bluuebunny/crossref_metadata_2025 huggingface dataset:
Pan-cancer subclonal mutation analysis of 7,827 tumors predicts clinical outcome
Authors: Jiang Yujie , Montierth Matthew D , Yu Kaixian , Ji Shuangxi , Guo Shuai , Tran Quang , Liu Xiaoqian , Shin Seung Jun , Cao Shaolong , Li Ruonan , Tang Yuxin , Lesluyes Tom , Kopetz Scott , Ajani Jaffer , Msaouel Pavlos , Subudhi Sumit K , Aparicio Ana , Sharma Padmanee , Shen John Paul , Sood Anil K , Tarabichi Maxime , Wang Jennifer R , Kimmel Marek , Loo Peter Van , Zhu Hongtu , Wang Wenyi
Published: July 2024

Intra-tumor heterogeneity is an important driver of tumor evolution and therapy response. Advances in precision cancer treatment will require understanding of mutation clonality and subclonal architecture. Currently the slow computational speed of subclonal reconstruction hinders large cohort studies. To overcome this bottleneck, we developed Clonal structure identification through Pairwise Penalization, or CliPP, which clusters subclonal mutations using a regularized likelihood model. CliPP reliably processed whole-genome and whole-exome sequencing data from over 12,000 tumor samples within 24 hours, thus enabling large-scale downstream association analyses between subclonal structures and clinical outcomes. Through a pan-cancer investigation of 7,827 tumors from 32 cancer types, we found that high subclonal mutational load (sML), a measure of latency time in tumor evolution, was significantly associated with better patient outcomes in 16 cancer types with low to moderate tumor mutation burden (TMB). In a cohort of prostate cancer patients participating in an immunotherapy clinical trial, high sML was indicative of favorable response to immune checkpoint blockade. This comprehensive study using CliPP underscores sML as a key feature of cancer. sML may be essential for linking mutation dynamics with immunotherapy response in the large population of non-high TMB cancers.
Recommendations for Population and Individual Diagnostic SNP Selection in Non-Model Species
Authors: Armstrong Ellie E. , Li Chenyang , Campana Michael G. , Ferrari Tessa , Kelley Joanna L. , Petrov Dmitri A. , Solari Katherine A. , Mooney Jazlyn A.
Published: July 2024

Despite substantial reductions in the cost of sequencing over the last decade, genetic panels remain relevant due to their cost-effectiveness and flexibility across a variety of sample types. In particular, single nucleotide polymorphism (SNP) panels are increasingly favored for conservation applications. SNP panels are often used because of their adaptability, effectiveness with low-quality samples, and cost-efficiency for use in population monitoring and forensics. However, the selection of diagnostic SNPs for population assignment and individual identification can be challenging. The consequences of poor SNP selection are under-powered panels, inaccurate results, and monetary loss. Here, we develop a novel user-friendly SNP selection pipeline for population assignment and individual identification, mPCRselect. mPCRselect allows any researcher, who has sufficient SNP-level data, to design a successful and cost-effective SNP panel for species of conservation concern.
Cloud-Connected Patch-Worn Auscultation Device for Chest Sound Monitoring
Authors: Downen R. Scott , Li Baichen , Dong Quan , Li Zhenyu
Published: July 2024

In recent years, wearable electrocardiograms have risen in popularity as a solution for personal monitoring of heart activity. However, this technology has limitations in diagnostic capability and structural function monitoring. Meanwhile, auscultation of the heart remains a fundamental tool for physicians in diagnosis and monitoring of heart disease largely unaddressed in a convenient wearable format. The present work outlines a promising system currently under investigation, allowing user-initiated 10-second chest-sound recordings to be transmitted over Bluetooth-Low-Energy, with an innovative package design providing inherent noise reduction and a high signal-to-noise ratio. The device has been tested on healthy individuals, and system response has been validated against calibrated electrocardiogram recording equipment to analyze signal capture fidelity.

Abstract Figure
Ultra-high temporal resolution 4D angiography using arterial spin labeling with subspace reconstruction
Authors: Shen Qijia , Wu Wenchuan , Chiew Mark , Ji Yang , Woods Joseph G. , Okell Thomas W.
Published: July 2024

Purpose

To achieve ultra-high temporal resolution non-contrast 4D angiography with improved spatiotemporal fidelity.

Methods

Continuous data acquisition using 3D golden-angle sampling following an arterial spin labeling preparation allows for flexibly reconstructing 4D dynamic angiograms at arbitrary temporal resolutions. However, k-space data is often temporally “binned” before image reconstruction, negatively affecting spatiotemporal fidelity and limiting temporal resolution. In this work, a subspace was extracted by linearly compressing a dictionary constructed from simulated curves of an angiographic kinetic model. The subspace was used to represent and reconstruct the voxelwise signal timecourse at the same temporal resolution as the data acquisition without temporal binning. Physiological parameters were estimated from the resulting images using a Bayesian fitting approach. A group of 8 healthy subjects were scanned and the in-vivo results reconstructed by different methods were compared. Due to the difficulty of obtaining ground truth 4D angiograms with ultra-high temporal resolution, the in-vivo results were cross-validated with numerical simulations.

Results

The proposed method enables 4D time-resolved angiography with much higher temporal resolution (14.7 ms) than previously reported (∼50 ms) whilst maintaining high spatial resolution (1.1 mm isotropic). Blood flow dynamics were depicted in greater detail, thin vessel visibility was improved, and the estimated physiological parameters also exhibited more realistic spatial patterns with the proposed method.

Conclusion

Incorporating a subspace compressed kinetic model into the reconstruction of 4D ASL angiograms notably improved the temporal resolution and spatiotemporal fidelity, which was subsequently beneficial for accurate physiological modeling.
Quantifying Forest Biomass and Genetic Contribution using Light Detection and Ranging
Authors: Sano Haruka , Miura Naoko , Inamori Minoru , Unno Yamato , Guo Wei , Isobe Sachiko , Kusunoki Kazutaka , Iwata Hiroyoshi
Published: July 2024

The growing focus on the role of forests in carbon sequestration highlights the importance of accurately and efficiently measuring biophysical traits, such as diameter at breast height (DBH) and tree height. Understanding genetic contributions to trait variation is crucial for enhancing carbon storage through genetic improvement of forest trees. Light detection and ranging (LiDAR) has been used to estimate DBH and tree height; however, few studies have explored the heritability of these traits or assessed the accuracy of biomass increment selections based on these traits. Therefore, this study aimed to leverage LiDAR to measure DBH and tree height, estimate tree heritability, and evaluate the accuracy of timber volume selections based on these traits using 60-year-old larch as the study material. Unmanned aerial vehicle (UAV) and backpack LiDAR were compared against hand-measured values. The accuracy of DBH estimations using backpack LiDAR resulted in a root mean square error (RMSE) of 2.7 cm and a coefficient of determination of 0.67. Conversely, the accuracy achieved with UAV LiDAR was 4.0 cm in RMSE and a 0.24 coefficient of determination. The heritability of DBH was found to be higher for backpack LiDAR than for UAV LiDAR and even exceeded that of hand measurements. Comparisons of the accuracy of timber volume selections based on the measured traits demonstrated comparable performances between the backpack and UAV LiDAR. Overall, these findings underscore the potential of using LiDAR remote sensing to quantitatively measure forest tree biomass and facilitate their genetic improvement of carbon-sequestration ability based on these measurements.
Human-Mouse Chimeric Brain Models to Study Human Glial-Neuronal and Macroglial-Microglial Interactions
Authors: Jin Mengmeng , Ma Ziyuan , Zhang Haiwei , Papetti Ava V. , Dang Rui , Stillitano Alessandro C. , Zou Lisa , Goldman Steven A. , Jiang Peng
Published: July 2024

Human-mouse chimeric brain models, generated by transplanting human induced pluripotent stem cell (hiPSC)-derived neural cells, are valuable for studying the development and function of human neural cells in vivo. Understanding glial-glial and glial-neuronal interactions is essential for unraveling the complexities of brain function and developing treatments for neurological disorders. To explore these interactions between human neural cells in vivo, we co-engrafted hiPSC-derived neural progenitor cells together with primitive macrophage progenitors into the neonatal mouse brain. This approach creates human-mouse chimeric brains containing human microglia, macroglia (astroglia and oligodendroglia), and neurons. Using super-resolution imaging and 3D reconstruction techniques, we examine the dynamics between human neurons and glia, and observe human microglia pruning synapses of human neurons, and often engulfing neurons themselves. Single-cell RNA sequencing analysis of the chimeric brain uncovers a close recapitulation of the human glial progenitor cell population, along with a dynamic stage in astroglial development that mirrors the processes found in the human brain. Furthermore, cell-cell communication analysis highlights significant neuronal-glial and macroglial-microglial interactions, especially the interaction between adhesion molecules neurexins and neuroligins between neurons and astroglia, emphasizing their key role in synaptogenesis. We also observed interactions between microglia and astroglia mediated by SPP1, crucial for promoting microglia growth and astrogliosis, and the PTN-MK pathways, instrumental in homeostatic maintenance and development in macroglial progenitors. This innovative co-transplantation model opens up new avenues for exploring the complex pathophysiological mechanisms underlying human neurological diseases. It holds particular promise for studying disorders where glial-neuronal interactions and non-cell-autonomous effects play crucial roles.
Copy number variation contributes to parallel local adaptation in an invasive plant
Authors: Wilson Jonathan , Bieker Vanessa C. , van Boheemen Lotte , Connallon Tim , Martin Michael D. , Battlay Paul , Hodgins Kathryn A.
Published: July 2024

Adaptation is a critical determinant of the diversification, persistence, and geographic range limits of species. Yet the genetic basis of adaptation is often unknown and potentially underpinned by a wide range of mutational types – from single nucleotide changes to large-scale alterations of chromosome structure. Copy number variation (CNV) is thought to be an important source of adaptive genetic variation, as indicated by decades of candidate gene studies that point to CNVs underlying rapid adaptation to strong selective pressures. Nevertheless, population- genomic studies of CNVs face unique logistical challenges not encountered by other forms of genetic variation. Consequently, few studies have systematically investigated the contributions of CNVs to adaptation at a genome-wide scale. We present a genome-wide analysis of CNV contributing to the adaptation of an invasive weed,Ambrosia artemisiifolia– the first such study in an invasive pest. CNVs show clear signatures of parallel local adaptation between North American (native) and European (invaded) ranges, implying widespread reuse of CNVs during adaptation to shared heterogeneous patterns of selection. We used a local principal component analysis to genotype CNV regions in whole-genome sequences of samples collected over the last two centuries. We identified 16 large CNV regions of up to 11.85 megabases in length, six of which show signals of rapid evolutionary change, with pronounced frequency shifts between historic and modern populations. Our results provide compelling genome-wide evidence that copy number variation underlies rapid adaptation over contemporary timescales of natural populations.

Significance Statement

Using a population-genomic approach, we identified copy number variants – stretches of DNA that can be either present, absent, or in multiple copies – displaying parallel signatures of local adaptation across the native and introduced ranges of the invasive weedAmbrosia artemisiifolia. We further identified 16 large copy number variants, some associated with ecologically important traits including sex allocation and height, that show strong signatures of selection over space, along with dramatic temporal changes over the past several decades. These results highlight the importance of an often-overlooked form of genomic variation in both local adaptation and rapid adaptation of invasive species.
Transposable Element Expression and Sub-cellular Dynamics During hPSC Differentiation to Endoderm, Mesoderm, and Ectoderm Lineages
Authors: Babarinde Isaac A. , Fu Xiuling , Ma Gang , Li Yuhao , Akinwole Mobolaji T. , Zhou Xuemeng , Hutchins Andrew P.
Published: July 2024

Transposable elements (TEs) are genomic elements that are found in multiple copies in mammalian genomes. TEs were previously thought to have little functional relevance but recent studies have reported TE roles in multiple biological processes, particularly in embryonic development. To investigate the expression dynamics of TEs during human early development, we used long-read sequence data generated fromin vitrodifferentiation of human pluripotent stem cells (hPSCs) to endoderm, mesoderm, and ectoderm lineages to construct lineage-specific transcriptome assemblies and accurately place TE sequences in their transcript context. Our analysis revealed that specific TE types, such as LINEs and LTRs, exhibit distinct expression patterns across different lineages. Notably, an expression outburst was observed in the ectoderm lineage, with multiple TE types showing dynamic expression trajectories. Additionally, certain LTRs, including HERVH and LTR7Y, were highly expressed in hPSCs and endodermal cells, but these HERVH and LTR7Y sequences originated from completely different transcripts. Interestingly, TE-containing transcripts exhibit distinct levels of transcript stability and subcellular localization across different lineages. Moreover, we showed a consistent trend of increased chromatin association of TE-containing transcripts in germ lineage cells compared to hPSCs. This study suggests that TEs contribute to human embryonic development through dynamic chromatin interaction.

Key findings

Different loci of the same TEs are independently regulated in different cell states

Ectoderm has the highest frequency of TE-containing transcripts

The presence of TEs dynamically drives transcripts to different sub-cellular compartments in different cell states

hPSCs have the least stable TE transcripts with the weakest TE chromatin association, highlighting loose hPSC chromatin and potential roles in cell differentiation
Postprandial sleep in short-sleeping Mexican cavefish
Authors: Gallman Kathryn , Rastogi Aakriti , North Owen , O’Gorman Morgan , Hutton Pierce , Lloyd Evan , Warren Wes , Kowalko Johanna E. , Duboue Erik R. , Rohner Nicolas , Keene Alex C.
Published: July 2024

Interaction between sleep and feeding behaviors are critical for adaptive fitness. Diverse species suppress sleep when food is scarce to increase the time spent foraging. Post-prandial sleep, an increase in sleep time following a feeding event, has been documented in vertebrate and invertebrate animals. While interactions between sleep and feeding appear to be highly conserved, the evolution of postprandial sleep in response to changes in food availability remains poorly understood. Multiple populations of the Mexican cavefish,Astyanax mexicanus,have independently evolved sleep loss and increased food consumption compared to surface-dwelling fish of the same species, providing the opportunity to investigate the evolution of interactions between sleep and feeding. Here, we investigate effects of feeding on sleep in larval and adult surface fish, and two parallelly evolved cave populations ofA. mexicanus.Larval surface and cave populations ofA. mexicanusincrease sleep immediately following a meal, providing the first evidence of postprandial sleep in a fish model. The amount of sleep was not correlated to meal size and occurred independently of feeding time. In contrast to larvae, postprandial sleep was not detected in adult surface or cavefish, that can survive for months without food. Together, these findings reveal that postprandial sleep is present in multiple short-sleeping populations of cavefish, suggesting sleep-feeding interactions are retained despite the evolution of sleep loss. These findings raise the possibility that postprandial sleep is critical for energy conservation and survival in larvae that are highly sensitive to food deprivation.
Mechanism of Proton Release during Water Oxidation in Photosystem II
Authors: Allgöwer Friederike , Pöverlein Maximilian C. , Rutherford A. William , Kaila Ville R. I.
Published: July 2024

Photosystem II (PSII) catalyzes the light-driven water oxidation that releases dioxygen into our atmosphere and provides the electrons needed for the synthesis of biomass. The catalysis occurs in the oxygen-evolving oxo-manganese-calcium (Mn4O5Ca) cluster that drives the stepwise oxidation and deprotonation of substrate water molecules leading to the O2formation. However, despite recent advances, the mechanism of these reactions remains unclear and much debated. Here we show that the light-driven Tyr161D1oxidation adjacent to the Mn4O5Ca cluster, significantly decreases the barrier for proton transfer from the putative substrate water molecule (W3/Wx) to Glu310D2, which is accessible to the luminal bulk. By combining hybrid quantum/classical (QM/MM) free energy calculations with atomistic molecular dynamics (MD) simulations, we probe the energetics of the proton transfer along the Cl1 pathway. We demonstrate that the proton transfer occurs via water molecules and a cluster of conserved carboxylates, driven by redox-triggered electric fields directed along the pathway. Glu65D1establishes a local molecular gate that controls the proton transfer to the luminal bulk, whilst Glu312D2acts as a local proton storage site. The identified gating region could be important in preventing back-flow of protons to the Mn4O5Ca cluster. The structural changes, derived here based on the dark-state PSII structure, strongly support recent time-resolved XFEL data of the S3→S4transition (Nature617, 2023), and reveal the mechanistic basis underlying deprotonation of the substrate water molecules. Our combined findings provide insight into the water oxidation mechanism of PSII and show how the interplay between redox-triggered electric fields, ion-pairs, and hydration effects control proton transport reactions.

Significance Statement

Photosystem II is nature’s water splitting enzyme that produces the oxygen in the atmosphere and drives the synthesis of biomass. The water splitting reaction releases protons to the luminal bulk contributing to the protonmotive force that drives the synthesis of ATP. Key mechanistic principles of the light-driven water splitting reaction remain debated, amongst them the catalytically important deprotonation steps. Here we show how the oxygen-evolving oxo-manganese-calcium cluster transports protons via conserved carboxylates and water molecules in proton arrays that lead to the luminal bulk. We identify a local proton storage site and molecular gates that prevent wasteful back reactions by undergoing conformational changes, and we show how electric field effects control the protonation dynamics in Photosystem II.
PlasGO: enhancing GO-based function prediction for plasmid-encoded proteins based on genetic structure
Authors: Ji Yongxin , Shang Jiayu , Guan Jiaojiao , Zou Wei , Liao Herui , Tang Xubo , Sun Yanni
Published: July 2024

Plasmid, as a mobile genetic element, plays a pivotal role in facilitating the transfer of traits, such as antimicrobial resistance, among the bacterial community. Annotating plasmid-encoded proteins with the widely used Gene Ontology (GO) vocabulary is a fundamental step in various tasks, including plasmid mobility classification. However, GO prediction for plasmid-encoded proteins faces two major challenges: the high diversity of functions and the limited availability of high-quality GO annotations. Thus, we introduce PlasGO, a tool that leverages a hierarchical architecture to predict GO terms for plasmid proteins. PlasGO utilizes a powerful protein language model to learn the local context within protein sentences and a BERT model to capture the global context within plasmid sentences. Additionally, PlasGO allows users to control the precision by incorporating a self-attention confidence weighting mechanism. We rigorously evaluated PlasGO and benchmarked it against six state-of-the-art tools in a series of experiments. The experimental results collectively demonstrate that PlasGO has achieved commendable performance. PlasGO significantly expanded the annotations of the plasmid-encoded protein database by assigning high-confidence GO terms to over 95% of previously unannotated proteins, showcasing impressive precision of 0.8229, 0.7941, and 0.8870 for the three GO categories, respectively, as measured on the novel protein test set.
Bayesian phylodynamics of early vertebrate development in BEAST 2
Authors: Zwaans Antoine , Seidel Sophie , Manceau Marc , Stadler Tanja
Published: July 2024

Analysing single-cell lineage relationships of an organism is crucial towards understanding the fundamental cellular dynamics that drive development. CRISPR-based dynamic lineage tracing relies on recent advances in genome editing and sequencing technologies to generate inheritable, evolving genetic barcode sequences which enable reconstruction of such cell lineage trees, also referred to as phylogenetic trees. Recent work generated custom computational strategies to produce robust tree estimates from such data. We further capitalise on these advancements and introduce GABI (GESTALT Analysis using Bayesian Inference), which extends the analysis of GESTALT (Genome Editing of Synthetic Target Arrays for Lineage Tracing) data to a fully integrated Bayesian phylogenetic inference frame-work in the software BEAST 2. This implementation allows to represent the uncertainty in reconstructed tree reconstruction and enables their scaling in absolute time. Furthermore, based on such time-scaled lineage trees, the underlying processes of growth, differentiation and apoptosis are quantified through so-called phylodynamic inference, typically relying on a birthdeath or coalescent model. After validating the implementation, we demonstrate that the methodology results in robust estimates of lineage trees and growth dynamics characteristics of early zebrafishDanio reriodevelopment. GABI’s codebase is publicly available athttps://github.com/azwaans/GABI.
Cellular and molecular events organizing the assembly of tertiary lymphoid structures in glioblastoma
Authors: Vaccaro Alessandra , Yang Fan , van de Walle Tiarne , Franke Saskia , Lugano Roberta , Dénes Anna , Magoulopoulou Anastasia , Cid-Fariña Inés , Smits Anja , Uhrbom Lene , Libard Sylwia , Latini Francesco , Essand Magnus , Nilsson Mats , He Liqun , Olsson Bontell Thomas , Jakola Asgeir S , Ramachandran Mohanraj , Dimberg Anna
Published: July 2024

Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates associated with improved prognosis in numerous tumors. To date, their prognostic value in central nervous system cancers and the events underlying their formation remain unclear. Here, we find that TLS correlate with improved survival in glioblastoma patients. Furthermore, combining spatial transcriptomics of human tissues with longitudinal studies in murine glioma, we establish that the assembly of T cell-rich clusters is a prerequisite for the development of canonical TLS, and show that CD4 T cells play a central role in this process. Indeed, we provide evidence that IL7R+CCR7+Th1 lymphocytes with lymphoid tissue-inducing potential are recruited to TLS nucleation sites, where they can initiate TLS assembly by expressing lymphotoxin β. Our work defines TLS as a potential prognostic factor in glioblastoma and identifies cellular and molecular mechanisms of TLS formation. These findings have broader implications for the development of TLS-inducing therapies for cancer.

Graphical abstract
The Impact of TLR9 Expression Loss on Breast Cancer Tumor Cells
Authors: Sible Emily , Weitsman Gregory , Amoyal Salome , Roblot Guillaume , Marotel Marie , Pescarmona Rémi , Bendriss-Vermare Nathalie , Gillet Cheryl , Ceesay Amie , Michallet Marie Cecile , Caux Christophe , Cosset Francois-Loic , Al Alem Umaima , Ng Tony , Hasan Uzma Ayesha
Published: July 2024

Toll-like receptor 9 (TLR9), primarily expressed in human dendritic and B cells, recognizes double-stranded DNA motifs from pathogens, initiating an inflammatory response. Recent studies have revealed TLR9’s involvement beyond its conventional role in immune response, notably during the tumorigenesis of various cancers such as head and neck, cervical, and ovarian cancers. Here we show by immunohistochemistry analysis demonstrated significantly lower TLR9 levels in breast cancer tumors compared to normal breast tissue epithelium. Similarly, TLR9 downregulation was also observed in several transformed breast cancer cell lines versus untransformed breast epithelial cell lines. Furthermore, overexpression of TLR9 led to reduced proliferation potential of breast cancer cell associated with activation of senescence as was evident by upregulation of proinflammatory cytokine IL-6, chemokines IL-8 and CXCL1; and growth factor GM-CSF. These findings support TLR9’s regulatory role in mitigating breast cancer and highlight its critical connection between the inflammatory response and tumor immunity.
Fully-Automated Multicolour Structured Illumination Module for Super-resolution Microscopy
Authors: Wang Haoran , Brown Peter T. , Ullom Jessica , Shepherd Douglas P. , Heintzmann Rainer , Diederich Benedict
Published: July 2024

In the rapidly advancing field of biological imaging, there is a great need for high-resolution imaging techniques that are both cost-effective and accessible, for example to better observe and understand dynamics in intracellular processes. Structured illumination microscopy (SIM) is the method of choice to achieve high axial and lateral resolution in living samples due to its optical sectioning and minimal phototoxicity. However, the high cost and complexity of conventional SIM systems limit their wide application. In our work, we present an open-source, fully-automated, two-color structured illumination module that is compatible with commercially available microscope stands. The compact design, consisting of low-cost single-mode fiber-coupled lasers and a digital micromirror device (DMD), is integrated into the open-source acquisition and control software (ImSwitch) in order to realize real-time super-resolution imaging. This developed system achieves up to a 1.55-fold improvement in lateral resolution compared to conventional wide-field microscopy. To rationally design this module, we developed a model to ensure optimal DMD diffraction per-formance using tilt and roll pixels, thus covering a wide range of low-cost video projectors for use in coherent SIM setups. Our goal is to democratize SIM-based super-resolution microscopy by providing both comprehensive open-source documentation and a modular software framework that works with various hardware components (e.g. cameras, stages) and reconstruction algorithms. In this way, we try to upgrade as many devices as possible to the super-resolution realm.
Pan-cortical cellular imaging in freely behaving mice using a miniaturized micro-camera array microscope (mini-MCAM)
Authors: Hu Jia , Cherkkil Arun , Surinach Daniel A , Oladepo Ibrahim , Hossain Ridwan F , Fausner Skylar , Saxena Kapil , Ko Eunsong , Peters Ryan , Feldkamp Michael , Konda Pavan C , Pathak Vinayak , Horstmeyer Roarke , Kodandaramaiah Suhasa B
Published: July 2024

Understanding how circuits in the brain simultaneously coordinate their activity to mediate complex ethnologically relevant behaviors requires recording neural activities from distributed populations of neurons in freely behaving animals. Current miniaturized imaging microscopes are typically limited to imaging a relatively small field of view, precluding the measurement of neural activities across multiple brain regions. Here we present a miniaturized micro-camera array microscope (mini-MCAM) that consists of four fluorescence imaging micro-cameras, each capable of capturing neural activity across a 4.5 mm x 2.55 mm field of view (FOV). Cumulatively, the mini-MCAM images over 30 mm2area of sparsely expressed GCaMP6s neurons distributed throughout the dorsal cortex, in regions including the primary and secondary motor, somatosensory, visual, retrosplenial, and association cortices across both hemispheres. We demonstrate cortex-wide cellular resolution in vivo Calcium (Ca2+) imaging using the mini-MCAM in both head-fixed and freely behaving mice.
Repeat-rich regions cause false positive detection of NUMTs: a case study in amphibians using an improved cane toad reference genome
Authors: Cheung K , Rollins LA , Hammond JM , Barton K , Ferguson JM , Eyck HJF , Shine R , Edwards RJ
Published: July 2024

Mitochondrial DNA (mtDNA) has been widely used in genetics research for decades. Contamination from nuclear DNA of mitochondrial origin (NUMT) can confound studies of phylogenetic relationships and mtDNA heteroplasmy. Homology searches with mtDNA are widely used to detect NUMTs in the nuclear genome. Nevertheless, false positive detection of NUMTs is common when handling repeat-rich sequences, whilst fragmented genomes might result in missing true NUMTs. In this study, we investigated different NUMT detection methods and how the quality of the genome assembly affects them. We presented an improved nuclear genome assembly (aRhiMar1.3) of the invasive cane toad (Rhinella marina) with additional long-read Nanopore and 10x linked-read sequencing. The final assembly was 3.47 Gb in length with 91.3% of tetrapod universal single-copy orthologs (n=5,310), indicating the gene-containing regions were well assembled. We used three complementary methods (NUMTFinder,dinumtandPALMER) to study the NUMT landscape of the cane toad genome. All three methods yielded consistent results, showing very few NUMTs in the cane toad genome. Furthermore, we expanded NUMT detection analyses to other amphibians and confirmed a weak relationship between genome size and the number of NUMTs present in the nuclear genome. Amphibians are repeat-rich, and we show that the number of NUMTs found in highly repetitive genomes is prone to inflation when using homology-based detection without filters. Together, this study provides an exemplar of how to robustly identify NUMTs in complex genomes when confounding effects on mtDNA analyses are a concern.

Significance

This study uses an updated cane toad nuclear genome assembly and multiple NUMT detection methods to confirm a lack of NUMTs that might confound the use of mtDNA as a population genetic marker in the cane toad. We provide an exemplar study for NUMT detection accounting for genome assembly quality and composition, and highlight the risks of using BLASTN-based approaches in highly repetitive nuclear genomes.
Robustness of mitochondrial biogenesis and respiration explain aerobic glycolysis
Authors: Arunachalam Easun , Keber Felix C. , Law Richard C. , Kumar Chirag K. , Shen Yihui , Park Junyoung O. , Wühr Martin , Needleman Daniel J.
Published: July 2024

A long-standing observation is that in fast-growing cells, respiration rate declines with increasing growth rate and is compensated by an increase in fermentation, despite respiration being more efficient than fermentation. This apparent preference for fermentation even in the presence of oxygen is known as aerobic glycolysis, and occurs in bacteria, yeast, and cancer cells. Considerable work has focused on understanding the potential benefits that might justify this seemingly wasteful metabolic strategy, but its mechanistic basis remains unclear. Here we show that aerobic glycolysis results from the saturation of mitochondrial respiration and the decoupling of mitochondrial biogenesis from the production of other cellular components. Respiration rate is insensitive to acute perturbations of cellular energetic demands or nutrient supplies, and is explained simply by the amount of mitochondria per cell. Mitochondria accumulate at a nearly constant rate across different growth conditions, resulting in mitochondrial amount being largely determined by cell division time. In contrast, glucose uptake rate is not saturated, and is accurately predicted by the abundances and affinities of glucose transporters. Combining these models of glucose uptake and respiration provides a quantitative, mechanistic explanation for aerobic glycolysis. The robustness of specific respiration rate and mitochondrial biogenesis, paired with the flexibility of other bioenergetic and biosynthetic fluxes, may play a broad role in shaping eukaryotic cell metabolism.
Discovery of the missing cytochrome P450 monooxygenase cyclases that conclude glyceollin biosynthesis in soybean
Authors: Khatri Praveen , Kuflu Kuflom , McDowell Tim , Lin Jie , Kovinich Nikola , Dhaubhadel Sangeeta
Published: July 2024

Glyceollins are isoflavonoid-derived metabolites produced by soybean that hold great promise in improving human and animal health due to their antimicrobial, and other medicinal properties. They play important roles in agriculture by defending soybean against one of its most destructive pathogens,Phytophthora sojae. Longstanding research efforts have focused on improving accessibility to glyceollins, yet chemical synthesis remains uneconomical. The fact that some of the key genes involved in the final step of glyceollin biosynthesis have not been identified, engineering the accumulation of these important compounds in microbes is not yet possible. Although the activity of a P450 cyclase was inferred to catalyze the final committed step in glyceollin biosynthesis forty years ago, the enzyme in question has never been conclusively identified. This study reports, for the first time, the identification of three cytochrome P450 monooxygenase cyclases that catalyze the final steps of glyceollin biosynthesis. UtilizingP. sojae-soybean transcriptome data, along with genome mining tools and co-expression network analysis, we have identified 16 candidate glyceollin synthases (GmGS). Heterologous expression of these candidate genes in yeast, coupled within vitroenzyme assays, enabled us to discover three enzymes capable of producing two glyceollin isomers. GmGS11A and GmGS11B catalyzed the conversion of glyceollidin to glyceollin I, whereas GmGS13A converted glyceocarpin to glyceollin III. The functionality of these candidates was further confirmedin plantathrough gene silencing and overexpression in soybean hairy roots. This groundbreaking study not only contributes to the understanding of glyceollin biosynthesis, but also demonstrates a new synthetic biology strategy that could potentially be scaled up to produce valuable molecules for crop and disease management.
Migration of Kupffer’s vesicle derived cells is essential for tail morphogenesis in zebrafish embryos
Authors: Hoeksma Jelmer , Hertog Jeroen den
Published: July 2024

A phenotypic screen of fungal filtrates on developing zebrafish embryos identified metabolites from the fungusCeratocystis populicolato induce ectopic tail formation, including a split notochord and a duplicated caudal fin. Chemical analyses led to the identification of monoterpene alcohols, in particular geraniol, as active compounds inducing ectopic tail formation during a specific 4 h time window during tail bud stage. Embryos from Tüpfel long fin zebrafish (TL) were more susceptible to ectopic tail formation by geraniol than embryos from Wild Indian Karyotpe (WIK) zebrafish, indicating zebrafish strain specificity. RNA sequencing on tail buds of 15-somite stage embryos revealed downregulation of essential genes of the retinoic acid signaling pathway and differential expression ofcyp26a1andfgf8aand downstreamhox-genes was validated. Time-lapse imaging revealed that Kupffer’s vesicle derived cells failed to migrate shorty after Kupffer’s vesicle collapse upon geraniol treatment and these cells failed to merge with progenitors from the tail bud. Instead, these cells contributed to an ectopic tail, expressing markers for presomitic mesoderm, somite and notochord tissue. Taken together, our data suggests that Kupffer’s vesicle cells harbor tail progenitor capacity, and proper migration of these cells is essential for normal tail morphogenesis.

Summary Statement

Inhibition of Kupffer’s vesicle derived cell migration affected tail morphogenesis and resulted in ectopic tail formation in zebrafish embryos.
Integrated Analysis of Single-Cell and Bulk RNA-Seq Data reveals that Ferroptosis-Related Genes Mediated the Tumor Microenvironment predicts Prognosis, and guides Drug Selection in Triple-Negative Breast Cancer
Authors: Gong Xuantong , Gu Lishuang , Yang Di , He Yu , Li Qian , Qin Hao , Wang Yong
Published: July 2024

Background

Triple-negative breast cancer (TNBC) is aggressive, lacking methods to predict recurrence and drug sensitivity. Ferroptotic heterogeneity varies in TNBC subtypes. However, the tumor microenvironment (TME) mediated by ferroptosis genes is unclear. Our study aims to integrate single-cell and bulk RNA sequencing (RNA-seq) data to reveal the ferroptosis-mediated TME in TNBC, predicting prognosis and guiding treatment.

Methods

The single-cell and bulk RNA-seq data of TNBC were sourced from the Gene Expression Omnibus (GEO) database. Using these data, a machine learning algorithm was employed to integrate and analyze the characteristics of the TME mediated by ferroptosis-related genes in TNBC. Prediction models for TNBC survival prognosis and drug treatment response were established and then validated in an independent set.

Results

At the individual cell level, T cells were categorized into three distinct subpopulations, and local macrophages into two subpopulations. The infiltration degree of these different cell subpopulations was closely associated with prognosis and treatment outcomes. Based on this, the risk score model we developed effectively predicted recurrence-free survival in TNBC patients, with independently validated pooled predicted 3-, 4-, and 5-year Area Under the Curves (AUCs) of 0.65, 0.67, and 0.71, respectively. Additionally, we found that patients in the high-risk group may be more responsive to 27 drugs.

Conclusions

We have uncovered the tumor immune cell clusters in TNBC mediated by ferroptosis. A risk score model was constructed to identify high-risk TNBC patients, which can assist physicians in disease monitoring and precision therapy. The genes identified hold significant potential as therapeutic targets for TNBC patients.

Funding

This project is funded by the National Natural Science Foundation of China (81974268, 82472000, 82304151), the Talent Incentive Program of Cancer Hospital Chinese, Academy of Medical Sciences (801032247), the Cooperation Fund of CHCAMS (CFA202202023), and the open project of Beijing Key Laboratory of Tumor Invasion and Metastasis Mechanism, Capital Medical University(2023ZLKF03).

Impact Statement

Integrating single-cell and bulk RNA-seq data elucidates the role of ferroptosis in TNBC, offering a prognostic model and personalized therapeutic insights.
A novel HER2 protein identification methodology in breast cancer cells using Raman spectroscopy and Raman imaging: an analytical validation study
Authors: Abramczyk H. , Surmacki J. , Kopeć M.
Published: July 2024

Background

Conventional assays such as immunohistochemistry (IHC) andin situhybridization (ISH) used in clinical procedures for quantification of the human epidermal growth factor receptor-2 (HER2) status in breast cancer have many limitations. Our results suggest that a new Raman method may improve specificity that will result in better patients selection for HER2 targeted therapy. In the current study, we have used HER2 expression in a broad range of breast cancer phenotypes to explore the potential utility of a novel immunodetection technique, using Raman spectroscopy and Raman imaging combined with artificial intelligence models.

Methods

The expression of HER2 protein in different cancer subtypes was evaluated using Raman methodology to test correlations with currently used quantitative protein analysis for a broad range of cancer subtypes based on the expression levels of estrogen and progesterone receptors (ER and PR), HER2, cytokeratins and claudins. In the current study, five breast cancer cell lines: MCF-10A, MCF-7, MDA-MB-231, HTB-30 (SK-BR-3) and AU-565 were tested.

Results

The correlations between Raman method and conventional HER2 testing methodologies (IHC and ISH) have been tested. Raman measurements showed a strong linear correlation (p= 0.05, R2=0,9816) with IHC analysis in the studied breast cell lines: MCF-10A, MCF-7, MDA-MB- 231, HTB-30 (SK-BR-3) and AU-565 representing normal, non-tumorigenic epithelial cells, triple-positive breast carcinoma and triple-negative breast cancer cell lines.

Conclusions

Analytic testing of Raman spectroscopy and Raman imaging demonstrated that this method may offer advantages over currently used diagnostic methodologies. The broad range of HER2 expression on the surface of human breast normal and cancer cells from triple negative to triple positive cell lines were studied. Our data demonstrate that Raman based methods for HER2 quantitation of HER2 may offer significant progress in patient selection for HER2 targeted therapy over conventional HER2 identification. Further studies of HER2 determination by Raman methods within vivocells andex vivotissue are warranted.
Coordination of protein synthesis and decay in mammalian cells
Authors: Sun Michael Shoujie , Martin Benjamin , Dembska Joanna , Deluz Cédric , Suter David M.
Published: July 2024

Protein synthesis and decay rates must be tightly coordinated to ensure proteome homeostasis. How this coordination is established is unknown. Here we use quantitative live cell imaging combined with computational inference to determine how changes in global protein synthesis rates alter protein decay rates. We identified two modes of adaptation hinging on differential responses of the cell cycle to changes in protein synthesis rates. A passive adaptation of protein degradation and dilution rates operates in cells tightly coupling proliferation rate to protein synthesis rates, allowing to partially buffer changes in proteome concentrations with increased loss of short-lived proteins. In contrast, the proliferation rate of mouse embryonic stem cells (mESCs) is hyposensitive to moderate changes in protein synthesis. mESCs uncouple protein degradation and dilution rates and robustly maintain their protein levels. Our work illuminates the complex interplay between protein synthesis, degradation, and cell division to regulate proteome homeostasis.
Integrating tDNA Epigenomics and Expression with Codon Usage Unravel an Intricate Connection with Protein Expression Dynamics inTrypanosoma cruzi
Authors: Silva Herbert G. S. , Kimura Satoshi , Lima Pedro L. C. , Pires David S. , Waldor Matthew K. , da Cunha Julia P. C.
Published: July 2024

Codon usage bias impacts protein expression across all kingdoms of life, including trypanosomatids. These protozoa, such as theTrypanosoma cruzi, primarily regulate their protein-coding genes through posttranscriptional mechanisms. Here, we integrated analyses of codon usage with multiple high-throughput sequencing data to investigate whether codon usage bias is present into surface virulence factors (disruptive compartment), conserved housekeeping proteins (core compartment), and proteins involved in the developmental stages ofT. cruzi. For the first time in trypanosomatids, tRNA sequencing was employed to reveal coadaptation between codon usage and anticodon availability. Despite notable differences in the proteomes of infective and non-infective forms, they exhibited similar pools of tRNAs and similar codon usage preferences. We observed that open chromatin levels of tRNA genes correlate with tRNA expression in non-infective forms, but not in infective forms, suggesting chromatin states do not control the tRNA pool in the latter. Our analysis also revealed a relationship between anticodon:codon pairing modes and protein abundance. Highly expressed mRNAs favored Watson– Crick base pairing, whereas less expressed mRNAs displayed more wobble base pairing. Overall, our findings suggest that protein expression inT. cruziis influenced by a combination of codon usage bias, tRNA abundance, and anticodon:codon pairing modes.

Graphical abstract
VPS28 regulates triglyceride synthesis and is mediated by the ubiquitination pathway in a bovine mammary epithelial cell line and mouse model
Authors: Liu Lily , Wang Jinhai , Zheng Xianrui , Zhang Qin
Published: July 2024

VPS28 (vacuolar protein sorting 28) is a subunit of the endosomal sorting complexes required for transport (ESCRTs), and is involved in ubiquitination. Ubiquitination is a crucial system for protein degradation in eukaryotes. Considering the recent findings on the role of ubiquitination in regulating lipid metabolism, we hypothesized that VPS28 might affect the expression of genes involved in milk fat synthesis. To test this hypothesis, we modulated VPS28 expression in the bovine mammary epithelial cell (MAC-T) line and measured the effects on triglyceride (TG) synthesis using lentivirus-mediated techniques. The results indicated that VPS28 knockdown significantly upregulated the fatty acid transporter CD36 (CD36 molecule) and the adipose differentiation-related protein (ADFP), leading to increased TG and fatty acid production, alongside elevated expression of ubiquitin (UB) protein and reduced proteasome activity. In contrast, VPS28 overexpression increased CD36 levels without significantly affecting ADFP and TG levels, showing a trend toward reduced lipid droplets and increased UB expression and proteasome activity. Furthermore, the inhibition of the ubiquitin-proteasome system and endosomal-lysosomal pathway using epoxomicin and chloroquine, respectively, resulted in a further elevation of CD36, ADFP, and TG levels, thereby enhancing cell viability. These in vitro findings were validated in vivo by a mouse model, where VPS28 knockdown enhanced CD36, ADFP, UB expression, TG content, and lipid droplets in mammary glands, without pathological changes in mammary tissue or blood TG alterations. These results confirm the pivotal role of VPS28 in regulating TG synthesis via the ubiquitination pathway, offering novel insights into the molecular mechanisms of milk fat production in a bovine in vitro cell model.
Surveillance of H5 HPAI in Michigan using retail milk
Authors: Lauring Adam S. , Martin Emily Toth , Eisenberg Marisa C. , Fitzsimmons William J. , Salzman Elizabeth , Leis Aleda M. , Lyon-Callo Sarah , Bagdasarian Natasha
Published: July 2024

An outbreak of highly pathogenic avian influenza virus (HPAI) H5N1 in dairy cattle in the United States has spread to at least 136 dairy herds in 11 states. Due to a lack of testing, it has been difficult to determine the number of farms affected and the extent of intrastate spread. We report a pilot study of real time RT-PCR (rRT-PCR) detection of influenza A virus (IAV) in milk purchased from local markets across Michigan. Two out of 13 samples were positive for IAV nucleic acid, suggestive of HPAI H5N1 infection in the originating herds. This small study of a convenience sample suggests that milk-based surveillance might be useful if applied systematically either at dairy processors or point of sale.