Mutational scanning of CRX classifies clinical variants and reveals biochemical properties of the transcriptional effector domain.

Cone-Rod Homeobox, encoded by CRX, is a transcription factor (TF) essential for the terminal differentiation and maintenance of mammalian photoreceptors. Structurally, CRX comprises an ordered DNA-binding homeodomain and an intrinsically disordered transcriptional effector domain. Although a handful of human variants in CRX have been shown to cause several different degenerative retinopathies with varying cone and rod predominance, as with most human disease genes the vast majority of observed CRX genetic variants are uncharacterized variants of uncertain significance (VUS). We performed a deep mutational scan (DMS) of nearly all possible single amino acid substitution variants in CRX, using an engineered cell-based transcriptional reporter assay. We measured the ability of each CRX missense variant to transactivate a synthetic fluorescent reporter construct in a pooled fluorescence-activated cell sorting assay and compared the activation strength of each variant to that of wild-type CRX to compute an activity score, identifying thousands of variants with altered transcriptional activity. We calculated a statistical confidence for each activity score derived from multiple independent measurements of each variant marked by unique sequence barcodes, curating a high-confidence list of nearly 2,000 variants with significantly altered transcriptional activity compared to wild-type CRX. We evaluated the performance of the DMS assay as a clinical variant classification tool using gold-standard classified human variants from ClinVar, and determined that activity scores could be used to identify pathogenic variants with high specificity. That this performance could be achieved using a synthetic reporter assay in a foreign cell type, even for a highly cell type-specific TF like CRX, suggests that this approach shows promise for DMS of other TFs that function in cell types that are not easily accessible. Per-position average activity scores closely aligned to a predicted structure of the ordered homeodomain and demonstrated position-specific residue requirements. The intrinsically disordered transcriptional effector domain, by contrast, displayed a qualitatively different pattern of substitution effects, following compositional constraints without specific residue position requirements in the peptide chain. The observed compositional constraints of the effector domain were consistent with the acidic exposure model of transcriptional activation. Together, the results of the CRX DMS identify molecular features of the CRX effector domain and demonstrate clinical utility for variant classification.

Persistent Cutaneous Lesions of Darier Disease and Second-Hit Somatic Variants in ATP2A2 Gene.

Importance: Darier disease (DD) is a rare genetic skin disorder caused by heterozygous variants in the ATP2A2 gene. Clinical manifestations include recurrent hyperkeratotic papules and plaques that occur mainly in seborrheic areas. Although some of the lesions wax and wane in response to environmental factors, others are severe and respond poorly to therapy. Objective: To investigate the molecular mechanism underlying the persistency of skin lesions in DD. Design, Setting, and Participants: In this case series, DNA was extracted from unaffected skin, transient and persistent lesional skin, and blood from 9 patients with DD. Genetic analysis was used using paired-whole exome sequencing of affected skin and blood or by deep sequencing of ATP2A2 of affected skin. Chromosomal microarray analysis was used to reveal copy number variants and loss of heterozygosity. All variants were validated by Sanger sequencing or restriction fragment length polymorphism. Interventions or Exposures: Paired whole-exome sequencing and deep sequencing of ATP2A2 gene from blood and skin samples isolated from persistent, transient lesions and unaffected skin in patients with DD. Main Outcomes and Measures: Germline and somatic genomic characteristics of persistent and transient cutaneous lesions in DD. Results: Of 9 patients with DD, all had heterozygous pathogenic germline variants in the ATP2A2 gene, 6 were female. Participant age ranged from 40 to 69 years on enrollment. All 11 persistent skin lesions were associated with second-hit somatic variants in the ATP2A2 gene. The somatic variants were classified as highly deleterious via combined annotation-dependent depletion (CADD) scores or affect splicing, and 3 of them had been previously described in patients with DD and acrokeratosis verruciformis of Hopf. Second-hit variants in the ATP2A2 gene were not identified in the transient lesions (n = 2) or the normal skin (n = 2). Conclusions and Relevance: In this study, persistent DD lesions were associated with the presence of second-hit somatic variants in the ATP2A2 gene. Identification of these second-hit variants offers valuable insight into the underlying mechanisms that contribute to the lasting nature of persistent DD lesions.

Preoperative cognitive impairment and postoperative delirium among Holocaust survivors who achieved longevity.

BACKGROUND: Holocaust survivors (HS) experience higher rates of physiological and psychological morbidities, increasing their vulnerability to perioperative complications. Limited information exists regarding their perioperative neurocognitive disorders. This study aimed to assess the rates of preoperative cognitive impairment, postoperative delirium, and other complications among HS aged 75+ undergoing elective surgery. METHODS: This is a single-center retrospective cohort study that analyzed prospectively collected data. All surgical patients born before 1945 who underwent elective noncardiac surgery at a tertiary hospital in Israel during 2020-2021 were included. The HS group was identified through the hospital's information system. Preoperative cognitive impairment was assessed using the Mini-Cog test. Postoperative delirium was defined as a combined outcome, which included a positive 4A's-Test (4AT) result during the postanesthesia care unit stay up to the second postoperative day, a positive 3-Minute Diagnostic Confusion Assessment Method (3D-CAM) test administered by the geriatric team, and identification through the medical records using the Chart-based Delirium Identification Instrument (CHART-DEL). We used multivariable regression to assess the risk factors for postoperative delirium. RESULTS: Out of 1332 eligible patients, 422 (32%) were HS, while the others served as controls. Both groups had a similar rate of preoperative cognitive impairment (24%, p = 0.89) and postoperative delirium (16%, p = 0.95). HS exhibited a higher risk of the composite adverse events (24% vs. 20%, p = 0.05, aOR [95% CI] 1.3 [1.0-1.7]), driven mainly by falls during hospitalization (4% vs. 2%, p = 0.03, aOR 2.1 [1.1-4.1]). HS patients were more likely to be childless (33% vs. 11%, p = 0.001) and had higher rates of chronic antidepressant and benzodiazepine use. CONCLUSIONS: HS achieving longevity do not face increased risks of preoperative cognitive impairment and postoperative delirium. However, special care is still warranted due to their elevated rate of other complications during hospitalization.

The Prevalence of Post-traumatic Stress Disorder as Part of Post-ICU Syndrome among Israeli ICU Survivors.

BACKGROUND: Survivors of critical illness are at increased risk of long-term impairments, referred to as post-intensive care unit (ICU) syndrome (PICS). Post-traumatic stress disorder (PTSD) is common among ICU survivors with reported rates of up to 27%. The prevalence of PTSD among Israeli ICU survivors has not been reported to date. OBJECTIVES: To evaluate the prevalence of new onset PTSD diagnosed in a post-ICU clinic at a tertiary center in Israel. METHODS: We conducted a retrospective, single center, cohort study. Data were collected from medical records of all patients who visited the Tel Aviv Sourasky Medical Center post-ICU clinic between October 2017 and June 2020. New onset PTSD was defined as PTSD diagnosed by a certified board psychiatrist during the post-ICU clinic visit. Data were analyzed using descriptive statistics. RESULTS: Overall, 39 patients (mean age 51 ± 17 years, 15/39 females [38%]) attended the post-ICU clinic during the study period. They were evaluated 82 ± 57 days after hospital discharge. After excluding 7 patients due to missing proper psychiatric analysis, 32 patients remained eligible for the primary analysis. New PTSD was diagnosed in one patient (3%). CONCLUSIONS: We found lower incidence of PTSD in our cohort when compared to existing literature. Possible explanations include different diagnostic tools and low risk factors rate. Unique national, cultural, and/or religious perspectives might have contributed to the observed low PTSD rate. Further research in larger study populations is required to establish the prevalence of PTSD among Israeli ICU survivors.

Pathogenic variants in CRX have distinct cis-regulatory effects on enhancers and silencers in photoreceptors.

Dozens of variants in the gene for the homeodomain transcription factor (TF) cone-rod homeobox (CRX) are linked with human blinding diseases that vary in their severity and age of onset. How different variants in this single TF alter its function in ways that lead to a range of phenotypes is unclear. We characterized the effects of human disease-causing variants on CRX cis-regulatory function by deploying massively parallel reporter assays (MPRAs) in mouse retina explants carrying knock-ins of two variants, one in the DNA-binding domain (p.R90W) and the other in the transcriptional effector domain (p.E168d2). The degree of reporter gene dysregulation in these mutant Crx retinas corresponds with their phenotypic severity. The two variants affect similar sets of enhancers, and p.E168d2 has distinct effects on silencers. Cis-regulatory elements (CREs) near cone photoreceptor genes are enriched for silencers that are derepressed in the presence of p.E168d2. Chromatin environments of CRX-bound loci are partially predictive of episomal MPRA activity, and distal elements whose accessibility increases later in retinal development are enriched for CREs with silencer activity. We identified a set of potentially pleiotropic regulatory elements that convert from silencers to enhancers in retinas that lack a functional CRX effector domain. Our findings show that phenotypically distinct variants in different domains of CRX have partially overlapping effects on its cis-regulatory function, leading to misregulation of similar sets of enhancers while having a qualitatively different impact on silencers.

Lack of mitochondrial complex I assembly factor NDUFAF2 results in a distinctive infantile-onset brainstem neurodegenerative disease with early lethality.

BACKGROUND: Congenital disorders of the mitochondrial respiratory chain are a heterogeneous group of inborn errors of metabolism. Among them, NADH:ubiquinone oxidoreductase (complex I, CI) deficiency is the most common. Biallelic pathogenic variants in NDUFAF2, encoding the nuclear assembly CI factor NDUFAF2, were initially reported to cause progressive encephalopathy beginning in infancy. Since the initial report in 2005, less than a dozen patients with NDUFAF2-related disease have been reported. METHODS: Clinical, biochemical, and neuroradiological features of four new patients residing in Northern Israel were collected during 2016-2022 at Emek Medical Center. Enzymatic activities of the five respiratory-chain complexes were determined in isolated fibroblast mitochondria by spectrophotometric methods. Western blot analyses were conducted with anti-human NDUFAF2 antibody; antibody against the mitochondrial marker VDAC1 was used as a loading control. Genetic studies were performed by chromosome microarray analysis using Affymetrix CytoScan 750 K arrays. RESULTS: All four patients presented with infantile-onset growth retardation, ophthalmological impairments with nystagmus, strabismus (starting between 5 and 9 months), and further progressed to life-threatening episodes of apnea usually triggered by trivial febrile illnesses (between 10 and 18 months) with gradual loss of acquired developmental milestones (3 of 4 patients). Serial magnetic-resonance imaging studies in two of the four patients showed a progressive pattern of abnormal T2-weighted hyperintense signals involving primarily the brainstem, the upper cervical cord, and later, the basal ganglia and thalami. Magnetic-resonance spectroscopy in one patient showed an increased lactate peak. Disease progression was marked by ventilatory dependency and early lethality. 3 of the 4 patients tested, harbored a homozygous 142-kb partial interstitial deletion that omits exons 2-4 of NDUFAF2. Mitochondrial CI activity was significantly decreased in the only patient tested. Western blot analysis disclosed the absence of NDUFAF2 protein compared to normal controls. In addition, we reviewed all 10 previously reported NDUFAF2-deficient cases to better characterize the disease. CONCLUSIONS: Biallelic loss-of-function mutations in NDUFAF2 result in a distinctive phenotype in the spectrum of Leigh syndrome with clinical and neuroradiological features that are primarily attributed to progressive brainstem damage.

Trends of Diagnosis, Disease Course, and Treatment of Atopic Dermatitis 2012-2021: Real-World Data from a Large Healthcare Provider.

In the last decade, new treatments for atopic dermatitis (AD) have emerged. We aimed to describe trends of the diagnosis, disease course, and treatment of AD over a decade (2012-2021) using data from Maccabi Healthcare Services (a 2.7-million-member healthcare provider in Israel). The AD prevalence was stable (4.0% on 31 December 2021 vs. 4.3% on 31 December 2012). The annual AD incidence was also stable (5.8/1000 in 2012 and 5.7/1000 in 2021). AD-related treatment use was highest in the first year post-diagnosis, and it included, among children (n = 87,414) vs. adults (n = 36,865), low-potency topical corticosteroids (TCS) (41.8% vs. 27.1%), mid-potency TCS (30.1% vs. 28.1%), high-potency TCS (34.9% vs. 60.3%), topical calcineurin inhibitor (10.8% vs. 10.1%), phosphodiesterase-4-inhibitor (0.3% vs. 0.7% overall; approved in 2019), phototherapy (0.1% vs. 2.3%), and systemic/biologic treatments (13.0% vs. 13.3%). Among children diagnosed in 2012 and followed through to 2021 (n = 5248), 21.5% had ≥1 AD diagnosis/treatment 10 years later (among 3223 adults: 38.3%). We conclude that the incidence and prevalence rates of AD were comparable to those in similar database studies and remained relatively stable over the past decade. The results underscore the burden of medication use among children and adults, particularly in the first year after AD diagnosis, and the low rate of AD diagnosis among patients originally diagnosed as children 10 years earlier.

Transcription factor interactions explain the context-dependent activity of CRX binding sites.

The effects of transcription factor binding sites (TFBSs) on the activity of a cis-regulatory element (CRE) depend on the local sequence context. In rod photoreceptors, binding sites for the transcription factor (TF) Cone-rod homeobox (CRX) occur in both enhancers and silencers, but the sequence context that determines whether CRX binding sites contribute to activation or repression of transcription is not understood. To investigate the context-dependent activity of CRX sites, we fit neural network-based models to the activities of synthetic CREs composed of photoreceptor TFBSs. The models revealed that CRX binding sites consistently make positive, independent contributions to CRE activity, while negative homotypic interactions between sites cause CREs composed of multiple CRX sites to function as silencers. The effects of negative homotypic interactions can be overcome by the presence of other TFBSs that either interact cooperatively with CRX sites or make independent positive contributions to activity. The context-dependent activity of CRX sites is thus determined by the balance between positive heterotypic interactions, independent contributions of TFBSs, and negative homotypic interactions. Our findings explain observed patterns of activity among genomic CRX-bound enhancers and silencers, and suggest that enhancers may require diverse TFBSs to overcome negative homotypic interactions between TFBSs.

The association between midazolam premedication and postoperative delirium - a retrospective cohort study.

STUDY OBJECTIVE: To evaluate the association between midazolam premedication and postoperative delirium in a large retrospective cohort of patients ≥70 years. DESIGN: Retrospective cohort study. SETTING: A single tertiary academic medical center. PATIENTS: Patients ≥70 years having elective non-cardiac surgery under general anesthesia from 2020 to 2021. INTERVENTIONS: Midazolam premedication, defined as intravenous midazolam administration prior to induction of general anesthesia. MEASUREMENTS: The primary outcome, postoperative delirium, was a collapsed composite outcome including at least one of the following: a positive 4A's test during post-anesthesia care unit stay and/or the initial 2 postoperative days; physician or nursing records reporting new-onset confusion as captured by the CHART-DEL instrument; or a positive 3D-CAM test. The association between midazolam premedication and postoperative delirium was assessed using multivariable logistic regression, adjusting for potential confounding variables. As secondary analysis, we investigated the association between midazolam premedication and a composite of other postoperative complications. Several sensitivity analyses were performed using similar regression models. MAIN RESULTS: In total, 1973 patients were analyzed (median age 75 years, 47% women, 50% ASA score ≥ 3, 32% high risk surgery). The overall incidence of postoperative delirium was 15.3% (302/1973). Midazolam premedication was administered to 782 (40%) patients (median [IQR] dose 2 [1,2] mg). After adjustment for potential confounding variables, midazolam premedication was not associated with increased odds of postoperative delirium, with adjusted odds ratio of 1.09 (95% confidence interval 0.82-1.45; P = 0.538). Midazolam premedication was also not associated with the composite of other postoperative complications. Furthermore, no association was found between midazolam premedication and postoperative delirium in any of the sensitivity analyses preformed. CONCLUSIONS: Our results suggest that low doses of midazolam can be safely used to pre-medicate elective surgical patients 70 years or older before non-cardiac surgery, without significant effect on the risk of developing postoperative delirium.

Association between postoperative hypotension and acute kidney injury after noncardiac surgery: a historical cohort analysis.

PURPOSE: The extent to which postoperative hypotension contributes to renal injury remains unclear, much less what the harm thresholds might be. We therefore tested the primary hypothesis that there is an absolute hypotensive arterial pressure threshold for acute kidney injury during the initial seven days after noncardiac surgery. METHODS: We conducted a single-centre historical cohort analysis of adults who had noncardiac surgery and had creatinine recorded preoperatively and postoperatively. Our exposure was the lowest postoperative mean arterial pressure, defined as the average of the three lowest postoperative pressure measurements. Our primary analysis was the association between the lowest mean arterial pressure and acute kidney injury, defined according to Kidney Disease: Improving Global Outcomes initiative criteria. Our analysis was adjusted for potentially relevant confounding factors including intraoperative hypotension. RESULTS: Among 64,349 patients analyzed, 2,812 (4.4%) patients had postoperative acute kidney injury. Each 5-mm Hg decrease in the lowest mean arterial pressure was associated with a 28% (97.5% confidence interval [CI], 23 to 32; P < 0.001) increase in the odds of acute kidney injury for lowest mean arterial pressures < 80 mm Hg. Higher lowest pressures were not associated with acute kidney injury (odds ratio, 1.08; 97.5% CI, 0.99 to 1.17; P = 0.04) for each 5-mm Hg decrease in the lowest mean arterial pressure. CONCLUSION: Postoperative hypotension, defined as the lowest postoperative mean arterial pressure < 80 mm Hg, was associated with acute kidney injury after noncardiac surgery. A prospective trial will be required to determine whether the observed association is causal and thus amenable to modification.

RéSUMé: OBJECTIF: Nous ne savons pas dans quelle mesure l'hypotension postopératoire contribue aux lésions rénales, et nous connaissons encore moins les seuils de lésion. Nous avons donc testé l'hypothèse primaire selon laquelle il existerait un seuil absolu de tension artérielle hypotensive pour l'insuffisance rénale aiguë au cours des sept premiers jours suivant une chirurgie non cardiaque. MéTHODE: Nous avons mené une analyse de cohorte historique monocentrique auprès d'adultes ayant bénéficié d'une chirurgie non cardiaque et pour lesquel·les les taux de créatinine avant et après l'opération avaient été enregistrés. Notre exposition était la tension artérielle moyenne postopératoire la plus basse, définie comme la moyenne des trois mesures de tension postopératoire les plus basses. Notre analyse principale a porté sur l'association entre la tension artérielle moyenne la plus basse et l'insuffisance rénale aiguë, définies selon les critères de l'initiative KDIGO (Kidney Disease: Improving Global Outcomes). Notre analyse a été ajustée pour tenir compte des facteurs de confusion potentiellement pertinents, notamment de l'hypotension peropératoire. RéSULTATS: Parmi les 64 349 patient·es analysé·es, 2812 (4,4 %) ont présenté une insuffisance rénale aiguë postopératoire. Chaque diminution de 5 mm Hg de la tension artérielle moyenne la plus faible était associée à une augmentation de 28 % (intervalle de confiance [IC] de 97,5 %, 23 à 32; P < 0,001) des risques d'insuffisance rénale aiguë pour les tensions artérielles moyennes les plus faibles < 80 mm Hg. Des tensions les plus faibles plus hautes n'ont pas été associées à une insuffisance rénale aiguë (rapport de cotes, 1,08; IC 97,5 %, 0,99 à 1,17; P = 0,04) pour chaque diminution de 5 mm Hg de la tension artérielle moyenne la plus faible. CONCLUSION: L'hypotension postopératoire, définie comme la tension artérielle moyenne postopératoire < 80 mm Hg la plus basse, a été associée à une insuffisance rénale aiguë après une chirurgie non cardiaque. Une étude prospective sera nécessaire pour déterminer si l'association observée est causale et donc susceptible d'être modifiée.

A Cre-dependent massively parallel reporter assay allows for cell-type specific assessment of the functional effects of non-coding elements in vivo.

The function of regulatory elements is highly dependent on the cellular context, and thus for understanding the function of elements associated with psychiatric diseases these would ideally be studied in neurons in a living brain. Massively Parallel Reporter Assays (MPRAs) are molecular genetic tools that enable functional screening of hundreds of predefined sequences in a single experiment. These assays have not yet been adapted to query specific cell types in vivo in a complex tissue like the mouse brain. Here, using a test-case 3'UTR MPRA library with genomic elements containing variants from autism patients, we developed a method to achieve reproducible measurements of element effects in vivo in a cell type-specific manner, using excitatory cortical neurons and striatal medium spiny neurons as test cases. This targeted technique should enable robust, functional annotation of genetic elements in the cellular contexts most relevant to psychiatric disease.

Active learning of enhancer and silencer regulatory grammar in photoreceptors.

Cis-regulatory elements (CREs) direct gene expression in health and disease, and models that can accurately predict their activities from DNA sequences are crucial for biomedicine. Deep learning represents one emerging strategy to model the regulatory grammar that relates CRE sequence to function. However, these models require training data on a scale that exceeds the number of CREs in the genome. We address this problem using active machine learning to iteratively train models on multiple rounds of synthetic DNA sequences assayed in live mammalian retinas. During each round of training the model actively selects sequence perturbations to assay, thereby efficiently generating informative training data. We iteratively trained a model that predicts the activities of sequences containing binding motifs for the photoreceptor transcription factor Cone-rod homeobox (CRX) using an order of magnitude less training data than current approaches. The model's internal confidence estimates of its predictions are reliable guides for designing sequences with high activity. The model correctly identified critical sequence differences between active and inactive sequences with nearly identical transcription factor binding sites, and revealed order and spacing preferences for combinations of motifs. Our results establish active learning as an effective method to train accurate deep learning models of cis-regulatory function after exhausting naturally occurring training examples in the genome.

Treatment with Methotrexate in Infants and Toddlers with Atopic Dermatitis: A Retrospective Multi-Center Study.

Atopic dermatitis (AD) is a chronic inflammatory skin disease affecting up to 20% of children. Methotrexate (MTX) is used off-label as a systemic treatment for AD patients unresponsive to topical therapies, but limited data exist regarding its safety and efficacy in children, especially in those < 4 years old. To further investigate MTX in younger patients, we screened the medical records of three referral centers between 2016 and 2022 and identified 28 infants and toddlers < 4 years old with AD treated with MTX. Mean age upon MTX initiation was 2.7 ± 1.2 years and mean investigator global assessment (IGA) score was 3.78 ± 0.4. Median duration of MTX treatment was five months. Following 12 and 24 weeks of MTX treatment, the response rate was 50% and IGA 0/1 was achieved in 14.2% and 21.4% of patients, respectively. Most treatment cessations were attributed to a lack of efficacy or parental concern. Although adverse events were reported in 57.1% of patients, MTX was discontinued due to such adverse events only in two patients (7.1%). Taken together, MTX demonstrated a high safety profile in AD patients <4 years old. MTX efficacy was moderate and presumably underestimated by parents who opted for premature treatment cessation due to concerns associated with an immunomodulatory drug.

Virtual reality and 3D printing in clinical anesthesia: a case series of two years' experience in a single tertiary medical centre.

PURPOSE: Anatomically correct patient-specific models made from medical imaging can be printed on a three-dimensional (3D) printer or turned into a virtual reality (VR) program. Until recently, use in anesthesia has been limited. In 2019, the anesthesia department at Tel Aviv Medical Center launched a 3D program with the aim of using 3D modelling to assist in preoperative anesthesia planning. METHODS: A retrospective review of all relevant patients between July 2019 and June 2021 referred for preoperative airway planning with 3D modelling. Patient files were reviewed for correlation between the model-based airway plan and the actual airway plan, the type of model used, and any anesthetic complications related to airway management. RESULTS: Twenty patients were referred for 3D modelling. Of these, 15 models were printed, including 12 children requiring one lung ventilation. Five patients had VR reconstructions, including three with mediastinal masses. One patient had both a 3D-printed model and a VR reconstruction. There were two cases (10%) where the model plan did not correlate with the final airway plan and one case where a model could not be created because of poor underlying imaging. For the remaining 17 cases, the plan devised on the model matched the final airway plan. There were no anesthetic complications. CONCLUSIONS: Three-dimensional modelling and subsequent printing or VR reconstruction are feasible in clinical anesthesia. Its routine use for patients with challenging airway anatomy correlated well with the final clinical outcome in most cases. High-quality imaging is essential.

RéSUMé: OBJECTIF: Des modèles anatomiquement corrects spécifiques à un·e patient·e réalisés à partir de l'imagerie médicale peuvent être imprimés sur une imprimante tridimensionnelle (3D) ou transformés en programme de réalité virtuelle (RV). Jusqu'à récemment, l'utilisation de cette modalité était limitée en anesthésie. En 2019, le service d'anesthésie du centre médical de Tel Aviv a lancé un programme 3D dans le but d'utiliser la modélisation 3D pour faciliter la planification préopératoire de l'anesthésie. MéTHODE: Nous avons réalisé un examen rétrospectif de toute la patientèle concernée référée pour une planification préopératoire des voies aériennes avec modélisation 3D entre juillet 2019 et juin 2021. Les dossiers des patient·es ont été examinés pour déterminer la corrélation entre le plan de prise en charge des voies aériennes fondé sur le modèle et le plan fondé sur les voies aériennes réelles, le type de modèle utilisé et toute complication anesthésique liée à la prise en charge des voies aériennes. RéSULTATS: Vingt patient·es ont été référé·es pour la modélisation 3D. À partir de cette cohorte, 15 modèles ont été imprimés, dont 12 pour des enfants nécessitant une ventilation pulmonaire. Cinq patient·es ont bénéficié de reconstructions en RV, dont trois avec des masses médiastinales. Un modèle imprimé en 3D et une reconstruction en RV ont été créés pour une personne. Il y a eu deux cas (10 %) où le plan modèle n'était pas corrélé avec le plan des voies aériennes final et un cas où il n'a pas été possible de créer un modèle en raison d'une mauvaise imagerie sous-jacente. Pour les 17 cas restants, le plan conçu sur le modèle correspondait au plan final de prise en charge des voies aériennes. Il n'y a pas eu de complications anesthésiques. CONCLUSION: La modélisation tridimensionnelle et l'impression ultérieure ou la reconstruction en RV sont réalisables en anesthésie clinique. Leur utilisation systématique pour les patient·es présentant une anatomie difficile au niveau des voies aériennes était bien corrélée avec le résultat clinique final dans la plupart des cas. Une imagerie de haute qualité est essentielle.

Pathogenic variants in Crx have distinct cis-regulatory effects on enhancers and silencers in photoreceptors.

Dozens of variants in the photoreceptor-specific transcription factor (TF) CRX are linked with human blinding diseases that vary in their severity and age of onset. It is unclear how different variants in this single TF alter its function in ways that lead to a range of phenotypes. We examined the effects of human disease-causing variants on CRX cis-regulatory function by deploying massively parallel reporter assays (MPRAs) in live mouse retinas carrying knock-ins of two variants, one in the DNA binding domain (p.R90W) and the other in the transcriptional effector domain (p.E168d2). The degree of reporter gene dysregulation caused by the variants corresponds with their phenotypic severity. The two variants affect similar sets of enhancers, while p.E168d2 has stronger effects on silencers. Cis-regulatory elements (CREs) near cone photoreceptor genes are enriched for silencers that are de-repressed in the presence of p.E168d2. Chromatin environments of CRX-bound loci were partially predictive of episomal MPRA activity, and silencers were notably enriched among distal elements whose accessibility increases later in retinal development. We identified a set of potentially pleiotropic regulatory elements that convert from silencers to enhancers in retinas that lack a functional CRX effector domain. Our findings show that phenotypically distinct variants in different domains of CRX have partially overlapping effects on its cis-regulatory function, leading to misregulation of similar sets of enhancers, while having a qualitatively different impact on silencers.

Transcription factor fluctuations underlie cell-to-cell variability in a signaling pathway response.

Stochastic differences among clonal cells can initiate cell fate decisions in development or cause cell-to-cell differences in the responses to drugs or extracellular ligands. One hypothesis is that some of this phenotypic variability is caused by stochastic fluctuations in the activities of transcription factors (TFs). We tested this hypothesis in NIH3T3-CG cells using the response to Hedgehog signaling as a model cellular response. Here, we present evidence for the existence of distinct fast- and slow-responding substates in NIH3T3-CG cells. These two substates have distinct expression profiles, and fluctuations in the Prrx1 TF underlie some of the differences in expression and responsiveness between fast and slow cells. Our results show that fluctuations in TFs can contribute to cell-to-cell differences in Hedgehog signaling.

The origins and functional effects of postzygotic mutations throughout the human life span.

Postzygotic mutations (PZMs) begin to accrue in the human genome immediately after fertilization, but how and when PZMs affect development and lifetime health remain unclear. To study the origins and functional consequences of PZMs, we generated a multitissue atlas of PZMs spanning 54 tissue and cell types from 948 donors. Nearly half the variation in mutation burden among tissue samples can be explained by measured technical and biological effects, and 9% can be attributed to donor-specific effects. Through phylogenetic reconstruction of PZMs, we found that their type and predicted functional impact vary during prenatal development, across tissues, and through the germ cell life cycle. Thus, methods for interpreting effects across the body and the life span are needed to fully understand the consequences of genetic variants.

Preoperative Anxiety Levels and Postoperative Middle Ear Surgery Pain Levels.

OBJECTIVE: This study aimed to characterize self-reported postoperative pain after tympanoplasty and tympanomastoidectomy and correlate pain severity with the patient's preoperative anxiety state. STUDY DESIGN: Prospective cohort study. SETTING: Tertiary referral medical center. PATIENTS: Adult patients undergoing any middle ear surgery between July 2018 and July 2019. MAIN OUTCOME MEASURES: Patient responses to an otology questionnaire (OQ) for scoring pain intensity on a visual analog scale preoperatively and on postoperative days (PODs) 1-4, 21, and 63. The responses were correlated with anxiety state (assessed by State-Trait Personality Inventory [STPI] scores) and clinical and operative data, including surgical technique-related details. RESULTS: Sixty patients were enrolled (mean age ± standard deviation, 40 ± 19.7 yr, 26 men). Their median preoperative (baseline) visual analog scale pain score was 6 on POD1, 5 on POD3, and 1 at 3 and 7 weeks. Their median preoperative OQ score was 32 of 70 (45.7%), 37 of 70 (52.8%) on POD1, 33 of 70 (47.1%) on POD3, 6 of 70 (8.5%) at 3 weeks, and 6 of 70 at 7 weeks. Their overall mean preoperative anxiety level (STPI score) was 2.63 ± 1.50. STPI scores were significantly higher among patients who reported OQ scores equal to or higher than the median during PODs 1 to 4 in comparison to patients who reported OQ scores lower than the median. The α Cronbach correlation between anxiety and postoperative pain scores on POD1 was 0.97. CONCLUSION: Preoperative anxiety levels are closely associated with postoperative pain levels after any middle ear surgery. Measures to control preoperative anxiety are warranted to alleviate postoperative pain.

Assessment of Intra-Abdominal Pressure with a Novel Continuous Bladder Pressure Monitor-A Clinical Validation Study.

INTRODUCTION: Intra-abdominal hypertension and the resulting abdominal compartment syndrome are serious complications of severely ill patients. Diagnosis requires an intra-abdominal pressure (IAP) measurement, which is currently cumbersome and underused. We aimed to test the accuracy of a novel continuous IAP monitor. METHODS: Adults having laparoscopic surgery and requiring urinary catheter intra-operatively were recruited to this single-arm validation study. IAP measurements using the novel monitor and a gold-standard foley manometer were compared. After anesthesia induction, a pneumoperitoneum was induced through a laparoscopic insufflator, and five randomly pre-defined pressures (between 5 and 25 mmHg) were achieved and simultaneously measured via both methods in each participant. Measurements were compared using Bland-Altman analysis. RESULTS: In total, 29 participants completed the study and provided 144 distinct pairs of pressure measurements that were analyzed. A positive correlation between the two methods was found (R2 = 0.93). There was good agreement between the methods, with a mean bias (95% CI) of -0.4 (-0.6, -0.1) mmHg and a standard deviation of 1.3 mmHg, which was statistically significant but of no clinical importance. The limits of agreement (where 95% of the differences are expected to fall) were -2.9 and 2.2 mmHg. The proportional error was statistically insignificant (p = 0.85), suggesting a constant agreement between the methods across the range of values tested. The percentage error was 10.7%. CONCLUSIONS: Continuous IAP measurements using the novel monitor performed well in the clinical setup of controlled intra-abdominal hypertension across the evaluated range of pressures. Further studies should expand the range to more pathological values.

A single-cell massively parallel reporter assay detects cell-type-specific gene regulation.

Massively parallel reporter gene assays are key tools in regulatory genomics but cannot be used to identify cell-type-specific regulatory elements without performing assays serially across different cell types. To address this problem, we developed a single-cell massively parallel reporter assay (scMPRA) to measure the activity of libraries of cis-regulatory sequences (CRSs) across multiple cell types simultaneously. We assayed a library of core promoters in a mixture of HEK293 and K562 cells and showed that scMPRA is a reproducible, highly parallel, single-cell reporter gene assay that detects cell-type-specific cis-regulatory activity. We then measured a library of promoter variants across multiple cell types in live mouse retinas and showed that subtle genetic variants can produce cell-type-specific effects on cis-regulatory activity. We anticipate that scMPRA will be widely applicable for studying the role of CRSs across diverse cell types.