Unplugging lateral fenestrations of NALCN reveals a hidden drug binding site within the pore region.

The sodium (Na+) leak channel (NALCN) is a member of the four-domain voltage-gated cation channel family that includes the prototypical voltage-gated sodium and calcium channels (NaVs and CaVs, respectively). Unlike NaVs and CaVs, which have four lateral fenestrations that serve as routes for lipophilic compounds to enter the central cavity to modulate channel function, NALCN has bulky residues (W311, L588, M1145, and Y1436) that block these openings. Structural data suggest that occluded fenestrations underlie the pharmacological resistance of NALCN, but functional evidence is lacking. To test this hypothesis, we unplugged the fenestrations of NALCN by substituting the four aforementioned residues with alanine (AAAA) and compared the effects of NaV, CaV, and NALCN blockers on both wild-type (WT) and AAAA channels. Most compounds behaved in a similar manner on both channels, but phenytoin and 2-aminoethoxydiphenyl borate (2-APB) elicited additional, distinct responses on AAAA channels. Further experiments using single alanine mutants revealed that phenytoin and 2-APB enter the inner cavity through distinct fenestrations, implying structural specificity to their modes of access. Using a combination of computational and functional approaches, we identified amino acid residues critical for 2-APB activity, supporting the existence of drug binding site(s) within the pore region. Intrigued by the activity of 2-APB and its analogues, we tested compounds containing the diphenylmethane/amine moiety on WT channels. We identified clinically used drugs that exhibited diverse activity, thus expanding the pharmacological toolbox for NALCN. While the low potencies of active compounds reiterate the pharmacological resistance of NALCN, our findings lay the foundation for rational drug design to develop NALCN modulators with refined properties.

New horizons in dermatological education: Skin cancer screening with virtual reality.

BACKGROUND: Technological advances in the field of virtual reality (VR) offer new opportunities in many areas of life, including medical education. The University of Münster has been using VR scenarios in the education of medical students for several years, especially for situations that are difficult to reproduce in reality (e.g., brain death). Due to the consistently positive feedback from students, a dermatological VR scenario for skin cancer screening was developed. OBJECTIVES: Presentation and first evaluation of the skin cancer screening VR scenario to determine to what extent the technical implementation of the scenario was evaluated overall by the students and how their subjective competence to perform a skin cancer screening changed over the course of the teaching unit (theory seminar, VR scenario, theoretical debriefing). METHODS: Students (n = 140) participating in the curricular pilot project during the 2023 summer term were surveyed throughout the teaching unit using several established questionnaires (System Usability Scale, Simulation Task-Load-Index, Realism and Presence Questionnaire) as well as additional questions on cybersickness and subjective learning. RESULTS: (i) The use of VR is technically feasible, (ii) students evaluate the VR scenario as a useful curricular supplement, and (iii) from the students' subjective perspective, a good learning outcome is achieved. Although preparation and follow-up appear to be important for overall learning, the greatest increase in subjective competence to perform a skin cancer screening is achieved by the VR scenario. CONCLUSIONS: Technically feasible and positively evaluated by students, VR can already be a useful addition to dermatology education, although costs are still high. As a visual discipline, dermatology offers special opportunities to create VR scenarios that are not always available or comfortable for patients in reality. Additionally, VR scenarios guarantee the same conditions for all students, which is essential for a high-quality education.

Unplugging lateral fenestrations of NALCN reveals a hidden drug binding site within the pore module.

The sodium (Na + ) leak channel (NALCN) is a member of the four-domain voltage-gated cation channel family that includes the prototypical voltage-gated sodium and calcium channels (Na V s and Ca V s, respectively). Unlike Na V s and Ca V s, which have four lateral fenestrations that serve as routes for lipophilic compounds to enter the central cavity to modulate channel function, NALCN has bulky residues (W311, L588, M1145 and Y1436) that block these openings. Structural data suggest that oc-cluded lateral fenestrations underlie the pharmacological resistance of NALCN to lipophilic compounds, but functional evidence is lacking. To test this hypothesis, we unplugged the fenestrations of NALCN by substituting the four aforementioned resi-dues with alanine (AAAA) and compared the effects of Na V , Ca V and NALCN block-ers on both wild-type (WT) and AAAA channels. Most compounds behaved in a simi-lar manner on both channels, but phenytoin and 2-aminoethoxydiphenyl borate (2-APB) elicited additional, distinct responses on AAAA channels. Further experiments using single alanine mutants revealed that phenytoin and 2-APB enter the inner cav-ity through distinct fenestrations, implying structural specificity to their modes of ac-cess. Using a combination of computational and functional approaches, we identified amino acid residues critical for 2-APB activity, supporting the existence of drug bind-ing site(s) within the pore region. Intrigued by the activity of 2-APB and its ana-logues, we tested additional compounds containing the diphenylmethane/amine moiety on WT channels. We identified compounds from existing clinically used drugs that exhibited diverse activity, thus expanding the pharmacological toolbox for NALCN. While the low potencies of active compounds reiterate the resistance of NALCN to pharmacological targeting, our findings lay the foundation for rational drug design to develop NALCN modulators with refined properties. Significance statement: The sodium leak channel (NALCN) is essential for survival: mutations cause life-threatening developmental disorders in humans. However, no treatment is currently available due to the resistance of NALCN to pharmacological targeting. One likely reason is that the lateral fenestrations, a common route for clinically used drugs to enter and block related ion channels, are occluded in NALCN. Using a combination of computational and functional approaches, we unplugged the fenestrations of NALCN which led us to the first molecularly defined drug binding site within the pore region. Besides that, we also identified additional NALCN modulators from existing clinically used therapeutics, thus expanding the pharmacological toolbox for this leak channel.

A Novel Risk Score to Guide the Evaluation of Acute Hematogenous Osteomyelitis in Children.

OBJECTIVES: To identify independent predictors of and derive a risk score for acute hematogenous osteomyelitis (AHO) in children. METHODS: We conducted a retrospective matched case-control study of children >90 days to <18 years of age undergoing evaluation for a suspected musculoskeletal (MSK) infection from 2017 to 2019 at 23 pediatric emergency departments (EDs) affiliated with the Pediatric Emergency Medicine Collaborative Research Committee. Cases were identified by diagnosis codes and confirmed by chart review to meet accepted diagnostic criteria for AHO. Controls included patients who underwent laboratory and imaging tests to evaluate for a suspected MSK infection and received an alternate final diagnosis. RESULTS: We identified 1135 cases of AHO matched to 2270 controls. Multivariable logistic regression identified 10 clinical and laboratory factors independently associated with AHO. We derived a 4-point risk score for AHO using (1) duration of illness >3 days, (2) history of fever or highest ED temperature ≥38°C, (3) C-reactive protein >2.0 mg/dL, and (4) erythrocyte sedimentation rate >25 mm per hour (area under the curve: 0.892, 95% confidence interval [CI]: 0.881 to 0.901). Choosing to pursue definitive diagnostics for AHO when 3 or more factors are present maximizes diagnostic accuracy at 84% (95% CI: 82% to 85%), whereas children with 0 factors present are highly unlikely to have AHO (sensitivity: 0.99, 95% CI: 0.98 to 1.00). CONCLUSIONS: We identified 10 predictors for AHO in children undergoing evaluation for a suspected MSK infection in the pediatric ED and derived a novel 4-point risk score to guide clinical decision-making.

Protein semisynthesis underscores the role of a conserved lysine in activation and desensitization of acid-sensing ion channels.

Acid-sensing ion channels (ASICs) are trimeric ion channels that open a cation-conducting pore in response to proton binding. Excessive ASIC activation during prolonged acidosis in conditions such as inflammation and ischemia is linked to pain and stroke. A conserved lysine in the extracellular domain (Lys211 in mASIC1a) is suggested to play a key role in ASIC function. However, the precise contributions are difficult to dissect with conventional mutagenesis, as replacement of Lys211 with naturally occurring amino acids invariably changes multiple physico-chemical parameters. Here, we study the contribution of Lys211 to mASIC1a function using tandem protein trans-splicing (tPTS) to incorporate non-canonical lysine analogs. We conduct optimization efforts to improve splicing and functionally interrogate semisynthetic mASIC1a. In combination with molecular modeling, we show that Lys211 charge and side-chain length are crucial to activation and desensitization, thus emphasizing that tPTS can enable atomic-scale interrogations of membrane proteins in live cells.

Changes in colloid oncotic pressure during cardiac surgery with different prime fluid strategies.

OBJECTIVE: In cardiac surgery, colloid oncotic pressure (COP) is affected by haemodilution that results from composition and volume of prime fluid of cardiopulmonary bypass (CPB). However, the extent to which different priming strategies alter COP is largely unknown. Therefore, we investigated the effect of different priming strategies on COP in on-pump cardiac surgery. METHODS: Patients (n = 60) were divided into 3 groups (n = 20 each), based on the center in which they were operated and the specific prime fluid strategy used in that center during the inclusion period. CPB prime fluids were either gelofusine-, albumin-, or crystalloid based, the latter two with or without retrograde autologous priming. RESULTS: In all groups, COP was lowest after weaning from CPB and one hour after CPB. Between groups, COP was lowest with gelofusine prime fluid (16.4, 16.8 mmHg, respectively) compared with crystalloids (MD: -1.9; 95% CI:-3.6, -0.2; p = .02 and MD: -2.4, 95% CI: -4.2, -0.7; p = .002) and albumin (MD: -1.8, 95% CI: -3.5, -0.50; p = .041 and MD: -2.4, 95% CI: -4.1, -0.7; p = .002). In all groups, the decrease in COP one hour after bypass compared to baseline correlated positively with fluid balance at the end of surgery (p < .001). CONCLUSIONS: COP significantly decrease during CPB surgery with the largest decrease in COP at the end of surgery, while at the same time fluid balance increases. We suggest that prime fluid strategy should be carefully selected when maintenance of COP during cardiac surgery is desirable.

Histology-based classifier to distinguish early mycosis fungoides from atopic dermatitis.

BACKGROUND: Histopathological differentiation of early mycosis fungoides (MF) from benign chronic inflammatory dermatoses remains difficult and often impossible, despite the inclusion of all available diagnostic parameters. OBJECTIVE: To identify the most impactful histological criteria for a predictive diagnostic model to discriminate MF from atopic dermatitis (AD). METHODS: In this multicentre study, two cohorts of patients with either unequivocal AD or MF were evaluated by two independent dermatopathologists. Based on 32 histological attributes, a hypothesis-free prediction model was developed and validated on an independent patient's cohort. RESULTS: A reduced set of two histological features (presence of atypical lymphocytes in either epidermis or dermis) was trained. In an independent validation cohort, this model showed high predictive power (95% sensitivity and 100% specificity) to differentiate MF from AD and robustness against inter-individual investigator differences. LIMITATIONS: The study investigated a limited number of cases and the classifier is based on subjectively evaluated histological criteria. CONCLUSION: Aiming at distinguishing early MF from AD, the proposed binary classifier performed well in an independent cohort and across observers. Combining this histological classifier with immunohistochemical and/or molecular techniques (such as clonality analysis or molecular classifiers) could further promote differentiation of early MF and AD.

Impact of cerebral hypoperfusion-reperfusion on optic nerve integrity and visual function in the DBA/2J mouse model of glaucoma.

OBJECTIVE: One of the most important risk factors for developing a glaucomatous optic neuropathy is elevated intraocular pressure. Moreover, mechanisms such as altered perfusion have been postulated to injure the optical path. In a mouse model, we compare first negative effects of cerebral perfusion/reperfusion on the optic nerve structure versus alterations by elevated intraocular pressure. Second, we compare the alterations by isolated hypoperfusion-reperfusion and isolated intraocular pressure to the combination of both. METHODS AND ANALYSIS: Mice were divided in four groups: (1) controls; (2) perfusion altered mice that underwent transient bi-common carotid artery occlusion (BCCAO) for 40 min; (3) glaucoma group (DBA/2J mice); (4) combined glaucoma and altered perfusion (DBA/2J mice with transient BCCAO). Optic nerve sections were stereologically examined 10-12 weeks after intervention. RESULTS: All experimental groups showed a decreased total axon number per optic nerve compared with controls. In DBA/2J and combined DBA/2J & BCCAO mice the significant decrease was roughly 50%, while BCCAO leaded to a 23% reduction of axon number, however reaching significance only in the direct t-test. The difference in axon number between BCCAO and both DBA/2J mice was almost 30%, lacking statistical significance due to a remarkably high variation in both DBA/2J groups. CONCLUSION: Elevated intraocular pressure in the DBA/2J mouse model of glaucoma leads to a much more pronounced optic nerve atrophy compared with transient forebrain hypoperfusion and reperfusion by BCCAO. A supposed worsening effect of an altered perfusion added to the pressure-related damage could not be detected.

Global forest management data for 2015 at a 100 m resolution.

Spatially explicit information on forest management at a global scale is critical for understanding the status of forests, for planning sustainable forest management and restoration, and conservation activities. Here, we produce the first reference data set and a prototype of a globally consistent forest management map with high spatial detail on the most prevalent forest management classes such as intact forests, managed forests with natural regeneration, planted forests, plantation forest (rotation up to 15 years), oil palm plantations, and agroforestry. We developed the reference dataset of 226 K unique locations through a series of expert and crowdsourcing campaigns using Geo-Wiki ( https://www.geo-wiki.org/ ). We then combined the reference samples with time series from PROBA-V satellite imagery to create a global wall-to-wall map of forest management at a 100 m resolution for the year 2015, with forest management class accuracies ranging from 58% to 80%. The reference data set and the map present the status of forest ecosystems and can be used for investigating the value of forests for species, ecosystems and their services.

Effects of crystalloid and colloid priming strategies for cardiopulmonary bypass on colloid oncotic pressure and haemostasis: a meta-analysis.

OBJECTIVES: Colloid oncotic pressure (COP) is an important factor in cardiac surgery, owing to its role in haemodilution. The effect of cardiopulmonary bypass prime fluids on the COP is unknown. In this study, the effect of crystalloid and colloid prime fluids, with or without retrograde autologous priming (RAP), on the COP during elective cardiac surgery was evaluated. METHODS: Randomized controlled trials and prospective clinical trials comparing crystalloid and colloid priming fluids or with RAP were selected. The primary outcome was the COP; secondary outcomes were fluid balance, fluid requirements, weight gain, blood loss, platelet count and transfusion requirements. RESULTS: From 1582 records, 29 eligible studies were identified. COPs were comparable between gelofusine and hydroxyethyl starch (HES) during bypass [mean difference (MD): 0.69; 95% confidence interval (CI): -2.05, 3.43; P = 0.621], after bypass (MD: -0.11; 95% CI: -2.54, 2.32; P = 0.930) and postoperative (MD: -0.61; 95% CI: -1.60, 0.38; P = 0.228). Fluid balance was lower with HES than with crystalloids. RAP reduced transfusion requirements compared with crystalloids. Blood loss was comparable between groups. CONCLUSIONS: COPs did not differ between crystalloids and colloids. As a result of increased transcapillary fluid movement, fluid balance was lower with HES than with crystalloids. Haematocrit and transfusion requirements were comparable between groups. However, the latter was lower when RAP was applied to crystalloid priming compared with crystalloids alone. Finally, no differences in blood loss were observed between the groups.

P2X2 receptor subunit interfaces are missense variant hotspots, where mutations tend to increase apparent ATP affinity.

BACKGROUND AND PURPOSE: P2X receptors are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding to their large extracellular domain. The seven known P2X subtypes can assemble as homotrimeric or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2X receptors is well established, but little is known about the range and prevalence of human genetic variations and the functional implications of specific domains. EXPERIMENTAL APPROACH: Here, we examine the impact of P2X2 receptor inter-subunit interface missense variants identified in the human population or by structural predictions. We test both single and double mutants through electrophysiological and biochemical approaches. KEY RESULTS: We demonstrate that predicted extracellular domain inter-subunit interfaces display a higher-than-expected density of missense variations and that the majority of mutations that disrupt putative inter-subunit interactions result in channels with higher apparent ATP affinity. Lastly, we show that double mutants at the subunit interface show significant energetic coupling, especially if located in close proximity. CONCLUSION AND IMPLICATIONS: We provide the first structural mapping of the mutational distribution across the human population in a ligand-gated ion channel and show that the density of missense mutations is constrained between protein domains, indicating evolutionary selection at the domain level. Our data may indicate that, unlike other ligand-gated ion channels, P2X2 receptors have evolved an intrinsically high threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation.

Structure-guided unlocking of NaX reveals a non-selective tetrodotoxin-sensitive cation channel.

Unlike classical voltage-gated sodium (NaV) channels, NaX has been characterized as a voltage-insensitive, tetrodotoxin-resistant, sodium (Na+)-activated channel involved in regulating Na+ homeostasis. However, NaX remains refractory to functional characterization in traditional heterologous systems. Here, to gain insight into its atypical physiology, we determine structures of the human NaX channel in complex with the auxiliary ß3-subunit. NaX reveals structural alterations within the selectivity filter, voltage sensor-like domains, and pore module. We do not identify an extracellular Na+-sensor or any evidence for a Na+-based activation mechanism in NaX. Instead, the S6-gate remains closed, membrane lipids fill the central cavity, and the domain III-IV linker restricts S6-dilation. We use protein engineering to identify three pore-wetting mutations targeting the hydrophobic S6-gate that unlock a robust voltage-insensitive leak conductance. This constitutively active NaX-QTT channel construct is non-selective among monovalent cations, inhibited by extracellular calcium, and sensitive to classical NaV channel blockers, including tetrodotoxin. Our findings highlight a functional diversity across the NaV channel scaffold, reshape our understanding of NaX physiology, and provide a template to demystify recalcitrant ion channels.

ExAID: A multimodal explanation framework for computer-aided diagnosis of skin lesions.

BACKGROUND AND OBJECTIVES: One principal impediment in the successful deployment of Artificial Intelligence (AI) based Computer-Aided Diagnosis (CAD) systems in everyday clinical workflows is their lack of transparent decision-making. Although commonly used eXplainable AI (XAI) methods provide insights into these largely opaque algorithms, such explanations are usually convoluted and not readily comprehensible. The explanation of decisions regarding the malignancy of skin lesions from dermoscopic images demands particular clarity, as the underlying medical problem definition is ambiguous in itself. This work presents ExAID (Explainable AI for Dermatology), a novel XAI framework for biomedical image analysis that provides multi-modal concept-based explanations, consisting of easy-to-understand textual explanations and visual maps, to justify the predictions. METHODS: Our framework relies on Concept Activation Vectors to map human-understandable concepts to those learned by an arbitrary Deep Learning (DL) based algorithm, and Concept Localisation Maps to highlight those concepts in the input space. This identification of relevant concepts is then used to construct fine-grained textual explanations supplemented by concept-wise location information to provide comprehensive and coherent multi-modal explanations. All decision-related information is presented in a diagnostic interface for use in clinical routines. Moreover, the framework includes an educational mode providing dataset-level explanation statistics as well as tools for data and model exploration to aid medical research and education processes. RESULTS: Through rigorous quantitative and qualitative evaluation of our framework on a range of publicly available dermoscopic image datasets, we show the utility of multi-modal explanations for CAD-assisted scenarios even in case of wrong disease predictions. We demonstrate that concept detectors for the explanation of pre-trained networks reach accuracies of up to 81.46%, which is comparable to supervised networks trained end-to-end. CONCLUSIONS: We present a new end-to-end framework for the multi-modal explanation of DL-based biomedical image analysis in Melanoma classification and evaluate its utility on an array of datasets. Since perspicuous explanation is one of the cornerstones of any CAD system, we believe that ExAID will accelerate the transition from AI research to practice by providing dermatologists and researchers with an effective tool that they can both understand and trust. ExAID can also serve as the basis for similar applications in other biomedical fields.

Clinical Presentation of Acute Osteomyelitis in the Pediatric Emergency Department.

OBJECTIVES: Acute osteomyelitis is a challenging diagnosis to make in the pediatric emergency department (ED), in part because of variability in its presentation. There are limited data detailing the presenting features of pediatric osteomyelitis, factors that are essential to understand to inform diagnostic decision making. We sought to describe relevant clinical data that contributed to the diagnosis of acute osteomyelitis in children presenting to a pediatric ED. METHODS: This was a 10-year retrospective cohort study of patients 18 years or younger diagnosed with acute osteomyelitis in the ED of a large tertiary care children's hospital. Collected data included demographics, clinical history, patient-reported symptoms, vital signs, physical examination findings, and results of basic laboratory, microbiologic, and imaging studies. Descriptive statistics were used to summarize key findings. RESULTS: Two hundred eleven cases of acute osteomyelitis were identified during the study period. The median age was 8.4 years, with 61.1% male. One hundred twenty-seven patients (60.2%) presented to care more than once before being diagnosed. Common symptoms included pain (94.3%), functional limitation (83.9%), and fever (76.3%). Common examination findings included functional limitation (78.2%), focal tenderness (73.5%), and swelling (52.1%). One hundred seventeen patients (55.5%) were febrile during their ED evaluation. Elevated C-reactive protein (>0.8 mg/dL, 92.9%) and erythrocyte sedimentation rate (>10 mm/h, 94.3%) were the most sensitive laboratory markers. CONCLUSIONS: Fever may be absent in up to a quarter of pediatric patients with acute osteomyelitis. Although highly sensitive, inflammatory marker elevations were more modest than those reported previously in cases of pediatric septic arthritis.