Reconstructing extrachromosomal DNA structural heterogeneity from long-read sequencing data using Decoil.

Circular extrachromosomal DNA (ecDNA) is a form of oncogene amplification found across cancer types and associated with poor outcome in patients. ecDNA can be structurally complex and contain rearranged DNA sequences derived from multiple chromosome locations. As the structure of ecDNA can impact oncogene regulation and may indicate mechanisms of its formation, disentangling it at high resolution from sequencing data is essential. Even though methods have been developed to identify and reconstruct ecDNA in cancer genome sequencing, it remains challenging to resolve complex ecDNA structures, in particular amplicons with shared genomic footprints. We here introduce Decoil, a computational method which combines a breakpoint-graph approach with regression to reconstruct complex ecDNA and deconvolve co-occurring ecDNA elements with overlapping genomic footprints from long-read nanopore sequencing. Decoil outperforms de novo assembly and alignment-based methods in simulated long-read sequencing data for both simple and complex ecDNAs. Applying Decoil on whole genome sequencing data uncovered different ecDNA topologies and explored ecDNA structure heterogeneity in neuroblastoma tumors and cell lines, indicating that this method may improve ecDNA structural analyzes in cancer.

Observation of Seven Astrophysical Tau Neutrino Candidates with IceCube.

We report on a measurement of astrophysical tau neutrinos with 9.7 yr of IceCube data. Using convolutional neural networks trained on images derived from simulated events, seven candidate ν_{τ} events were found with visible energies ranging from roughly 20 TeV to 1 PeV and a median expected parent ν_{τ} energy of about 200 TeV. Considering backgrounds from astrophysical and atmospheric neutrinos, and muons from π^{±}/K^{±} decays in atmospheric air showers, we obtain a total estimated background of about 0.5 events, dominated by non-ν_{τ} astrophysical neutrinos. Thus, we rule out the absence of astrophysical ν_{τ} at the 5σ level. The measured astrophysical ν_{τ} flux is consistent with expectations based on previously published IceCube astrophysical neutrino flux measurements and neutrino oscillations.

Safari with an Electron Gun: Visualization of Protein and Membrane Interactions in Mitochondria in Natural Environment.

This paper presents new structural data about mitochondria using correlative light and electron microscopy (CLEM) and cryo-electron tomography. These state-of-the-art structural biology methods allow studying biological objects at nanometer scales under natural conditions. Non-invasiveness of these methods makes them comparable to observing animals in their natural environment on a safari. The paper highlights two areas of research that can only be accomplished using these methods. The study visualized location of the Aß42 amyloid aggregates in relation to mitochondria to test a hypothesis of development of mitochondrial dysfunction in Alzheimer's disease. The results showed that the Aß42 aggregates do not interact with mitochondria, although some of them are closely located. Therefore, the study demonstrated that mitochondrial dysfunction is not directly associated with the effects of aggregates on mitochondrial structure. Other processes should be considered as sources of mitochondrial dysfunction. Second unique area presented in this work is high-resolution visualization of the mitochondrial membranes and proteins in them. Analysis of the cryo-ET data reveals toroidal holes in the lamellar structures of cardiac mitochondrial cristae, where ATP synthases are located. The study proposes a new mechanism for sorting and clustering protein complexes in the membrane based on topology. According to this suggestion, position of the OXPHOS system proteins in the membrane is determined by its curvature. High-resolution tomography expands and complements existing ideas about the structural and functional organization of mitochondria. This makes it possible to study the previously inaccessible structural interactions of proteins with each other and with membranes in vivo.

SkQ3 Exhibits the Most Pronounced Antioxidant Effect on Isolated Rat Liver Mitochondria and Yeast Cells.

Oxidative stress is involved in a wide range of age-related diseases. A critical role has been proposed for mitochondrial oxidative stress in initiating or promoting these pathologies and the potential for mitochondria-targeted antioxidants to fight them, making their search and testing a very urgent task. In this study, the mitochondria-targeted antioxidants SkQ1, SkQ3 and MitoQ were examined as they affected isolated rat liver mitochondria and yeast cells, comparing SkQ3 with clinically tested SkQ1 and MitoQ. At low concentrations, all three substances stimulated the oxidation of respiratory substrates in state 4 respiration (no ADP addition); at higher concentrations, they inhibited the ADP-triggered state 3 respiration and the uncoupled state, depolarized the inner mitochondrial membrane, contributed to the opening of the mPTP (mitochondrial permeability transition pore), did not specifically affect ATP synthase, and had a pronounced antioxidant effect. SkQ3 was the most active antioxidant, not possessing, unlike SkQ1 or MitoQ, prooxidant activity with increasing concentrations. In yeast cells, all three substances reduced prooxidant-induced intracellular oxidative stress and cell death and prevented and reversed mitochondrial fragmentation, with SkQ3 being the most efficient. These data allow us to consider SkQ3 as a promising potential therapeutic agent to mitigate pathologies associated with oxidative stress.

Enhancing the Spin Hall Effect of Cylindrically Polarized Beams.

Two linked gear wheels in a micromachine can be simultaneously rotated in opposite directions by using a laser beam that has in its section areas the spin angular momentum (SAM) of the opposite sign. However, for instance, a cylindrical vector beam has zero SAM in the focus. We alter a cylindrical vector beam so as to generate areas in its focus where the SAM is of opposite signs. The first alteration is adding to the cylindrical vector beam a linearly polarized beam. Thus, we study superposition of two rotationally symmetric beams: those with cylindrical and linear polarization. We obtain an expression for the SAM and prove two of its properties. The first property is that changing superposition coefficients does not change the shape of the SAM density distribution, whereas the intensity changes. The second property is that maximal SAM density is achieved when both beams in the superposition have the same energy. The second perturbation is adding a spatial carrier frequency. We study the SAM density of a cylindrical vector beam with a spatial carrier frequency. Due to periodic modulation, upon propagation in space, such a beam is split into two beams, having left and right elliptic polarization. Thus, in the beam transverse section, areas with the spin of different signs are separated in space, which is a manifestation of the spin Hall effect. We demonstrate that such light beams can be generated by metasurfaces, with the transmittance depending periodically on one coordinate.

Genomic background sequences systematically outperform synthetic ones in de novo motif discovery for ChIP-seq data.

Efficient de novo motif discovery from the results of wide-genome mapping of transcription factor binding sites (ChIP-seq) is dependent on the choice of background nucleotide sequences. The foreground sequences (ChIP-seq peaks) represent not only specific motifs of target transcription factors, but also the motifs overrepresented throughout the genome, such as simple sequence repeats. We performed a massive comparison of the 'synthetic' and 'genomic' approaches to generate background sequences for de novo motif discovery. The 'synthetic' approach shuffled nucleotides in peaks, while in the 'genomic' approach selected sequences from the reference genome randomly or only from gene promoters according to the fraction of A/T nucleotides in each sequence. We compiled the benchmark collections of ChIP-seq datasets for mouse, human and Arabidopsis, and performed de novo motif discovery. We showed that the genomic approach has both more robust detection of the known motifs of target transcription factors and more stringent exclusion of the simple sequence repeats as possible non-specific motifs. The advantage of the genomic approach over the synthetic approach was greater in plants compared to mammals. We developed the AntiNoise web service (https://denovosea.icgbio.ru/antinoise/) that implements a genomic approach to extract genomic background sequences for twelve eukaryotic genomes.

An enteric ultrastructural surface atlas of the model insect Manducasexta.

The tobacco hornworm is a laboratory model that is particularly suitable for analyzing gut inflammation, but a physiological reference standard is currently unavailable. Here, we present a surface atlas of the healthy hornworm gut generated by scanning electron microscopy and nano-computed tomography. This comprehensive overview of the gut surface reveals morphological differences between the anterior, middle, and posterior midgut, allowing the screening of aberrant gut phenotypes while accommodating normal physiological variations. We estimated a total resorptive midgut surface of 0.42 m2 for L5d6 larvae, revealing its remarkable size. Our data will support allometric scaling and dose conversion from Manduca sexta to mammals in preclinical research, embracing the 3R principles. We also observed non-uniform gut colonization by enterococci, characterized by dense biofilms in the pyloric cone and downstream of the pylorus associated with pore and spine structures in the hindgut intima, indicating a putative immunosurveillance function in the lepidopteran hindgut.

Computational Reconstruction of the Transcription Factor Regulatory Network Induced by Auxin in Arabidopsis thaliana L.

In plant hormone signaling, transcription factor regulatory networks (TFRNs), which link the master transcription factors to the biological processes under their control, remain insufficiently characterized despite their crucial function. Here, we identify a TFRN involved in the response to the key plant hormone auxin and define its impact on auxin-driven biological processes. To reconstruct the TFRN, we developed a three-step procedure, which is based on the integrated analysis of differentially expressed gene lists and a representative collection of transcription factor binding profiles. Its implementation is available as a part of the CisCross web server. With the new method, we distinguished two transcription factor subnetworks. The first operates before auxin treatment and is switched off upon hormone application, the second is switched on by the hormone. Moreover, we characterized the functioning of the auxin-regulated TFRN in control of chlorophyll and lignin biosynthesis, abscisic acid signaling, and ribosome biogenesis.

ML-driven segmentation of microvascular features during histological examination of tissue-engineered vascular grafts.

Introduction: The development of next-generation tissue-engineered medical devices such as tissue-engineered vascular grafts (TEVGs) is a leading trend in translational medicine. Microscopic examination is an indispensable part of animal experimentation, and histopathological analysis of regenerated tissue is crucial for assessing the outcomes of implanted medical devices. However, the objective quantification of regenerated tissues can be challenging due to their unusual and complex architecture. To address these challenges, research and development of advanced ML-driven tools for performing adequate histological analysis appears to be an extremely promising direction. Methods: We compiled a dataset of 104 representative whole slide images (WSIs) of TEVGs which were collected after a 6-month implantation into the sheep carotid artery. The histological examination aimed to analyze the patterns of vascular tissue regeneration in TEVGs in situ. Having performed an automated slicing of these WSIs by the Entropy Masker algorithm, we filtered and then manually annotated 1,401 patches to identify 9 histological features: arteriole lumen, arteriole media, arteriole adventitia, venule lumen, venule wall, capillary lumen, capillary wall, immune cells, and nerve trunks. To segment and quantify these features, we rigorously tuned and evaluated the performance of six deep learning models (U-Net, LinkNet, FPN, PSPNet, DeepLabV3, and MA-Net). Results: After rigorous hyperparameter optimization, all six deep learning models achieved mean Dice Similarity Coefficients (DSC) exceeding 0.823. Notably, FPN and PSPNet exhibited the fastest convergence rates. MA-Net stood out with the highest mean DSC of 0.875, demonstrating superior performance in arteriole segmentation. DeepLabV3 performed well in segmenting venous and capillary structures, while FPN exhibited proficiency in identifying immune cells and nerve trunks. An ensemble of these three models attained an average DSC of 0.889, surpassing their individual performances. Conclusion: This study showcases the potential of ML-driven segmentation in the analysis of histological images of tissue-engineered vascular grafts. Through the creation of a unique dataset and the optimization of deep neural network hyperparameters, we developed and validated an ensemble model, establishing an effective tool for detecting key histological features essential for understanding vascular tissue regeneration. These advances herald a significant improvement in ML-assisted workflows for tissue engineering research and development.

Impact of primary care system interventions on healthcare worker satisfaction and intention to stay in the Philippines: a follow-up study.

BACKGROUND: The Philippines has a shortage and uneven distribution of healthcare workers (HCWs). Job satisfaction is an important element to HCW retention and attracting new HCWs into the health system. OBJECTIVE: This study measured HCWs' intent to stay and HCWs' satisfaction after implementation of multiple interventions intended to strengthen the primary care system, and determine factors significantly associated with HCWs' intent to stay. METHODOLOGY: This is a serial cross-sectional study in urban, rural and remote primary care sites in the Philippines. All physicians, nurses, midwives, dentists, community health workers and support staff were invited to participate. Baseline HCWs' intent to stay and satisfaction were obtained using a self-administered questionnaire prior to implementation of interventions. The same survey was again conducted in the years 2021 and 2022, corresponding to 5 and 6 years after initial implementation for the urban site, and 2 and 3 years for the rural and remote sites. We used multiple logistic regression to determine factors associated with intent to stay. RESULTS: There were 430 survey respondents (89.4% response rate) for year 2021, and 417 survey respondents (97.4% response rate) for year 2022. The urban and rural sites had significant increase in several HCW satisfaction domains, while the remote site had significant decrease in several HCW satisfaction domains. There was no significant difference in the intent to stay in the three sites. Factors that decreased intent to stay included length of employment, job involvement and employment as a nurse, while factors that increased intent to stay included job satisfaction, enjoyment and working in the urban site. CONCLUSION: HCW satisfaction improved in the urban site and rural site, while HCW satisfaction declined in the remote site. Intention to stay of primary care HCWs did not significantly change.

Elimusertib has Antitumor Activity in Preclinical Patient-Derived Pediatric Solid Tumor Models.

The small-molecule inhibitor of ataxia telangiectasia and Rad3-related protein (ATR), elimusertib, is currently being tested clinically in various cancer entities in adults and children. Its preclinical antitumor activity in pediatric malignancies, however, is largely unknown. We here assessed the preclinical activity of elimusertib in 38 cell lines and 32 patient-derived xenograft (PDX) models derived from common pediatric solid tumor entities. Detailed in vitro and in vivo molecular characterization of the treated models enabled the evaluation of response biomarkers. Pronounced objective response rates were observed for elimusertib monotherapy in PDX, when treated with a regimen currently used in clinical trials. Strikingly, elimusertib showed stronger antitumor effects than some standard-of-care chemotherapies, particularly in alveolar rhabdomysarcoma PDX. Thus, elimusertib has strong preclinical antitumor activity in pediatric solid tumor models, which may translate to clinically meaningful responses in patients.

Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer.

DNA amplifications in cancer do not only harbor oncogenes. We sought to determine whether passenger coamplifications could create collateral therapeutic vulnerabilities. Through an analysis of >3,000 cancer genomes followed by the interrogation of CRISPR-Cas9 loss-of-function screens across >700 cancer cell lines, we determined that passenger coamplifications are accompanied by distinct dependency profiles. In a proof-of-principle study, we demonstrate that the coamplification of the bona fide passenger gene DEAD-Box Helicase 1 (DDX1) creates an increased dependency on the mTOR pathway. Interaction proteomics identified tricarboxylic acid (TCA) cycle components as previously unrecognized DDX1 interaction partners. Live-cell metabolomics highlighted that this interaction could impair TCA activity, which in turn resulted in enhanced mTORC1 activity. Consequently, genetic and pharmacologic disruption of mTORC1 resulted in pronounced cell death in vitro and in vivo. Thus, structurally linked coamplification of a passenger gene and an oncogene can result in collateral vulnerabilities. SIGNIFICANCE: We demonstrate that coamplification of passenger genes, which were largely neglected in cancer biology in the past, can create distinct cancer dependencies. Because passenger coamplifications are frequent in cancer, this principle has the potential to expand target discovery in oncology. This article is featured in Selected Articles from This Issue, p. 384.

Evaluation of Mechanical Versus Manual Root Canal Preparation in Primary Molars-A Comparative In Vitro Study.

The endodontic treatment of primary teeth is to maintain the function of the tooth free of symptoms until its physiological exfoliation. A critical factor for success is how quickly and effectively the root canal preparation can be performed. Therefore, the aim of this comparative in vitro study was to analyze the efficiency of two mechanical root canal preparation systems FM (FlexMaster) and HF (HyFlex EDM) to manual KF (K-file) on extracted primary molars. A total of 45 teeth were divided into three groups (n = 15): KF (#15-35), FM (04#30) and HF (25/~ OneFile). Root canal preparation was performed, and the preparation time was measured. All root canals were non-destructively analyzed by micro-computed tomography in the cervical, middle and apical thirds before and after preparation with regard to the parameters of canal transport (in µm) and centering ratio (0-1). Statistical analysis was performed at a 5% significance level using non-parametric tests. HF caused the lowest canal transport in the apical third (p = 0.008). The centering ratio value of HF was significantly higher in the middle third of the root canals than in the other two groups (p < 0.01). The mean instrumentation time was significantly higher for KF (6.67 min) than for FM (4.69 min) and HF (4.03 min, p < 0.01). HF can be recommended for primary molar root canal treatment.

Extradural spinal cord hemangioblastoma: a case report and literature review / Hemangioblastoma extradural medular: relato de caso e revisão de literatura / Hemangioblastoma extradural de la médula espinal: informe de un caso y revisión de la literatura

ABSTRACT Objective: Hemangioblastoma is a rare vascular sporadically occurring CNS tumor that can be associated with von Hippel-Lindau disease. Hemangioblastomas account for 2-6% of all spinal cord neoplasms and rank third among intramedullary space-occupying lesions. Methods: This was the first time in our practice that we had dealt with paravertebral hemangioblastoma with the sandglass growth pattern. The world literature describes only 3 case of a tumor with this growth pattern. Surgical and diagnostic aspects of patient treatment are considered. Results: During the operation, we adhered to the following stages: localization of the feeding vessel and of the poles of the tumor, surface dissection of the tumor, en bloc resection of the tumor, and hemostasis of the tumor cavity. Conclusions: Hemangioblastoma of extradural localization is a very rare pathology. However, when MRI signs characteristic of a vascular lesion are identified, it is necessary to carry out additional examinations, which may include CT perfusion study and, if required, selective angiography. Level of Evidence 5; Case report.

RESUMO Objetivos: Hemangioblastoma é um tumor vascular raro do SNC, que ocorre esporadicamente e pode ser associada à doença de Von Hippel-Lindau. Hemangioblastomas são responsáveis por 2-6% de todas as neoplasias da medula espinal e na terceira posição entre lesões, ocupando espaço intramedulares da medula espinal. Métodos: Foi a primeira vez em nossa prática que lidamos com hemangioblastoma paravertebral com o padrão de crescimento ampulheta. As fontes da literatura mundial descrevem apenas 3 casos de um tumor com esse padrão de crescimento. Aspectos cirúrgicos e diagnósticos de tratamentos do paciente são considerados. Resultados: Durante a operação, aderiu-se às seguintes fases: o recipiente de alimentação foi encontrado, os pólos do tumor foram encontrados, a dissecação da superfície do tumor foi feita, o tumor foi removido por um único bloco e a hemostasia da cavidade do tumor foi realizada. Conclusões: Hemangioblastoma de localização extradural é uma patologia muito rara, no entanto, quando sinais de MRI característicos de uma lesão vascular são identificados, o que é necessário para levar a cabo um exame suplementar, o qual pode incluir o estudo de perfusão CT e, se necessário, angiografia seletiva. Nível de Evidência V; Relato de caso.

RESUMEN Objetivo: El hemangioblastoma es un tumor vascular raro del SNC que ocurre esporádicamente y que puede asociarse con la enfermedad de von Hippel-Lindau. Los hemangioblastomas representan el 2%-6% de todas las neoplasias de la médula espinal y están en el tercer lugar entre las lesiones intramedulares que ocupan espacio. Métodos: Esta fue la primera vez en nuestra práctica que tratamos el hemangioblastoma paravertebral con patrón de crecimiento de reloj de arena. La literatura mundial describe solo tres casos de un tumor con este patrón de crecimiento. Se consideran aspectos quirúrgicos y de diagnóstico del tratamiento del paciente. Resultados: Durante la operación, elegimos las siguientes etapas: localización del vaso de irrigación y de los polos del tumor, disección superficial del tumor resección en bloque del tumor y hemostasia de la cavidad tumoral. Conclusiones: El hemangioblastoma de localización extradural es una patología muy rara. Sin embargo, cuando se identifican signos característicos de lesión vascular en la RM, es necesario realizar exámenes adicionales, que pueden incluir estudio de perfusión por TC y, si es necesario, una angiografía selectiva. Nivel de Evidencia V; Reporte de caso.