Search

RNA helicases drive necessary rearrangements and ensure fidelity during the pre-mRNA splicing cycle. DEAD-box helicase DDX41 has been linked to human disease and has recently been shown to interact with DEAH-box helicase PRP22 in the spliceosomal C* complex, yet its function in splicing remains unknown. Depletion of DDX41 homolog SACY-1 from somatic cells has been previously shown to lead to changes in alternative 3' splice site (3'ss) usage. Here, we show by transcriptomic analysis of published and novel data sets that SACY-1 perturbation causes a previously unreported pattern in alternative 3' splicing in introns with pairs of 3' splice sites ≤18 nt away from each other. We find that both SACY-1 depletion and the allele sacy-1(G533R) lead to a striking unidirectional increase in the usage of the proximal (upstream) 3'ss. We previously discovered a similar alternative splicing pattern between germline tissue and somatic tissue, in which there is a unidirectional increase in proximal 3'ss usage in the germline for ∼200 events; many of the somatic SACY-1 alternative 3' splicing events overlap with these developmentally regulated events. We generated targeted mutant alleles of the Caenorhabditis elegans homolog of PRP22, mog-5, in the region of MOG-5 that is predicted to interact with SACY-1 based on the human C* structure. These viable alleles, and a mimic of the myelodysplastic syndrome-associated allele DDX41(R525H), all promote the usage of proximal alternative adjacent 3' splice sites. We show that PRP22/MOG-5 and DDX41/SACY-1 have overlapping roles in proofreading the 3'ss., (© 2024 Osterhoudt et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The spliceosome undergoes extensive rearrangements as it assembles onto precursor messenger RNAs. In the earliest assembly step, U1snRNA identifies the 5' splice site. However, U1snRNA leaves the spliceosome relatively early in assembly, and 5' splice site identity is subsequently maintained through interactions with U6snRNA, protein factor PRP8, and other components during the rearrangements that build the catalytic site. Using a forward genetic screen in Caenorhabditis elegans, we have identified suppressors of a locomotion defect caused by a 5'ss mutation. Here we report three new suppressor alleles from this screen, two in PRP8 and one in SNRNP200/BRR2. mRNASeq studies of these suppressor strains indicate that they also affect specific native alternative 5'ss, especially for suppressor PRP8 D1549N. A strong suppressor at the unstructured N-terminus of SNRNP200, N18K, indicates a novel role for this region. By examining distinct changes in the splicing of native genes, examining double mutants between suppressors, comparing these new suppressors to previously identified splicing suppressors from yeast, and mapping conserved suppressor residues onto cryoEM structural models of assembling human spliceosomes, we conclude that there are multiple interactions at multiple stages in spliceosome assembly responsible for maintaining the initial 5'ss identified by U1snRNA for entry into the catalytic core., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pre-mRNA splicing is an essential step of eukaryotic gene expression carried out by a series of dynamic macromolecular protein/RNA complexes, known collectively and individually as the spliceosome. This series of spliceosomal complexes define, assemble on, and catalyze the removal of introns. Molecular model snapshots of intermediates in the process have been created from cryo-EM data, however, many aspects of the dynamic changes that occur in the spliceosome are not fully understood. Caenorhabditis elegans follow the GU-AG rule of splicing, with almost all introns beginning with 5' GU and ending with 3' AG. These splice sites are identified early in the splicing cycle, but as the cycle progresses and "custody" of the pre-mRNA splice sites is passed from factor to factor as the catalytic site is built, the mechanism by which splice site identity is maintained or re-established through these dynamic changes is unclear. We performed a genetic screen in C. elegans for factors that are capable of changing 5' splice site choice. We report that KIN17 and PRCC are involved in splice site choice, the first functional splicing role proposed for either of these proteins. Previously identified suppressors of cryptic 5' splicing promote distal cryptic GU splice sites, however, mutations in KIN17 and PRCC instead promote usage of an unusual proximal 5' splice site which defines an intron beginning with UU, separated by 1nt from a GU donor. We performed high-throughput mRNA sequencing analysis and found that mutations in PRCC, and to a lesser extent KIN17, changed alternative 5' splice site usage at native sites genome-wide, often promoting usage of nearby non-consensus sites. Our work has uncovered both fine and coarse mechanisms by which the spliceosome maintains splice site identity during the complex assembly process., Competing Interests: The authors have declared that no competing interests exist.

A safe and healthy natural and built environment is fundamental to children's health and represents a significant determinant of community well-being. We aimed to identify and prioritize environmental health concerns within resource-poor neighborhoods in the Dominican Republic using free-listing and semi-structured focus groups composed of parents and caregivers in the perirural community of Consuelo, Dominican Republic. Transcripts were coded and relevant themes identified using qualitative content analysis. Demographic data and information regarding trash disposal practices were also collected. Participants described common health concerns, including respiratory infections, asthma, vector-borne illnesses, and diarrheal diseases and linked them to environmental hazards in their communities, such as air quality and sanitation. Interventional priorities that emerged included reduction of trash accumulation and trash burning as well as improvement of sanitation facilities., Competing Interests: The authors have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Methemoglobinemia occurs when the heme moiety of hemoglobin (Hb) is oxidized from the ferrous to ferric state, leading to impairments in oxygen transport and delivery. Methemoglobinemia is rare in pediatric patients but has been described in the setting of congenital abnormalities in the Hb structure, inherited enzyme deficiencies, oxidative Hb injury in response to illness, and oxidative Hb injury due to toxicants. We present a 1-week-old infant born with a cervical lymphangioma who developed persistent desaturations that were unresponsive to oxygen after sclerotherapy with doxycycline. Arterial blood gas revealed a high Pao 2 despite low saturations being found on pulse oximetry and a methemoglobin level that was found to be elevated. Further sclerotherapy was discontinued, the saturations eventually normalized, and the methemoglobin level decreased. This is a novel report of sclerotherapy with doxycycline associated with the development of methemoglobinemia., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2019 by the American Academy of Pediatrics.)

A 27-month-old girl with a history of congenital myopathy presented with two indurated, pink plaques involving the right arm and left thigh. Closer examination identified central puncta within these plaques, which reportedly occurred at sites of witnessed arachnid bites. After confirmation of the spider species as Trachelas tranquillus, she was treated to address cutaneous inflammation and suspected superinfection using oral and topical antibiotics as well as topical corticosteroid resulting in prompt resolution of her lesions. Trachelas tranquillus should be considered as a possible source of inflammatory spider bites that can become superinfected., (© 2018 Wiley Periodicals, Inc.)

Control of the agitated patient in the emergency department is challenging. Many options exist for chemical sedation, but most have suboptimal pharmacodynamic action, and many have undesirable adverse effects. There are reports of ketamine administration for control of agitation prehospital and in traumatically injured patients. Ketamine is a noncompetitive N-methyl-D-aspartic acid receptor antagonist, making it an effective dissociative agent. We present 5 cases of ketamine administration to manage agitated adolescent patients with underlying psychiatric disease and/or drug intoxication. Ketamine, as a dissociative agent, may be an alternative pharmacological consideration for the control of agitation in patients with undifferentiated agitated delirium.

Background: Intravenous lipid emulsion (ILE) has gained favor as a rescue treatment for cardiovascular collapse due to intravenous local anesthetic overdose, however, goals of ILE therapy are still being defined. We describe a case of a girl given 66 mL/kg of 20% lipid emulsion (ILE) in the treatment of presumed mepivacaine toxicity., Case Report: An 11-year-old girl weighing 55.6 kg developed pallor, rolling back of the eyes, and rhythmic muscle twitching after receiving a mandibular nerve block injection with a 1.8 mL ampule of 3% mepivacaine. With concern for persistent seizures she was given three 1 mL/kg boluses of ILE, followed by an infusion of 0.25 mL/kg/min. The total dose ultimately administered was 3670 mL (66 mL/kg) over 7 h. A serum triglyceride concentration, drawn 2 h after cessation of ILE infusion, was estimated to be 16,583 mg/dL (429 mmol/L) after several dilutions; her blood was grossly lipemic. Notable signs included hypersomnolence, tachypnea, and tachycardia. Other complications included apparent metabolic acidosis (serum bicarbonate of 5 mmol/L) with hyperlactatemia (lactate 7.0 mmol/L), difficulty with serum laboratory interpretation, and a non-contrast brain magnetic resonance imaging showing high signal in the dural venous sinuses. The lipemia cleared over three days and the patient recovered uneventfully. Case discussion: This case demonstrates a unique neurologic and metabolic toxicity associated with ILE given as an antidote in a high total dose, and highlights the need for cautious antidotal application of lipid emulsion infusions. Until more data is available, clinicians are advised to take great care if considering a dose in excess of 12.5 mL/kg/day, the maximum daily dosage recommended by the U.S. Food and Drug Administration for nutritional supplementation. Careful monitoring of total doses administered across institutions and hospital wards during transfers is paramount to avoid inadvertent overdose of antidotes.

Background: The role of the surveyor in trauma resuscitations is to identify life-threatening injuries and is meant to be conducted by a set protocol for every patient. Optimal performance of the trauma survey is known to be a challenge in pediatric trauma resuscitation. A postulated reason for this observation is that many trainees, such as pediatric residents, who perform the trauma survey have minimal experience and do not have formal advanced trauma life support training. The assessment of factors that may be obstacles in performing the trauma survey has not been studied robustly., Objective: The objective of this retrospective cohort study was to use video review of resuscitation of real-life traumatically injured children to (1) describe the characteristics of the trauma patient, the surveyor, and the trauma response team in its current state of function at a tertiary level I trauma center, (2) describe current performance of primary and secondary surveys, as measured by an assessment tool, and (3) determine whether there are specific characteristics associated with reduced quality, completeness, or timeliness of the assessment of an injured child., Methods: Retrospective review of emergency department (ED) trauma activations captured by video recording between June 2009 and January 2012. Video-recorded resuscitations were reviewed, and survey performance was scored using a novel assessment tool applying a scoring system (0, 1, or 2 points) for each essential element (airway, breathing, circulation, etc.) accounting for quality, sequence, and timing of assessments. Maximum score was 8 points for the primary survey and 22 points for the secondary survey. Time to completion of survey elements was recorded. Chart review identified surveyor characteristics (level of training and type of training program) and patient data fields (age, mechanism of injury, trauma level, Glasgow Coma Score, time of encounter, disposition, and number of procedures). Descriptive statistics and univariate analysis were performed., Results: Of 749 eligible trauma activations, 228 activations were enrolled in the study with complete data for 202 patients. Most activations met level II criteria and involved blunt trauma. Most patients had a Glasgow Coma Score of 15 and were non-ICU inpatient admissions. PGY-3 residents performed the most surveys (53% of surveys done by residents). Pediatric residents performed 46% of surveys; emergency medicine (EM) residents, 41%; and pediatric EM fellows, 6%. Median scores on primary and secondary surveys were 7 and 12, respectively; median time to completion was 82 seconds and 265 seconds, respectively. Only 22% of primary surveys and 0% of secondary surveys were performed completely. Pediatric EM fellows had the highest mean score on primary and secondary survey. Pediatric EM fellows took longest to perform primary survey and shortest to complete secondary survey. Mean scores on primary and secondary survey were not significantly different between pediatric and EM residents (6.7 vs 6.7; 12.5 vs 11.6). There was no association between survey scores and level or type of training. Emergency medicine residents spent less time on the trauma survey, but this difference did not reach statistical significance., Conclusions: Primary and secondary surveys are frequently performed incompletely and inefficiently regardless of level of training or type of training program. There is no difference in measured performance among different types of residency programs. The impact of trauma resuscitation education on improved survey performance should be studied prospectively.

To date, there appear to be no studies that assess Medical Toxicologists' (MTs) practice improvement (PI) activities in their Medical Toxicology practice settings. The MT Assessment of Practice Performance (APP) Taskforce queried all MT diplomates about (1) activities currently available in their practice settings that potentially would meet the requirements of APP, (2) potential APP activities that best fit with current MT practice, and (3) the relationship between MT practice patterns and APP requirements. One hundred twenty-seven surveys were completed. Participation in MT practice improvement activities is not universal, with approximately a third of the survey participants reporting that they are not involved in any practice improvement activity. Few respondents reported that they collected performance improvement-related data. Most who did so participated in CME, case, or chart reviews. Peer reviews, self-improvement plans based on chart reviews, and population research were considered the most valid measures of MT practice improvement. Communication skills were considered important topics for patient surveys. Suggested outcomes for peer assessment included accuracy of information provided, understanding medical staff concerns, timeliness of feedback, and helpfulness. Most respondents rated all of the APP options as being somewhat very intrusive. Access to those with sufficient knowledge of the diplomate's practice improvement program to verify APP could pose a challenge to a successful completion of APP requirements. Optimal settings for the APP program administration are hospitals and poison centers. While barriers to MT APP activities exist, studying current MT diplomates' opinions and practices could inform the future development and administration of such programs.

Uncertainty exists regarding the ability of catnip (Nepeta cataria) to affect human consciousness. We report a case of a toddler exhibiting central nervous system depression after consuming a large quantity of catnip. His obtundation was not attributable to another cause. We review the published literature describing the alleged psychoactive capabilities of catnip and present our case as further information for use in this ongoing controversy.

Chronic, intermittent isovaleric acidemia was undiagnosed in a boy with an increased anion gap metabolic acidosis until the boy was 5 years of age. This case emphasizes the importance of maintaining a high index of suspicion for inborn errors of metabolism in patients with metabolic acidosis, even in late childhood.

Cytochalasins enhanced surfactant secretion from primary cultures of [3H]choline-labeled type II epithelial cells from the rat. Cytochalasins A, B, C, D and dihydrocytochalasin B enhanced secretion of phosphatidyl-[3H]choline [(3H]PC) in a dose-dependent manner with EC50 values of 1, 2, 0.5, 0.1 and 1 microM for cytochalasins A, B, C, D and dihydrocytochalasin B, respectively. Only cytochalasin A caused significant cytotoxicity as determined by release of the intracellular enzyme lactate dehydrogenase (EC 1.1.1.17). Dose responses of surfactant release induced by cytochalasins B, C and D were biphasic; maximal release was observed between 0.1-1.0 microM for cytochalasins C and D between 1 and 10 microM for cytochalasin B. Secretion decreased toward control levels at concentrations of cytochalasin above these maximal concentrations. Increased rates of [3H]PC release were noted between 1 and 3 h after exposure to cytochalasin D. Increased rates of surfactant release induced by cytochalasin D were additive to release induced by the beta-adrenergic agonist, terbutaline, or forskolin, although cytochalasin D had no direct effect on cytosolic cyclic AMP levels. Changes in cell shape and microfilament organization were observed by phase-contrast microscopy and fluorescence microscopy using rhodamine-conjugated phalloidin after exposure of the isolated type II cells to cytochalasin D. Disruption of microfilaments associated with lamellar bodies of the purified type II cells occurred after treatment with cytochalasin D. Cytochalasin D augmented surfactant release from purified type II cells and disrupted the microfilament structure of those cells, supporting the hypothesis that alterations in microfilaments are associated with surfactant release.

The mechanism of cellular uptake of cadmium, a highly toxic metal ion, is not known. We have studied cadmium uptake and toxicity in an established secretory cell line, GH4C1, which has well characterized calcium channels. Nimodipine, an antagonist of voltage-sensitive calcium channels, protected cells against cadmium toxicity by increasing the LD50 for CdCl2 from 15 to 45 microM, whereas the calcium channel agonist BAY K8644 decreased the LD50. Organic calcium channel blockers of three classes protected cells from cadmium toxicity at concentrations previously shown to block high K+-induced 45Ca2+ influx and secretion. Half-maximal protective effects were obtained at 20 nM nifedipine, 4 microM verapamil, and 7 microM diltiazem. Increasing the extracellular calcium concentration from 20 microM to 10 mM also protected cells from cadmium by causing a 5-fold increase in the LD50 for CdCl2. Neither the calcium channel antagonist nimodipine nor the agonist BAY K8644 altered intracellular metallothionein concentrations, while cadmium caused a 9-20-fold increase in metallothionein over 18 h. Cadmium was a potent blocker of depolarization-stimulated 45Ca2+ uptake (IC50 = 4 microM), and the net uptake of cadmium measured with 109Cd2+ was less than 0.3% that of calcium. Although the rate of cadmium uptake was low relative to that of calcium, entry via voltage-sensitive calcium channels appeared to account for a significant portion of cadmium uptake; 109Cd2+ uptake at 30 min was increased 57% by high K+/BAY K8644, which facilitates entry through channels. Furthermore, calcium channel blockade with 100 nM nimodipine decreased total cell 109Cd2+ accumulation after 24 h by 63%. These data indicate that flux of cadmium through dihydropyridine-sensitive, voltage-sensitive calcium channels is a major mechanism for cadmium uptake by GH4C1 cells, and that pharmacologic blockade of calcium channels can afford dramatic protection against cadmium toxicity.