Reoperation rates were similar between groups. In summary, we identified an intraoperative anesthesia protocol for pediatric tonsillectomy and adenotonsillectomy surgery utilizing dexmedetomidine and ketorolac that provides effective analgesia without increasing recovery times or reoperation rates.

Positioning Europe for the EPITRANSCRIPTOMICS challenge.

The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.

Ultrasound diagnosis of tracheal cartilaginous sleeve in a patient with Pfeiffer syndrome.

There is an association between tracheal cartilaginous sleeve and syndromic craniosynostosis. We present a case of tracheal cartilaginous sleeve diagnosed by ultrasound (US) in a patient with Pfeiffer syndrome. The patient developed respiratory failure and was suspected at bronchoscopy to have tracheal cartilaginous sleeve. US performed before tracheostomy placement demonstrated continuous hypoechoic cartilage along the anterior surface of the trachea, confirming the diagnosis. Our report shows that US can make a definitive diagnosis of tracheal cartilaginous sleeve and raises the possibility of using US to screen for the condition in patients with syndromic craniosynostosis without the need for anesthesia or ionizing radiation.

Tracheal cartilaginous sleeves in children with syndromic craniosynostosis.

PURPOSE: Because a tracheal cartilaginous sleeve (TCS) confers a significant mortality risk that can be mitigated with appropriate intervention, we sought to describe the prevalence and associated genotypes in a large cohort of children with syndromic craniosynostosis. METHODS: Chart review of patients with syndromic craniosynostosis across two institutions. RESULTS: In a cohort of 86 patients with syndromic craniosynostosis, 31 required airway evaluation under anesthesia. TCS was found in 19, for an overall prevalence of 22%. FGFR2, TWIST1, and FGFR3 mutations were identified in children with TCS. All five children with a W290C mutation in FGFR2 had TCS, and most previously reported children with W290C had identification of TCS or early death. In contrast, TCS was not associated with other mutations at residue 290. CONCLUSION: There is an association between TCS and syndromic craniosynostosis, and it appears to be particularly high in individuals with the W290C mutation in FGFR2. Referral to a pediatric otolaryngologist and consideration of operative airway evaluation (i.e., bronchoscopy or rigid endoscopy) in all patients with syndromic craniosynostosis should be considered to evaluate for TCS. Results from genetic testing may help providers weigh the risks and benefits of early airway evaluation and intervention in children with higher-risk genotypes.Genet Med 19 1, 62-68.

Mycobacterium sarraceniae sp. nov. and Mycobacterium helvum sp. nov., isolated from the pitcher plant Sarracenia purpurea.

Several fast- to intermediate-growing, acid-fast, scotochromogenic bacteria were isolated from Sarracenia purpurea pitcher waters in Minnesota sphagnum peat bogs. Two strains (DL734T and DL739T) were among these isolates. On the basis of 16S rRNA gene sequences, the phylogenetic positions of both strains is in the genus Mycobacterium with no obvious relation to any characterized type strains of mycobacteria. Phenotypic characterization revealed that neither strain was similar to the type strains of known species of the genus Mycobacterium in the collective properties of growth, pigmentation or fatty acid composition. Strain DL734T grew at temperatures between 28 and 32 °C, was positive for 3-day arylsulfatase production, and was negative for Tween 80 hydrolysis, urease and nitrate reduction. Strain DL739T grew at temperatures between 28 and 37 °C, and was positive for Tween 80 hydrolysis, urea, nitrate reduction and 3-day arylsulfatase production. Both strains were catalase-negative while only DL739T grew with 5 % NaCl. Fatty acid methyl ester profiles were unique for each strain. DL739T showed an ability to survive at 8 °C with little to no cellular replication and is thus considered to be psychrotolerant. Therefore, strains DL734T and DL739T represent two novel species of the genus Mycobacterium with the proposed names Mycobacterium sarraceniae sp. nov. and Mycobacterium helvum sp. nov., respectively. The type strains are DL734T (=JCM 30395T=NCCB 100519T) and DL739T (=JCM 30396T=NCCB 100520T), respectively.

Biochemical properties of tissue-engineered cartilage.

OBJECTIVE: Microtia is treated with rib cartilage sculpting and staged procedures; though aesthetically pleasing, these constructs lack native ear flexibility. Tissue-engineered (TE) elastic cartilage may bridge this gap; however, TE cartilage implants lead to hypertrophic changes with calcification and loss of flexibility. Retaining flexibility in TE cartilage must focus on increased elastin, maintained collagen II, decreased collagen X, with prevention of calcification. This study compares biochemical properties of human cartilage to TE cartilage from umbilical cord mesenchymal stem cells (UCMSCs). Our goal is to establish a baseline for clinically useful TE cartilage. METHODS: Discarded cartilage from conchal bowl, microtic ears, preauricular tags, rib, and TE cartilage were evaluated for collagen I, II, X, calcium, glycosaminoglycans, elastin, and fibrillin I and III. Human UCMSCs were chondroinduced on 2D surfaces and 3D D,L-lactide-co-glycolic acid (PLGA) fibers. RESULTS: Cartilage samples demonstrated similar staining for collagens I, II, and X, elastin, and fibrillin I and III, but differed from rib. TE pellets and PLGA-supported cartilage were similar to auricular samples in elastin and fibrillin I staining. TE samples were exclusively stained for fibrillin III. Only microtic samples demonstrated calcium staining. CONCLUSIONS: TE cartilage expressed similar levels of elastin, fibrillin I, and collagens I and X when compared to native cartilage. Microtic cartilage demonstrated elevated calcium, suggesting this abnormal tissue may not be a viable cell source for TE cartilage. TE cartilage appears to recapitulate the embryonic development of fibrillin III, which is not expressed in adult tissue, possibly providing a strategy to control TE elastic cartilage phenotype.