A platform for rapid patient-derived cutaneous neurofibroma organoid establishment and screening.

Localized cutaneous neurofibromas (cNFs) are benign tumors that arise in the dermis of patients affected by neurofibromatosis type 1 syndrome. cNFs are benign lesions: they do not undergo malignant transformation or metastasize. Nevertheless, they can cover a significant proportion of the body, with some individuals developing hundreds to thousands of lesions. cNFs can cause pain, itching, and disfigurement resulting in substantial socio-emotional repercussions. Currently, surgery and laser desiccation are the sole treatment options but may result in scarring and potential regrowth from incomplete removal. To identify effective systemic therapies, we introduce an approach to establish and screen cNF organoids. We optimized conditions to support the ex vivo growth of genomically diverse cNFs. Patient-derived cNF organoids closely recapitulate cellular and molecular features of parental tumors as measured by immunohistopathology, methylation, RNA sequencing, and flow cytometry. Our cNF organoid platform enables rapid screening of hundreds of compounds in a patient- and tumor-specific manner.

Anti-Plexin-D1 Seropositive Small Fiber Neuropathy: Clinical Phenotype, Demographics, and Literature Review.

OBJECTIVES: Small fiber neuropathy (SFN) is a subtype of painful neuropathies defined by dysfunction of the Aδ and unmyelinated C fibers. It presents with both neuropathic pain and dysautonomia symptoms, posing a significant diagnostic and therapeutic challenge. To address this challenge, research has been conducted to identify autoantibodies and define their association with phenotypes. METHODS: Eleven cases of anti-plexin-D1 seropositive SFN were reviewed, along with relevant literature, in attempt to better define anti-plexin-D1 SFN demographics, symptoms, associated medical conditions, and therapeutics. RESULTS: Anti-plexin-D1 SFN typically presents in female patients, with neuropathic pain, normal skin biopsy findings, and normal nerve conduction studies. Anti-plexin-D1 shows an association with concurrent chronic pain, with almost half of the patients undergoing an interventional procedure. CONCLUSIONS: Anti-plexin-D1 represents a unique subgroup of SFN, defined by distinct demographics, phenotype, biopsy findings, and therapeutic management.

Accelerated Fabrication of Fiber-Welded Mesoporous Cotton Composites.

Natural fiber-welded (NFW) biopolymer composites are rapidly garnering industrial and commercial attention in the textile sector, and a recent disclosure demonstrating the production of mesoporous NFW materials suggests a bright future as sorbents, filters, and nanoparticle scaffolds. A significant roadblock in the mass production of mesoporous NFW composites for research and development is their lengthy preparation time: 24 h of water rinses to remove the ionic liquid (IL) serving as a welding medium and then 72 h of solvent exchanges (polar to nonpolar), followed by oven drying to attain a mesoporous composite. In this work, the rinsing procedure is systematically truncated using the solution conductivity as a yardstick to monitor IL removal. The traditional water immersion rinses are replaced by a flow-through system (i.e., infinite dilution) using a peristaltic pump, reducing the required water rinse time for the maximum removal of IL to 30 min. This procedure also allows for easy in-line monitoring of solution conductivity and reclamation of an expensive welding solvent. Further, the organic solvent exchange is minimized to 10 min per solvent (from 24 h), resulting in a total combined rinse time of 1 h. This process acceleration reduces the overall solvent exposure time from 96 to 1 h, an almost 99% temporal improvement.

Functional promiscuity of small multidrug resistance transporters from Staphylococcus aureus, Pseudomonas aeruginosa, and Francisella tularensis.

Small multidrug resistance transporters efflux toxic compounds from bacteria and are a minimal system to understand multidrug transport. Most previous studies have focused on EmrE, the model SMR from Escherichia coli, finding that EmrE has a broader substrate profile than previously thought and that EmrE may perform multiple types of transport, resulting in substrate-dependent resistance or susceptibility. Here, we performed a broad screen to identify potential substrates of three other SMRs: PAsmr from Pseudomonas aeruginosa; FTsmr from Francisella tularensis; and SAsmr from Staphylococcus aureus. This screen tested metabolic differences in E. coli expressing each transporter versus an inactive mutant, for a clean comparison of sequence and substrate-specific differences in transporter function, and identified many substrates for each transporter. In general, resistance compounds were charged, and susceptibility substrates were uncharged, but hydrophobicity was not correlated with phenotype. Two resistance hits and two susceptibility hits were validated via growth assays and IC50 calculations. Susceptibility is proposed to occur via substrate-gated proton leak, and the addition of bicarbonate antagonizes the susceptibility phenotype, consistent with this hypothesis.

Airway topicalization in pediatric anesthesia: An international cross-sectional study.

BACKGROUND: There is no national or international consensus or guideline on recommended dosing of lidocaine for airway topicalization in children. Doses quoted in the literature vary substantially. AIMS: The primary aim of the study was to ascertain current international dosing practices (mg.kg-1 and concentration of solution) for lidocaine airway topicalization in children. The secondary aims included examining aftercare instructions for those receiving lidocaine airway topicalization and instances of local anesthetic systemic toxicity secondary to the use of lidocaine for airway topicalization in pediatric patients. METHODS: This cross-sectional study consisted of 11-20 questions across three domains-population demographics, clinical practice, and local anesthetic systemic toxicity. It adhered to the consensus-based checklist for reporting of survey studies. Responses were collected over 14 weeks using a combination of probability (cluster and simple random) and nonprobability (purposive, convenience and snowball) sampling. Data were analyzed based on the response rate per question with proportions expressed as percentages and nonparametric data expressed as median (interquartile range [range]) in an effort to minimize nonresponse error. No weighting of items or propensity scoring was applied. RESULTS: After initial exclusions, 1501 participants from 69 countries, across six continents, were included. Consultant anesthetists or those with an equivalent level of experience accounted for 1262/1501 (84.1%) of responses. Results showed heterogeneity in dosing and timing regimens and evidence that dosing may contribute to adverse outcomes. The maximum dose reported by participants who use lidocaine for airway topicalization as part of their normal practice was 5 mg.kg-1 (4-6 mg.kg-1 [0.5-50]) median (interquartile range [range]) over 2 h (1-4 h [0-30]). CONCLUSION: The results support the need for further research and consensus in this area, in order to provide safe provision of lidocaine airway topicalization in children. It is hoped the results of this study can support future collaborative work in this area.

Overexpression of manganese superoxide dismutase mitigates ACL injury-induced muscle atrophy, weakness and oxidative damage.

Oxidative stress has been implicated in the etiology of skeletal muscle weakness following joint injury. We investigated longitudinal patient muscle samples following knee injury (anterior cruciate ligament tear). Following injury, transcriptomic analysis revealed downregulation of mitochondrial metabolism-related gene networks, which were supported by reduced mitochondrial respiratory flux rates. Additionally, enrichment of reactive oxygen species (ROS)-related pathways were upregulated in muscle following knee injury, and further investigation unveiled marked oxidative damage in a progressive manner following injury and surgical reconstruction. We then investigated whether antioxidant protection is effective in preventing muscle atrophy and weakness after knee injury in mice that overexpress Mn-superoxide dismutase (MnSOD+/-). MnSOD+/- mice showed attenuated oxidative damage, atrophy, and muscle weakness compared to wild type littermate controls following ACL transection surgery. Taken together, our results indicate that ROS-related damage is a causative mechanism of muscle dysfunction after knee injury, and that mitochondrial antioxidant protection may hold promise as a therapeutic target to prevent weakness and development of disability.

The landscape of drug sensitivity and resistance in sarcoma.

Sarcomas are a family of rare malignancies composed of over 100 distinct histological subtypes. The rarity of sarcoma poses significant challenges in conducting clinical trials to identify effective therapies, to the point that many rarer subtypes of sarcoma do not have standard-of-care treatment. Even for established regimens, there can be substantial heterogeneity in responses. Overall, novel, personalized approaches for identifying effective treatments are needed to improve patient out-comes. Patient-derived tumor organoids (PDTOs) are clinically relevant models representative of the physiological behavior of tumors across an array of malignancies. Here, we use PDTOs as a tool to better understand the biology of individual tumors and characterize the landscape of drug resistance and sensitivity in sarcoma. We collected n=194 specimens from n=126 sarcoma patients, spanning 24 distinct subtypes. We characterized PDTOs established from over 120 biopsy, resection, and metastasectomy samples. We leveraged our organoid high-throughput drug screening pipeline to test the efficacy of chemotherapeutics, targeted agents, and combination therapies, with results available within a week from tissue collection. Sarcoma PDTOs showed patient-specific growth characteristics and subtype-specific histopathology. Organoid sensitivity correlated with diagnostic subtype, patient age at diagnosis, lesion type, prior treatment history, and disease trajectory for a subset of the compounds screened. We found 90 biological pathways that were implicated in response to treatment of bone and soft tissue sarcoma organoids. By comparing functional responses of organoids and genetic features of the tumors, we show how PDTO drug screening can provide an orthogonal set of information to facilitate optimal drug selection, avoid ineffective therapies, and mirror patient outcomes in sarcoma. In aggregate, we were able to identify at least one effective FDA-approved or NCCN-recommended regimen for 59% of the specimens tested, providing an estimate of the proportion of immediately actionable information identified through our pipeline. Highlights: Standardized organoid culture preserve unique sarcoma histopathological featuresDrug screening on patient-derived sarcoma organoids provides sensitivity information that correlates with clinical features and yields actionable information for treatment guidanceHigh-throughput screenings provide orthogonal information to genetic sequencingSarcoma organoid response to treatment correlates with patient response to therapyLarge scale, functional precision medicine programs for rare cancers are feasible within a single institution.

Drug screening at single-organoid resolution via bioprinting and interferometry.

High throughput drug screening is an established approach to investigate tumor biology and identify therapeutic leads. Traditional platforms use two-dimensional cultures which do not accurately reflect the biology of human tumors. More clinically relevant model systems such as three-dimensional tumor organoids can be difficult to scale and screen. Manually seeded organoids coupled to destructive endpoint assays allow for the characterization of treatment response, but do not capture transitory changes and intra-sample heterogeneity underlying clinically observed resistance to therapy. We present a pipeline to generate bioprinted tumor organoids linked to label-free, time-resolved imaging via high-speed live cell interferometry (HSLCI) and machine learning-based quantitation of individual organoids. Bioprinting cells gives rise to 3D structures with unaltered tumor histology and gene expression profiles. HSLCI imaging in tandem with machine learning-based segmentation and classification tools enables accurate, label-free parallel mass measurements for thousands of organoids. We demonstrate that this strategy identifies organoids transiently or persistently sensitive or resistant to specific therapies, information that could be used to guide rapid therapy selection.

Efficacy of a hybrid technique of simultaneous videolaryngoscopy with flexible bronchoscopy in children with difficult direct laryngoscopy in the Pediatric Difficult Intubation Registry.

Children with difficult tracheal intubation are at increased risk of severe complications, including hypoxaemia and cardiac arrest. Increasing experience with the simultaneous use of videolaryngoscopy and flexible bronchoscopy (hybrid) in adults led us to hypothesise that this hybrid technique could be used safely and effectively in children under general anaesthesia. We reviewed observational data from the international Pediatric Difficult Intubation Registry from 2017 to 2021 to assess the safety and efficacy of hybrid tracheal intubation approaches in paediatric patients. In total, 140 patients who underwent 180 attempts at tracheal intubation with the hybrid technique were propensity score-matched 4:1 with 560 patients who underwent 800 attempts with a flexible bronchoscope. In the hybrid group, first attempt success was 70% (98/140) compared with 63% (352/560) in the flexible bronchoscope group (odds ratio (95%CI) 1.4 (0.9-2.1), p = 0.1). Eventual success rates in the matched groups were 90% (126/140) for hybrid vs. 89% (499/560) for flexible bronchoscope (1.1 (0.6-2.1), p = 0.8). Complication rates were similar in both groups (15% (28 complications in 182 attempts) hybrid; 13% (102 complications in 800 attempts) flexible bronchoscope, p = 0.3). The hybrid technique was more likely than flexible bronchoscopy to be used as a rescue technique following the failure of another technique (39% (55/140) vs. 25% (138/560), 2.1 (1.4-3.2) p < 0.001). While technically challenging, the hybrid technique has success rates similar to other advanced airway techniques, few complications and may be considered an alternative technique when developing an airway plan for paediatric patients whose tracheas are difficult to intubate under general anaesthesia.

The "Syringe Hickey": An Alternative Skin Marking Method for Lumbar Puncture.

BACKGROUND: Lumbar puncture is a procedure that is commonly performed in emergency departments. Despite their absence from procedure kits, emergency physicians often use skin markers to delineate landmarks for a lumbar puncture. We prefer to create a temporary indentation in the skin using the suction of a syringe. This "syringe hickey" eliminates the need for a skin marker. DISCUSSION: We created a photo demonstration comparing the syringe hickey to a skin marker for site marking. The syringe hickey was created using a 10-mL syringe aspirated to 5 mL on the forearm for 1 min. The syringe hickey lasted over 30 min on a range of skin tones across the Fitzpatrick Scale. The skin marker faded but the syringe hickey maintained its definition after application of ultrasound gel and sterilization with either chlorhexidine or betadine. CONCLUSIONS: The syringe hickey is a simple skin marking technique that is resistant to antiseptic agents and ultrasound gel. The syringe hickey may be useful for other procedures that require puncture site marking.

Charge neutralization of the active site glutamates does not limit substrate binding and transport by small multidrug resistance transporter EmrE.

EmrE, a small multidrug resistance transporter from Escherichia coli, confers broad-spectrum resistance to polyaromatic cations and quaternary ammonium compounds. Previous transport assays demonstrate that EmrE transports a +1 and a +2 substrate with the same stoichiometry of two protons:one cationic substrate. This suggests that EmrE substrate binding capacity is limited to neutralization of the two essential glutamates, E14A and E14B (one from each subunit in the antiparallel homodimer), in the primary binding site. Here, we explicitly test this hypothesis, since EmrE has repeatedly broken expectations for membrane protein structure and transport mechanism. We previously showed that EmrE can bind a +1 cationic substrate and proton simultaneously, with cationic substrate strongly associated with one E14 residue, whereas the other remains accessible to bind and transport a proton. Here, we demonstrate that EmrE can bind a +2 cation substrate and a proton simultaneously using NMR pH titrations of EmrE saturated with divalent substrates, for a net +1 charge in the transport pore. Furthermore, we find that EmrE can alternate access and transport a +2 substrate and proton at the same time. Together, these results lead us to conclude that E14 charge neutralization does not limit the binding and transport capacity of EmrE.

Activating alternative transport modes in a multidrug resistance efflux pump to confer chemical susceptibility.

Small multidrug resistance (SMR) transporters contribute to antibiotic resistance through proton-coupled efflux of toxic compounds. Previous biophysical studies of the E. coli SMR transporter EmrE suggest that it should also be able to perform proton/toxin symport or uniport, leading to toxin susceptibility rather than resistance in vivo. Here we show EmrE does confer susceptibility to several previously uncharacterized small-molecule substrates in E. coli, including harmane. In vitro electrophysiology assays demonstrate that harmane binding triggers uncoupled proton flux through EmrE. Assays in E. coli are consistent with EmrE-mediated dissipation of the transmembrane pH gradient as the mechanism underlying the in vivo phenotype of harmane susceptibility. Furthermore, checkerboard assays show this alternative EmrE transport mode can synergize with some existing antibiotics, such as kanamycin. These results demonstrate that it is possible to not just inhibit multidrug efflux, but to activate alternative transport modes detrimental to bacteria, suggesting a strategy to address antibiotic resistance.

Using expert-elicitation to deliver biodiversity monitoring priorities on a Mediterranean island.

Biodiversity monitoring plays an essential role in tracking changes in ecosystems, species distributions and abundances across the globe. Data collected through both structured and unstructured biodiversity recording can inform conservation measures designed to reduce, prevent, and reverse declines in valued biodiversity of many types. However, given that resources for biodiversity monitoring are limited, it is important that funding bodies prioritise investments relative to the requirements in any given region. We addressed this prioritisation requirement for a biodiverse Mediterranean island (Cyprus) using a three-stage process of expert-elicitation. This resulted in a structured list of twenty biodiversity monitoring needs; specifically, a hierarchy of three groups of these needs was created using a consensus approach. The most highly prioritised biodiversity monitoring needs were those related to the development of robust survey methodologies, and those ensuring that sufficiently skilled citizens are available to contribute. We discuss ways that the results of our expert-elicitation process could be used to support current and future biodiversity monitoring in Cyprus.

High-pH structure of EmrE reveals the mechanism of proton-coupled substrate transport.

The homo-dimeric bacterial membrane protein EmrE effluxes polyaromatic cationic substrates in a proton-coupled manner to cause multidrug resistance. We recently determined the structure of substrate-bound EmrE in phospholipid bilayers by measuring hundreds of protein-ligand HN-F distances for a fluorinated substrate, 4-fluoro-tetraphenylphosphonium (F4-TPP+), using solid-state NMR. This structure was solved at low pH where one of the two proton-binding Glu14 residues is protonated. Here, to understand how substrate transport depends on pH, we determine the structure of the EmrE-TPP complex at high pH, where both Glu14 residues are deprotonated. The high-pH complex exhibits an elongated and hydrated binding pocket in which the substrate is similarly exposed to the two sides of the membrane. In contrast, the low-pH complex asymmetrically exposes the substrate to one side of the membrane. These pH-dependent EmrE conformations provide detailed insights into the alternating-access model, and suggest that the high-pH conformation may facilitate proton binding in the presence of the substrate, thus accelerating the conformational change of EmrE to export the substrate.

Recent advances in 3D bioprinting of musculoskeletal tissues.

The musculoskeletal system is essential for maintaining posture, protecting organs, facilitating locomotion, and regulating various cellular and metabolic functions. Injury to this system due to trauma or wear is common, and severe damage may require surgery to restore function and prevent further harm. Autografts are the current gold standard for the replacement of lost or damaged tissues. However, these grafts are constrained by limited supply and donor site morbidity. Allografts, xenografts, and alloplastic materials represent viable alternatives, but each of these methods also has its own problems and limitations. Technological advances in three-dimensional (3D) printing and its biomedical adaptation, 3D bioprinting, have the potential to provide viable, autologous tissue-like constructs that can be used to repair musculoskeletal defects. Though bioprinting is currently unable to develop mature, implantable tissues, it can pattern cells in 3D constructs with features facilitating maturation and vascularization. Further advances in the field may enable the manufacture of constructs that can mimic native tissues in complexity, spatial heterogeneity, and ultimately, clinical utility. This review studies the use of 3D bioprinting for engineering bone, cartilage, muscle, tendon, ligament, and their interface tissues. Additionally, the current limitations and challenges in the field are discussed and the prospects for future progress are highlighted.

The evolution and genomic basis of beetle diversity.

The order Coleoptera (beetles) is arguably the most speciose group of animals, but the evolutionary history of beetles, including the impacts of plant feeding (herbivory) on beetle diversification, remain poorly understood. We inferred the phylogeny of beetles using 4,818 genes for 146 species, estimated timing and rates of beetle diversification using 89 genes for 521 species representing all major lineages and traced the evolution of beetle genes enabling symbiont-independent digestion of lignocellulose using 154 genomes or transcriptomes. Phylogenomic analyses of these uniquely comprehensive datasets resolved previously controversial beetle relationships, dated the origin of Coleoptera to the Carboniferous, and supported the codiversification of beetles and angiosperms. Moreover, plant cell wall-degrading enzymes (PCWDEs) obtained from bacteria and fungi via horizontal gene transfers may have been key to the Mesozoic diversification of herbivorous beetles-remarkably, both major independent origins of specialized herbivory in beetles coincide with the first appearances of an arsenal of PCWDEs encoded in their genomes. Furthermore, corresponding (Jurassic) diversification rate increases suggest that these novel genes triggered adaptive radiations that resulted in nearly half of all living beetle species. We propose that PCWDEs enabled efficient digestion of plant tissues, including lignocellulose in cell walls, facilitating the evolution of uniquely specialized plant-feeding habits, such as leaf mining and stem and wood boring. Beetle diversity thus appears to have resulted from multiple factors, including low extinction rates over a long evolutionary history, codiversification with angiosperms, and adaptive radiations of specialized herbivorous beetles following convergent horizontal transfers of microbial genes encoding PCWDEs.

Chemical Inhibition of Pre-mRNA Splicing in Living Saccharomyces cerevisiae.

The spliceosome mediates precursor mRNA splicing in eukaryotes, including the model organism Saccharomyces cerevisiae (yeast). Despite decades of study, no chemical inhibitors of yeast splicing in vivo are available. We have developed a system to efficiently inhibit splicing and block proliferation in living yeast cells using compounds that target the human spliceosome protein SF3B1. Potent inhibition is observed in yeast expressing a chimeric protein containing portions of human SF3B1. However, only a single point mutation in the yeast homolog of SF3B1 is needed for selective inhibition of splicing by pladienolide B, herboxidiene, or meayamycin in liquid culture. Mutations that enable inhibition also improve splicing of branch sites containing mismatches between the intron and small nuclear RNA-suggesting a link between inhibitor sensitivity and usage of weak branch sites in humans. This approach provides powerful new tools for manipulating splicing in live yeast and studies of spliceosome inhibitors.

The efficacy of GlideScope® videolaryngoscopy compared with direct laryngoscopy in children who are difficult to intubate: an analysis from the paediatric difficult intubation registry.

BACKGROUND: We analysed data from the Paediatric Difficult Intubation Registry examining the use of direct laryngoscopy and GlideScope® videolaryngoscopy. METHODS: Data collected by a multicentre, paediatric difficult intubation registry from 1295 patients were analysed. Rates of success and complications between direct laryngoscopy and GlideScope videolaryngoscopy were analysed. RESULTS: Initial (464/877 = 53% vs 33/828 = 4%, Z-test = 22.2, P < 0.001) and eventual (720/877 = 82% vs. 174/828 = 21%, Z-test = 25.2, P < 0.001) success rates for GlideScope were significantly higher than direct laryngoscopy. Children weighing <10 kg had lower success rates with the GlideScope than the group as a whole. There were no differences in complication rates per attempt between direct laryngoscopy and GlideScope. The direct laryngoscopy group had more complications associated with the greater number of attempts needed to intubate. There were no increased risks of hypoxia or trauma with GlideScope use. Each additional attempt at intubation with either device resulted in a two-fold increase in complications (odds ratio: 2.0, 95% confidence interval: 1.5-2.5, P < 0.001). CONCLUSIONS: During difficult tracheal intubation in children, direct laryngoscopy is an overly used technique with a low chance of success. GlideScope use was associated with a higher chance of success with no increased risk of complications. GlideScope use in children with difficult tracheal intubation has a lower success rate than in adults with difficult tracheal intubation. Children weighing less than 10 kilograms had lower success rates with either device. Attempts should be minimized with either device to decrease complications.