Structural architecture of the human NALCN channelosome.

Depolarizing sodium (Na+) leak currents carried by the NALCN channel regulate the resting membrane potential of many neurons to modulate respiration, circadian rhythm, locomotion and pain sensitivity1-8. NALCN requires FAM155A, UNC79 and UNC80 to function, but the role of these auxiliary subunits is not understood3,7,9-12. NALCN, UNC79 and UNC80 are essential in rodents2,9,13, and mutations in human NALCN and UNC80 cause severe developmental and neurological disease14,15. Here we determined the structure of the NALCN channelosome, an approximately 1-MDa complex, as fundamental aspects about the composition, assembly and gating of this channelosome remain obscure. UNC79 and UNC80 are massive HEAT-repeat proteins that form an intertwined anti-parallel superhelical assembly, which docks intracellularly onto the NALCN-FAM155A pore-forming subcomplex. Calmodulin copurifies bound to the carboxy-terminal domain of NALCN, identifying this region as a putative modulatory hub. Single-channel analyses uncovered a low open probability for the wild-type complex, highlighting the tightly closed S6 gate in the structure, and providing a basis to interpret the altered gating properties of disease-causing variants. Key constraints between the UNC79-UNC80 subcomplex and the NALCN DI-DII and DII-DIII linkers were identified, leading to a model of channelosome gating. Our results provide a structural blueprint to understand the physiology of the NALCN channelosome and a template for drug discovery to modulate the resting membrane potential.

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.

A population of dust-enshrouded objects orbiting the Galactic black hole.

The central 0.1 parsecs of the Milky Way host a supermassive black hole identified with the position of the radio and infrared source Sagittarius A* (refs. 1,2), a cluster of young, massive stars (the S stars3) and various gaseous features4,5. Recently, two unusual objects have been found to be closely orbiting Sagittarius A*: the so-called G sources, G1 and G2. These objects are unresolved (having a size of the order of 100 astronomical units, except at periapse, where the tidal interaction with the black hole stretches them along the orbit) and they show both thermal dust emission and line emission from ionized gas6-10. G1 and G2 have generated attention because they appear to be tidally interacting with the supermassive Galactic black hole, possibly enhancing its accretion activity. No broad consensus has yet been reached concerning their nature: the G objects show the characteristics of gas and dust clouds but display the dynamical properties of stellar-mass objects. Here we report observations of four additional G objects, all lying within 0.04 parsecs of the black hole and forming a class that is probably unique to this environment. The widely varying orbits derived for the six G objects demonstrate that they were commonly but separately formed.

Structure of the human sodium leak channel NALCN.

Persistently depolarizing sodium (Na+) leak currents enhance electrical excitability1,2. The ion channel responsible for the major background Na+ conductance in neurons is the Na+ leak channel, non-selective (NALCN)3,4. NALCN-mediated currents regulate neuronal excitability linked to respiration, locomotion and circadian rhythm4-10. NALCN activity is under tight regulation11-14 and mutations in NALCN cause severe neurological disorders and early death15,16. NALCN is an orphan channel in humans, and fundamental aspects of channel assembly, gating, ion selectivity and pharmacology remain obscure. Here we investigate this essential leak channel and determined the structure of NALCN in complex with a distinct auxiliary subunit, family with sequence similarity 155 member A (FAM155A). FAM155A forms an extracellular dome that shields the ion-selectivity filter from neurotoxin attack. The pharmacology of NALCN is further delineated by a walled-off central cavity with occluded lateral pore fenestrations. Unusual voltage-sensor domains with asymmetric linkages to the pore suggest mechanisms by which NALCN activity is modulated. We found a tightly closed pore gate in NALCN where the majority of missense patient mutations cause gain-of-function phenotypes that cluster around the S6 gate and distinctive π-bulges. Our findings provide a framework to further study the physiology of NALCN and a foundation for discovery of treatments for NALCN channelopathies and other electrical disorders.

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.

Functional cross-talk between phosphorylation and disease-causing mutations in the cardiac sodium channel Nav1.5.

The voltage-gated sodium channel Nav1.5 initiates the cardiac action potential. Alterations of its activation and inactivation properties due to mutations can cause severe, life-threatening arrhythmias. Yet despite intensive research efforts, many functional aspects of this cardiac channel remain poorly understood. For instance, Nav1.5 undergoes extensive posttranslational modification in vivo, but the functional significance of these modifications is largely unexplored, especially under pathological conditions. This is because most conventional approaches are unable to insert metabolically stable posttranslational modification mimics, thus preventing a precise elucidation of the contribution by these modifications to channel function. Here, we overcome this limitation by using protein semisynthesis of Nav1.5 in live cells and carry out complementary molecular dynamics simulations. We introduce metabolically stable phosphorylation mimics on both wild-type (WT) and two pathogenic long-QT mutant channel backgrounds and decipher functional and pharmacological effects with unique precision. We elucidate the mechanism by which phosphorylation of Y1495 impairs steady-state inactivation in WT Nav1.5. Surprisingly, we find that while the Q1476R patient mutation does not affect inactivation on its own, it enhances the impairment of steady-state inactivation caused by phosphorylation of Y1495 through enhanced unbinding of the inactivation particle. We also show that both phosphorylation and patient mutations can impact Nav1.5 sensitivity toward the clinically used antiarrhythmic drugs quinidine and ranolazine, but not flecainide. The data highlight that functional effects of Nav1.5 phosphorylation can be dramatically amplified by patient mutations. Our work is thus likely to have implications for the interpretation of mutational phenotypes and the design of future drug regimens.

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.

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.

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.

Delayed Subaponeurotic Fluid Collections: A Benign Cause of Scalp Swelling in Infancy.

ABSTRACT: Delayed subaponeurotic fluid collections (DSFCs) are a recently described cause of idiopathic scalp swelling in young infants. Associated with traumatic birth and the use of vacuum-assisted devices or fetal scalp electrodes, DSFCs can be differentiated from similar causes of neonatal scalp swelling based on characteristic history and physical examination findings. In this article, we highlight 2 cases of young infants presenting to a pediatric emergency department with large, fluctuant scalp swellings, which were appropriately diagnosed as DSFCs; clinical data on an additional eight cases are presented in table format. In cases of isolated DSFCs in otherwise well-appearing infants, further laboratory work, imaging, subspecialty consultation, and child abuse investigation is not necessary. Recommendations for the diagnosis and management of DSFCs presented reflect the clinical experience of the authors in conjunction with a review of the available medical literature on this topic.

Dietary soybean oil modulates fatty acid composition of pork.

This study was conducted to evaluate the impact of soybean oil level on performance and fatty acid profile of backfat and longissimus lumborum muscle of gilts. Forty-eight gilts with an initial weight of 21.75 ± 0.138 kg and final weight of 98.65 ± 2.106 kg were subjected to one of the following six dietary soybean oil inclusions (0.00, 1.086, 2.173, 3.259, 4.345, and 5.432%). Experimental design was completely randomized block with six treatments and four replicates of two animals each. Lipid profile of backfat and longissimus lumborum muscle was analyzed by gas chromatography. Increasing dietary soybean oil levels did not influence final weight, daily weight gain, and feed intake (P > 0.05) but improved feed to gain ratio (P < 0.05). The inclusion of soybean oil modified the lipid profile of backfat and muscle, reduced saturated and monounsaturated fatty acids, and increased polyunsaturated fatty acids concentration, mainly linoleic and α-linolenic acids (P < 0.05). Increasing dietary soybean oil inclusion decreased atherogenic and thrombogenic indexes, and the omega-6:omega-3 ratio of the backfat and longissimus lumborum muscle (P < 0.05). The level of soybean oil in swine diets influenced backfat and longissimus lumborum lipid profile.

Evolutionarily Conserved Interactions within the Pore Domain of Acid-Sensing Ion Channels.

Despite the sequence homology between acid-sensing ion channels (ASICs) and epithelial sodium channel (ENaCs), these channel families display very different functional characteristics. Whereas ASICs are gated by protons and show a relatively low degree of selectivity for sodium over potassium, ENaCs are constitutively active and display a remarkably high degree of sodium selectivity. To decipher if some of the functional diversity originates from differences within the transmembrane helices (M1 and M2) of both channel families, we turned to a combination of computational and functional interrogations, using statistical coupling analysis and mutational studies on mouse ASIC1a. The coupling analysis suggests that the relative position of M1 and M2 in the upper part of the pore domain is likely to remain constant during the ASIC gating cycle, whereas they may undergo relative movements in the lower part. Interestingly, our data suggest that to account for coupled residue pairs being in close structural proximity, both domain-swapped and nondomain-swapped ASIC M2 conformations need to be considered. Such conformational flexibility is consistent with structural work, which suggested that the lower part of M2 can adopt both domain-swapped and nondomain-swapped conformations. Overall, mutations to residues in the middle and lower pore were more likely to affect gating and/or ion selectivity than those in the upper pore. Indeed, disrupting the putative interaction between a highly conserved Trp/Glu residue pair in the lower pore is detrimental to gating and selectivity, although this interaction might occur in both domain-swapped and nonswapped conformations. Finally, our results suggest that the greater number of larger, aromatic side chains in the ENaC M2 helix may contribute to the constitutive activity of these channels at a resting pH. Together, the data highlight differences in the transmembrane domains of these closely related ion channels that may help explain some of their distinct functional properties.

EGFR/Ras-induced CCL20 production modulates the tumour microenvironment.

BACKGROUND: The activation of the EGFR/Ras-signalling pathway in tumour cells induces a distinct chemokine repertoire, which in turn modulates the tumour microenvironment. METHODS: The effects of EGFR/Ras on the expression and translation of CCL20 were analysed in a large set of epithelial cancer cell lines and tumour tissues by RT-qPCR and ELISA in vitro. CCL20 production was verified by immunohistochemistry in different tumour tissues and correlated with clinical data. The effects of CCL20 on endothelial cell migration and tumour-associated vascularisation were comprehensively analysed with chemotaxis assays in vitro and in CCR6-deficient mice in vivo. RESULTS: Tumours facilitate progression by the EGFR/Ras-induced production of CCL20. Expression of the chemokine CCL20 in tumours correlates with advanced tumour stage, increased lymph node metastasis and decreased survival in patients. Microvascular endothelial cells abundantly express the specific CCL20 receptor CCR6. CCR6 signalling in endothelial cells induces angiogenesis. CCR6-deficient mice show significantly decreased tumour growth and tumour-associated vascularisation. The observed phenotype is dependent on CCR6 deficiency in stromal cells but not within the immune system. CONCLUSION: We propose that the chemokine axis CCL20-CCR6 represents a novel and promising target to interfere with the tumour microenvironment, and opens an innovative multimodal strategy for cancer therapy.

The complement system: an unexpected role in synaptic pruning during development and disease.

An unexpected role for the classical complement cascade in the elimination of central nervous system (CNS) synapses has recently been discovered. Complement proteins are localized to developing CNS synapses during periods of active synapse elimination and are required for normal brain wiring. The function of complement proteins in the brain appears analogous to their function in the immune system: clearance of cellular material that has been tagged for elimination. Similarly, synapses tagged with complement proteins may be eliminated by microglial cells expressing complement receptors. In addition, developing astrocytes release signals that induce the expression of complement components in the CNS. In the mature brain, early synapse loss is a hallmark of several neurodegenerative diseases. Complement proteins are profoundly upregulated in many CNS diseases prior to signs of neuron loss, suggesting a reactivation of similar developmental mechanisms of complement-mediated synapse elimination potentially driving disease progression.

Advances in airway management and mechanical ventilation in inhalation injury.

PURPOSE OF REVIEW: Airway management, mechanical ventilation, and treatment of systemic poisoning in burn patients with inhalation injury remains challenging. This review summarizes new concepts as well as open questions. RECENT FINDINGS: Several life-threatening complications, such as airway patency impairment and respiratory insufficiency, can arise in burn patients and require adequate and timely airway management. However, unnecessary endotracheal intubation should be avoided. Direct visual inspection via nasolaryngoscopy can guide appropriate airway management decisions. In cases of lower airway injury, bronchoscopy is recommended to remove casts and estimate the extent of the injury in intubated patients. Several mechanical ventilation strategies have been studied. An interesting modality might be high-frequency percussive ventilation. However, to date, there is no sound evidence that patients with inhalation injury should be ventilated with modes other than those applied to non-burn patients. In all burn patients exposed to enclosed fire, carbon monoxide as well as cyanide poisoning should be suspected. Carbon monoxide poisoning should be treated with an inspiratory oxygen fraction of 100%, whereas cyanide poisoning should be treated with hydroxocobalamin. SUMMARY: Burn patients need specialized care that requires specific knowledge about airway management, mechanical ventilation, and carbon monoxide and cyanide poisoning.

Time-lapse imaging points towards a non-toxic, mainly immune-driven mode of action of ingenol mebutate in the treatment of anogenital warts.

Recently, it has been reported that ingenol mebutate (IM) is an effective treatment option for anogenital warts (AGW), inducing fast wart necrosis within 24 hours in vivo. With regard to its mode of action, IM is thought to act both as an inducer of direct cytotoxic effects and immunologic mechanisms. To distinguish whether the wart necrosis is mainly caused by cytotoxic effects, or whether immune mechanisms are leading, we used time-lapse imaging to analyse IM-treated warts ex vivo over 24 hours. Ex vivo IM-treated warts, which have been detached from the immune system, did not show destructive necrosis, pointing towards a primarily immune-driven mode of action of IM in the treatment of AGW.

Imatinib Triggers Phagolysosome Acidification and Antimicrobial Activity against Mycobacterium bovis Bacille Calmette-Guérin in Glucocorticoid-Treated Human Macrophages.

Glucocorticoids are extensively used to treat inflammatory diseases; however, their chronic intake increases the risk for mycobacterial infections. Meanwhile, the effects of glucocorticoids on innate host responses are incompletely understood. In this study, we investigated the direct effects of glucocorticoids on antimycobacterial host defense in primary human macrophages. We found that glucocorticoids triggered the expression of cathelicidin, an antimicrobial critical for antimycobacterial responses, independent of the intracellular vitamin D metabolism. Despite upregulating cathelicidin, glucocorticoids failed to promote macrophage antimycobacterial activity. Gene expression profiles of human macrophages treated with glucocorticoids and/or IFN-γ, which promotes induction of cathelicidin, as well as antimycobacterial activity, were investigated. Using weighted gene coexpression network analysis, we identified a module of highly connected genes that was strongly inversely correlated with glucocorticoid treatment and associated with IFN-γ stimulation. This module was linked to the biological functions autophagy, phagosome maturation, and lytic vacuole/lysosome, and contained the vacuolar H(+)-ATPase subunit a3, alias TCIRG1, a known antimycobacterial host defense gene, as a top hub gene. We next found that glucocorticoids, in contrast with IFN-γ, failed to trigger expression and phagolysosome recruitment of TCIRG1, as well as to promote lysosome acidification. Finally, we demonstrated that the tyrosine kinase inhibitor imatinib induces lysosome acidification and antimicrobial activity in glucocorticoid-treated macrophages without reversing the anti-inflammatory effects of glucocorticoids. Taken together, we provide evidence that the induction of cathelicidin by glucocorticoids is not sufficient for macrophage antimicrobial activity, and identify the vacuolar H(+)-ATPase as a potential target for host-directed therapy in the context of glucocorticoid therapy.