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Nina Hein,1 Conner Callaway,2 Devin Ford,2 John C Carlson1 1Ochsner Health System, Department of Allergy and Clinical Immunology, New Orleans, LA, USA; 2Tulane University, School of Medicine, New Orleans, LA, USACorrespondence: John C Carlson, Department of Pediatrics, Ochsner Health System, 1315 Jefferson Hwy, New Orleans, LA, 20121, USA, Tel +1-504-842-3900, Fax +1-504-842-5848, Email john.carlson@ochsner.org

John C Carlson,1 Monica Hajirawala,2 Nina Hein3 1Department of Pediatrics, Ochsner Health System, New Orleans, LA, USA; 2Department of Pediatric Allergy and Immunology, University of South Florida, St. Petersburg, FL, USA; 3Department of Allergy and Clinical Immunology, Tulane University, New Orleans, LA, USACorrespondence: John C Carlson, Department of Pediatrics, Ochsner Health System, 1315 Jefferson Hwy, New Orleans, LA, 20121, USA, Tel +1-504-842-3900, Fax +1-504-842-5848, Email john.carlson@ochsner.orgAbstract: While guidelines recommend testing and treatment for patients with venom-induced anaphylaxis to prevent morbidity and mortality, significant barriers prevent most patients from receiving the evaluation and treatments that they need. This review examines these barriers in the United States along with the facilitators that can be used to overcome them.Keywords: insect hypersensitivity, venom, anaphylaxis, immunotherapy, social determinants of health

Classifications of forest vegetation types and characterization of related species assemblages are important analytical tools for mapping and diversity monitoring of forest communities. The discrimination of forest communities is often based on β-diversity, which can be quantified via numerous indices to derive compositional dissimilarity between samples. This study aims to evaluate the applicability of unsupervised classification for National Forest Inventory data from Georgia by comparing two cluster hierarchies. We calculated the mean basal area per hectare for each woody species across 1059 plot observations and quantified interspecies distances for all 87 species. Following an unspuervised cluster analysis, we compared the results derived from the species-neutral dissimilarity (Bray-Curtis) with those based on the Discriminating Avalanche dissimilarity, which incorporates interspecies phylogenetic variation. Incorporating genetic variation in the dissimilarity quantification resulted in a more nuanced discrimination of woody species assemblages and increased cluster coherence. Favorable statistics include the total number of clusters (23 vs. 20), mean distance within clusters (0.773 vs. 0.343), and within sum of squares (344.13 vs. 112.92). Clusters derived from dissimilarities that account for genetic variation showed a more robust alignment with biogeographical units, such as elevation and known habitats. We demonstrate that the applicability of unsupervised classification of species assemblages to large-scale forest inventory data strongly depends on the underlying quantification of dissimilarity. Our results indicate that by incorporating phylogenetic variation, a more precise classification aligned with biogeographic units is attained. This supports the concept that the genetic signal of species assemblages reflects biogeographical patterns and facilitates more precise analyses for mapping, monitoring, and management of forest diversity., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s). Ecology and Evolution published by John Wiley & Sons Ltd.)

While genotoxic chemotherapeutic agents are among the most effective tools to combat cancer, they are often associated with severe adverse effects caused by indiscriminate DNA damage in non-tumor tissue as well as increased risk of secondary carcinogenesis. This study builds on our previous work demonstrating that the RNA Polymerase I (Pol I) transcription inhibitor CX-5461 elicits a non-canonical DNA damage response and our discovery of a critical role for Topoisomerase 2α (Top2α) in the initiation of Pol I-dependent transcription. Here, we identify Top2α as a mediator of CX-5461 response in the murine Eµ- Myc B lymphoma model whereby sensitivity to CX-5461 is dependent on cellular Top2α expression/activity. Most strikingly, and in contrast to canonical Top2α poisons, we found that the Top2α-dependent DNA damage induced by CX-5461 is preferentially localized at the ribosomal DNA (rDNA) promoter region, thereby highlighting CX-5461 as a loci-specific DNA damaging agent. This mechanism underpins the efficacy of CX-5461 against certain types of cancer and can be used to develop effective non-genotoxic anticancer drugs.

DNA methylation plays essential roles in regulating physiological processes, from tissue and organ development to gene expression and aging processes and has emerged as a widely used biomarker for the identification of body fluids and age prediction. Currently, methylation markers are targeted independently at specific CpG sites as part of a multiplexed assay rather than through a unified assay. Methylation detection is also dependent on divergent methodologies, ranging from enzyme digestion and affinity enrichment to bisulfite treatment, alongside various technologies for high-throughput profiling, including microarray and sequencing. In this pilot study, we test the simultaneous identification of age-associated and body fluid-specific methylation markers using a single technology, nanopore adaptive sampling. This innovative approach enables the profiling of multiple CpG marker sites across entire gene regions from a single sample without the need for specialized DNA preparation or additional biochemical treatments. Our study demonstrates that adaptive sampling achieves sufficient coverage in regions of interest to accurately determine the methylation status, shows a robust consistency with whole-genome bisulfite sequencing data, and corroborates known CpG markers of age and body fluids. Our work also resulted in the identification of new sites strongly correlated with age, suggesting new possible age methylation markers. This study lays the groundwork for the systematic development of nanopore-based methodologies in both age prediction and body fluid identification, highlighting the feasibility and potential of nanopore adaptive sampling while acknowledging the need for further validation and expansion in future research., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The heterogeneous composition of cellular transcriptomes poses a major challenge for detecting weakly expressed RNA classes, as they can be obscured by abundant RNAs. Although biochemical protocols can enrich or deplete specified RNAs, they are time-consuming, expensive and can compromise RNA integrity. Here we introduce RISER, a biochemical-free technology for the real-time enrichment or depletion of RNA classes. RISER performs selective rejection of molecules during direct RNA sequencing by identifying RNA classes directly from nanopore signals with deep learning and communicating with the sequencing hardware in real time. By targeting the dominant messenger and mitochondrial RNA classes for depletion, RISER reduces their respective read counts by more than 85%, resulting in an increase in sequencing depth of 47% on average for long non-coding RNAs. We also apply RISER for the depletion of globin mRNA in whole blood, achieving a decrease in globin reads by more than 90% as well as an increase in non-globin reads by 16% on average. Furthermore, using a GPU or a CPU, RISER is faster than GPU-accelerated basecalling and mapping. RISER's modular and retrainable software and intuitive command-line interface allow easy adaptation to other RNA classes. RISER is available at https://github.com/comprna/riser ., (© 2024. The Author(s).)

Large-scale barcoding projects help to aggregate information on genetic variability of multiple species throughout their ranges. Comparing DNA sequences of both non-conspecific and conspecific individuals from distant parts of their ranges helps to compare level of genetic isolation-by-distance patterns in different species and adaptive types. We compared mitochondrial CO1 gene sequences of 223 spiders from Georgia (Caucasus), representing 124 species and eight families, with 3097 homological sequences from spiders mostly from Europe, but also from other parts of the World. In most families, a significant isolation-by distance pattern was observed on family level. On species level, a significant isolation-by-distance was observed in 40 species, although this low proportion is most likely related to a lack of data. Simultaneously, remarkable differences in spatial structure were shown for different species. Although the majority of the studied species have a broad western Palearctic range, web-building spiders from families Araneidae, Theridiidae, and Linyphiidae are less isolated spatially than flower spiders (Thomisidae), jumping spiders (Salticidae), wolf spiders (Lycosidae), sac spiders (Clubionidae), and ground spiders (Gnaphosidae). This pattern is related with more common ballooning in web building than in actively hunting spiders, which commonly remain isolated since preglacial time. Ground spiders build the most isolated populations in the Caucasus., Competing Interests: None declared., (© 2024 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Combination treatments have been of increasing importance in drug development across therapeutic areas to improve treatment response, minimize the development of resistance, and/or minimize adverse events. Pre-clinical in-vitro combination experiments aim to explore the potential of such drug combinations during drug discovery by comparing the observed effect of the combination with the expected treatment effect under the assumption of no interaction (i.e., null model). This tutorial will address important design aspects of such experiments to allow proper statistical evaluation. Additionally, it will highlight the Biochemically Intuitive Generalized Loewe methodology (BIGL R package available on CRAN) to statistically detect deviations from the expectation under different null models. A clear advantage of the methodology is the quantification of the effect sizes, together with confidence interval while controlling the directional false coverage rate. Finally, a case study will showcase the workflow in analyzing combination experiments., (© 2024 John Wiley & Sons Ltd.)

On December 5, 2022, an indirect drive fusion implosion on the National Ignition Facility (NIF) achieved a target gain G_{target} of 1.5. This is the first laboratory demonstration of exceeding "scientific breakeven" (or G_{target}>1) where 2.05 MJ of 351 nm laser light produced 3.1 MJ of total fusion yield, a result which significantly exceeds the Lawson criterion for fusion ignition as reported in a previous NIF implosion [H. Abu-Shawareb et al. (Indirect Drive ICF Collaboration), Phys. Rev. Lett. 129, 075001 (2022)PRLTAO0031-900710.1103/PhysRevLett.129.075001]. This achievement is the culmination of more than five decades of research and gives proof that laboratory fusion, based on fundamental physics principles, is possible. This Letter reports on the target, laser, design, and experimental advancements that led to this result.

All ecosystems face ecological challenges in this century. Therefore, it is becoming increasingly important to understand the ecology and degree of local adaptation of functionally important Arctic-alpine biomes by looking at the most diverse taxon of metazoans: the Arthropoda. This is the first study to utilize metabarcoding in the Alpine tundra, providing insights into the effects of micro-environmental parameters on alpha- and beta-diversity of arthropods in such unique environments. To characterize arthropod diversity, pitfall traps were set at three middle-alpine sampling sites in the Scandinavian mountain range in Norway during the snow-free season in 2015. A metabarcoding approach was then used to determine the small-scale biodiversity patterns of arthropods in the Alpine tundra. All DNA was extracted directly from the preservative EtOH from 27 pitfall traps. In order to identify the controlling environmental conditions, all sampling locations were equipped with automatic data loggers for permanent measurement of the microenvironmental conditions. The variables measured were: air temperature [°C] at 15 cm height, soil temperature [°C] at 15 cm depth, and soil moisture [vol.%] at 15 cm depth. A total of 233 Arthropoda OTUs were identified. The number of unique OTUs found per sampling location (ridge, south-facing slope, and depression) was generally higher than the OTUs shared between the sampling locations, demonstrating that niche features greatly impact arthropod community structure. Our findings emphasize the fine-scale heterogeneity of arctic-alpine ecosystems and provide evidence for trait-based and niche-driven adaptation. The spatial and temporal differences in arthropod diversity were best explained by soil moisture and soil temperature at the respective locations. Furthermore, our results show that arthropod diversity is underestimated in alpine-tundra ecosystems using classical approaches and highlight the importance of integrating long-term functional environmental data and modern taxonomic techniques into biodiversity research to expand our ecological understanding of fine- and meso-scale biogeographical patterns., Competing Interests: The authors declare no conflict of interest., (© 2024 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

The high rates of protein synthesis and processing render multiple myeloma (MM) cells vulnerable to perturbations in protein homeostasis. The induction of proteotoxic stress by targeting protein degradation with proteasome inhibitors (PIs) has revolutionized the treatment of MM. However, resistance to PIs is inevitable and represents an ongoing clinical challenge. Our first-in-human study of the selective inhibitor of RNA polymerase I transcription of ribosomal RNA genes, CX-5461, has demonstrated a potential signal for anti-tumor activity in three of six heavily pre-treated MM patients. Here, we show that CX-5461 has potent anti-myeloma activity in PI-resistant MM preclinical models in vitro and in vivo . In addition to inhibiting ribosome biogenesis, CX-5461 causes topoisomerase II trapping and replication-dependent DNA damage, leading to G2/M cell-cycle arrest and apoptotic cell death. Combining CX-5461 with PI does not further enhance the anti-myeloma activity of CX-5461 in vivo . In contrast, CX-5461 shows synergistic interaction with the histone deacetylase inhibitor panobinostat in both the Vk∗MYC and the 5T33-KaLwRij mouse models of MM by targeting ribosome biogenesis and protein synthesis through distinct mechanisms. Our findings thus provide strong evidence to facilitate the clinical development of targeting the ribosome to treat relapsed and refractory MM., Competing Interests: R.D.H. is a Chief Scientific Advisor to Pimera, Inc., (© 2024 The Author(s).)

Mouse embryonic fibroblasts (MEFs) are primary fibroblasts purified from mouse embryos at a defined time post-fertilization. MEFs have versatile applications, including use as feeder cell layers or sources of untransformed primary cells for a variety of biological assays. MEFs are most commonly isolated between embryonic day (E)12.5 and E13.5 but can be isolated from embryos as early as E8.5 and as late as E15.5. The individual embryos are harvested by carefully removing uterine tissue, yolk sac, and placenta. The embryos are euthanized, and non-mesenchymal tissues, such as the fetal liver and heart, are removed before tissue homogenization. The remaining fetal tissue is homogenized by mechanical mincing using a sterile blade, followed by enzymatic digestion and resuspension. During tissue dissociation, the duration of trypsin-EDTA/DNase digestion and enzyme concentration are critical parameters to produce high-quality MEFs with the highest rates of cell viability and proliferation potential. MEFs can be cryopreserved at passage (P) 0 if >80% confluent, passaged for further expansion before freezing down, or directly utilized for downstream applications, i.e., preparation as feeder cell layers. Primary MEFs possess a limited proliferation capacity of ∼20 cell divisions, beyond which the percentage of senescent cells rapidly increases; thus, cultures should only be expanded/passaged to a maximum of P5. Critical for cell viability during cryopreservation and thawing of MEFs is the slow decrease in temperature when freezing, the rapid increase when thawing, the use of a cryoprotective agent, and an optimal cell density. While it is critical to generate high-quality MEFs to standardize and optimize preparation procedures and utilize fresh reagents, some variability in proliferation capacity and cell viability between MEF preparations remains. Thus, MEF preparation, culture, and cryopreservation procedures are continuously being optimized. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Purification, passaging, and expansion of MEFs Supporting Protocol: Cryopreservation and thawing of MEFs., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)

This checklist reports 47 species of Psocoptera from 15 families and three suborders from Georgia, of which 31 species are recorded for the first time, increasing the known fauna of the country by more than 65%. Of these, 37 species have been barcoded, representing 210 Barcode Identification Numbers (BINs). An additional 14 species are expected to occur in Georgia but remain undiscovered, meaning that only ≈ 77% of the fauna is currently documented. Barcodes, comments on distributions, and images of voucher specimens are given followed by a map of the sampling sites., Competing Interests: No conflict of interest was declared., (Armen Seropian, Eka Arsenashvili, Natalia Bulbulashvili, Anano Shubashishvili, Giorgi Iankoshvili, Mariami Todua, Ana Ananiashvili, Shota Japarashvili, Tinatin Chkhartisvhili, Aleksi Memishishi, Sopio Balkhamishvili, Beka Chitadze, Elisabeth Karalashvili, Levan Mumladze, Nils Hein, Björn Rulik.)

The survival rate of patients with osteosarcoma (OS) has not improved over the last 30 years. Mutations in the genes TP53 , RB1 and c-Myc frequently occur in OS and enhance RNA Polymerase I (Pol I) activity, thus supporting uncontrolled cancer cell proliferation. We therefore hypothesised that Pol I inhibition may be an effective therapeutic strategy for this aggressive cancer. The Pol I inhibitor CX-5461 has demonstrated therapeutic efficacy in different cancers in pre-clinical and phase I clinical trials; thus, the effects were determined on ten human OS cell lines. Following characterisation using genome profiling and Western blotting, RNA Pol I activity, cell proliferation and cell cycle progression were evaluated in vitro, and the growth of TP53 wild-type and mutant tumours was measured in a murine allograft model and in two human xenograft OS models. CX-5461 treatment resulted in reduced ribosomal DNA (rDNA) transcription and Growth 2 (G2)-phase cell cycle arrest in all OS cell lines. Additionally, tumour growth in all allograft and xenograft OS models was effectively suppressed without apparent toxicity. Our study demonstrates the efficacy of Pol I inhibition against OS with varying genetic alterations. This study provides pre-clinical evidence to support this novel therapeutic approach in OS.

Introduction: Obesity is a major global health problem associated with comorbidities such as diabetes, cardiovascular disease, and cancer. Bariatric surgery is recognized to be the most effective weight loss intervention, but it is highly invasive and costly and can have serious side effects. Intragastric balloon (IGB) placement by endoscopy and hypocaloric diets are among a number of techniques that have been used in patients unsuitable for, or unwilling to undergo, obesity surgery. In this study, we compared the efficacy, safety, and cost-effectiveness of the hypocaloric OPTIFAST program (OPT) with endoscopic IGB placement for weight loss., Methods: In this retrospective observational cohort propensity score-weighted comparison (performed May 2014 to December 2020), participants with a BMI of 30-55 kg/m2, aged 18-70 years, were randomized to OPT or IGB for 26 weeks, followed by a weight maintenance phase. Patients were matched according to age, gender, and BMI. The study outcomes were percentage excess body weight lost, total body weight lost (TBWL), and percentage TBWL (%TBWL)., Results: A total of 148 participants (75% of those randomized; 74 OPT, 74 IGB) made up the ITT population. Mean age was 44.1 ± 10.4 years, and the patients were predominantly female (77%). Baseline BMI was 44.1 ± 10.4 kg/m2. At 26 weeks, %TBWL in the OPT group was 19.6 ± 6.8% versus 11.9 ± 6.7% for IGB (p < 0.001). At 52 weeks, %TBWL for OPT was 18.2 ± 9.0% versus 12.0 ± 6.6% for IGB (p < 0.001). The OPT cohort also experienced significantly fewer adverse events compared with the IGB group., Conclusion: IGB placement and OPT induce clinically meaningful weight loss. However, OPT appears to induce clinically superior weight loss and has economic advantages through lower rates of complications and adverse events., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

Sap flow measurement is one of the most effective methods for quantifying plant water use.A better understanding of sap flow dynamics can aid in more efficient water and crop management, particularly under unpredictable rainfall patterns and water scarcity resulting from climate change. In addition to detecting infected plants, sap flow measurement helps select plant species that could better cope with hotter and drier conditions. There exist multiple methods to measure sap flow including heat balance, dyes and radiolabeled tracers. Heat sensor-based techniques are the most popular and commercially available to study plant hydraulics, even though most of them are invasive and associated with multiple kinds of errors. Heat-based methods are prone to errors due to misalignment of probes and wounding, despite all the advances in this technology. Among existing methods for measuring sap flow, nuclear magnetic resonance (NMR) is an appropriate non-invasive approach. However, there are challenges associated with applications of NMR to measure sap flow in trees or field crops, such as producing homogeneous magnetic field, bulkiness and poor portable nature of the instruments, and operational complexity. Nonetheless, various advances have been recently made that allow the manufacture of portable NMR tools for measuring sap flow in plants. The basic concept of the portal NMR tool is based on an external magnetic field to measure the sap flow and hence advances in magnet types and magnet arrangements (e.g., C-type, U-type, and Halbach magnets) are critical components of NMR-based sap flow measuring tools. Developing a non-invasive, portable and inexpensive NMR tool that can be easily used under field conditions would significantly improve our ability to monitor vegetation responses to environmental change., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kumar, Hosseinzadehtaher, Hein, Shadmand, Jagadish and Ghanbarian.)

The nucleolar surveillance pathway monitors nucleolar integrity and responds to nucleolar stress by mediating binding of ribosomal proteins to MDM2, resulting in p53 accumulation. Inappropriate pathway activation is implicated in the pathogenesis of ribosomopathies, while drugs selectively activating the pathway are in trials for cancer. Despite this, the molecular mechanism(s) regulating this process are poorly understood. Using genome-wide loss-of-function screens, we demonstrate the ribosome biogenesis axis as the most potent class of genes whose disruption stabilizes p53. Mechanistically, we identify genes critical for regulation of this pathway, including HEATR3. By selectively disabling the nucleolar surveillance pathway, we demonstrate that it is essential for the ability of all nuclear-acting stresses, including DNA damage, to induce p53 accumulation. Our data support a paradigm whereby the nucleolar surveillance pathway is the central integrator of stresses that regulate nuclear p53 abundance, ensuring that ribosome biogenesis is hardwired to cellular proliferative capacity., Competing Interests: Declaration of interests R.D.H. is a Chief Scientific Advisor of Pimera, Inc. (San Diego, CA). All other authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Uterine leiomyosarcoma is a rare aggressive smooth muscle cancer with poor survival rates. RNA Polymerase I (Pol I) activity is elevated in many cancers supporting tumour growth and prior studies in uterine leiomyosarcoma revealed enlarged nucleoli and upregulated Pol I activity-related genes. This study aimed to investigate the anti-tumour potential of CX-5461, a Pol I transcription inhibitor currently being evaluated in clinical trials for several cancers, against the human uterine leiomyosarcoma cell line, SK-UT-1., Methods: SK-UT-1 was characterised using genome profiling and western blotting. The anti-tumour effects of CX-5461 were investigated using cell proliferation assays, expression analysis using qRT-PCR, and BrdU/PI based cell cycle analysis., Results: Genetic analysis of SK-UT-1 revealed mutations in TP53, RB1, PTEN, APC and TSC1 & 2, all potentially associated with increased Pol I activity. Protein expression analysis showed dysregulated p53, RB1 and c-Myc. CX-5461 treatment resulted in an anti-proliferation response, G2 phase cell-cycle arrest and on-target activity demonstrated by reduced ribosomal DNA transcription., Conclusions: SK-UT-1 was confirmed as a representative model of uterine leiomyosarcoma and CX-5461 has significant potential as a novel adjuvant for this rare cancer., (© 2022. The Author(s).)

Objective: To perform a systematic review of the health economic evidence on the care of children and adolescents with complex clinical conditions, comparing groups included and not included (control group) in palliative care at the end of life., Data Source: The seven databases searched were PubMed, Embase, Web of Science, Cochrane Library, Virtual Health Library-Latin American and Caribbean Health Sciences Literature (VHL-LILACS), EBSCOhost, and Paediatric Economic Database Evaluation, following recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statement, from January 1979 to November 2020. The review included studies of patients under 18 years of age with complex clinical conditions that compared a palliative care group with a control group. The economic outcomes analyzed were length and place of stay at the end of life (home, hospice, ward, intensive care unit, emergency room), diagnostic and therapeutic procedures performed, and health-related costs. The exclusion criteria were: studies without a matched control group, conference/congress abstracts, letters to the editor, editorials, comments, qualitative studies, narrative reviews, studies with ten or fewer participants in each group, articles published in languages other than English, Portuguese, or Spanish., Data Synthesis: Out of the 518 articles identified, 4 met the inclusion criteria. We found evidence of direct economic benefits, such as reduced health costs, indirect savings, and protection of patients from undergoing invasive procedures, surgeries, and costly therapies, which cause greater suffering at the end of life. Therefore, participating in a palliative care program saved financial and technological resources, besides increasing the frequency of deaths at home and improving the quality of life., Conclusions: Public and private policies to promote palliative care represent better efficiency when allocating available health care resources.