bluuebunny/crossref_metadata_2025 huggingface dataset:
Towards verifications of Krylov complexity
Authors: Sasaki Ryu
Published: May 2024

Krylov complexity is considered to provide a measure of the growth of operators evolving under Hamiltonian dynamics. The main strategy is the analysis of the structure of Krylov subspace $\mathcal {K}_M(\mathcal {H},\eta )$ spanned by the multiple applications of the Liouville operator $\mathcal {L}$ defined by the commutator in terms of a Hamiltonian $\mathcal {H}$, $\mathcal {L}:=[\mathcal {H},\cdot ]$ acting on an operator η, $\mathcal {K}_M(\mathcal {H},\eta )=\text{span}\lbrace \eta ,\mathcal {L}\eta ,\ldots ,\mathcal {L}^{M-1}\eta \rbrace$. For a given inner product ( ·, ·) of the operators, the orthonormal basis $\lbrace \mathcal {O}_n\rbrace$ is constructed from $\mathcal {O}_0=\eta /\sqrt{(\eta ,\eta )}$ by Lanczos algorithm. The moments $\mu _m=(\mathcal {O}_0,\mathcal {L}^m\mathcal {O}_0)$ are closely related to the important data {bn} called Lanczos coefficients. I present the exact and explicit expressions of the moments {μm} for 16 quantum mechanical systems which are exactly solvable both in the Schrödinger and Heisenberg pictures. The operator η is the variable of the eigenpolynomials. Among them six systems show a clear sign of ‘non-complexity’ as vanishing higher Lanczos coefficients bm = 0, m ≥ 3.
The forced battle between peer-review and scientometric research assessment: Why the CoARA initiative is unsound
Authors: Abramo Giovanni
Published: May 2024

Endorsed by the European Research Area, a Coalition for Advancing Research Assessment (CoARA), primarily composed of research institutions and funders, was established in 2022. Its mission is to reform research assessment, emphasizing a shift towards qualitative judgment. This study examines the CoARA initiative from theoretical, practical, and applicative perspectives, exploring its implications. The manuscript questions a standardized way to evaluate research suitable in every context. Through empirical evidence and internal logical arguments, it challenges the supremacy of qualitative over quantitative methods in terms of robustness, accuracy, validity, functionality, and time and cost convenience in STEMM research assessment. The aim is to clear up common misunderstandings and offer insights to policymakers and decision-makers in charge of defining how research should be evaluated.
Beyond minimum wage: Broader employment policies can significantly affect food insecurity
Authors: Piekarz-Porter Elizabeth , Oddo Vanessa M , Stiehl Emily , Kim Sage J
Published: May 2024

Although many have investigated the impacts of minimum wage on a broad array of health outcomes, innovative policies surrounding broader employment policies have largely not been studied. To that end, this paper contributes in three ways. First, it discusses the rise in precarious employment. Then, it turns to the current federal framework of employment policies, namely minimum wage. Finally, it explores what a broader definition of employment policies could include and how future studies could use state, county, and municipal policymaking in this space to investigate ways in which they might contribute to reducing food insecurity and in turn, improve health outcomes.
Heparan sulphate binding controlsin vivohalf-life of the HpARI protein family
Authors: Colomb Florent , Jamwal Abhishek , Ogunkanbi Adefunke , Frangova Tania , Savage Alice R. , Kelly Sarah , Wright Gavin J , Higgins Matthew K. , McSorley Henry J
Published: May 2024

The parasitic nematodeHeligmosomoides polygyrus bakerisecretes the HpARI family, which bind to IL-33, either suppressing (HpARI1 and HpARI2) or enhancing (HpARI3) responses to the cytokine. We previously showed that HpARI2 also bound to DNA via its first Complement Control Protein (CCP1) domain. Here, we find that HpARI1 can also bind DNA, while HpARI3 cannot. Through the production of HpARI2/HpARI3 CCP1 domain-swapped chimeras, DNA-binding ability can be transferred, and correlates with in vivo half-life of administered proteins. We found that HpARI1 and HpARI2 (but not HpARI3) also binds to the extracellular matrix component heparan sulphate (HS), and structural modelling showed a basic charged patch in the CCP1 domain of HpARI1 and HpARI2 (but not HpARI3) which could facilitate these interactions. Finally, a mutant of HpARI2 was produced which lacked DNA and HS binding, and was also shown to have a short half-life in vivo. Therefore, we propose that during infection the suppressive HpARI1 and HpARI2 proteins have long-lasting effects at the site of deposition due to DNA and/or extracellular matrix interactions, while HpARI3 has a shorter half-life due to a lack of these interactions.
Standardized Nomenclature and Reporting for PacBio HiFi Sequencing and Analysis of rAAV Gene Therapy Vectors
Authors: Talevich Eric , Tseng Elizabeth , Diallo Alpha , Sellami Nadia , Elliott Amicia , Cantarel Brandi L , Tonthat Nam , Chatterjee Pranam , Tai Phillip W.L. , Aldridge Claire
Published: May 2024

Despite recombinant adeno-associated viruses (rAAVs) being the leading platform for gene therapy, there is a lack of standardized computational analysis methods and reporting to assess the contents of each capsid through long-read sequencing. PacBio's highly accurate long-read HiFi sequencing enables comprehensive characterization of AAV genomes but requires bioinformatics expertise for analyzing, interpreting and comparing the results. To address this need and improve the understanding of functional viral payloads, our working group established standardized nomenclature and reporting for long-read sequencing data of rAAV vectors. The working group recommendations cover critical quality attributes (CQAs) related to vector purity (full-length vs. fragmented genomes) and identification of contaminants (host DNA, plasmid DNA). Our data analyses of de novo manufacturing runs by the recommended protocol revealed specificity of full and partially filled capsids and high-resolution characterization of partial/truncated vector species. Finally, we provide an open-source software implementing this standardized AAV analysis and reporting to promote transparency, facilitate data comparability, and improve rAAV vector design and quality control.
Metastasis founder cells activate immunosuppression early in human melanoma metastatic colonization
Authors: Guetter Severin , König Courtney , Koerkel-Qu Huiqin , Markiewicz Aleksandra , Scheitler Sebastian , Katzer Marie , Berneburg Mark , Renner Philipp , Cucuruz Beatrix , Guttenberger Leonhard , Naimer Veronika , Weidele Kathrin , Treitschke Steffi , Werno Christian , Jaser Hanna , Bargmann Tonia , Braun Armin , Weber Florian , Rachel Reinhard , Baumann Felix , Schmidleithner Lisa , Schambeck Kathrin , Mohammadi Parvaneh , Ulmer Anja , Haferkamp Sebastian , Klein Christoph A. , Werner-Klein Melanie
Published: May 2024

The earliest steps of lethal metastasis in patients are incompletely understood. To dissect them, we prospectively searched for the earliest detectable disseminated cancer cells (DCC) in sentinel lymph node biopsies of 492 stage I-III patients. By visually-controlled, micromanipulator-assisted isolation and single cell transcriptome analysis of these extremely rare DCC, we identified MCSP+melanoma cells as strong candidates for metastasis founder cells (MFC) in lymph nodes. Based on a median follow-up time of 6 years, their detection was the strongest predictor of systemic metastasis and death upon multivariable analysis. During transition from single cells to metastasis-initiating clusters, melanoma DCC were exposed to CD8 T cell attack, activated the extracellular vesicular exosomal pathway, and expressed the immunomodulatory proteins CD155 and CD276, but rarely PD-L1. CD155 and CD276-positive extracellular vesicles from patient-derived DCC models exhibited an immunosuppressive activity on CD8 T cells. Our data indicate that either direct targeting of MFC employing MCSP or their immune escape mechanisms might be key for cure of early-stage melanoma.
Draft genome analysis ofDelftia tsuruhatensisIICT-RSP4, a strain with uricase potential isolated from soil
Authors: Anumalla Mahesh , Batchu Uma Rajeswari , Bhukya Bhima , Shetty Prakasham Reddy
Published: May 2024

Delftia tsuruhatensisIICT-RSP4, an uricase producing bacterium was isolated usingi-chip method from soil and characterized. Here, we report the draft genome sequence ofD. tsuruhatensisIICT-RSP4. The genome data comprised of 6,627,718bp (6.6 MB) with a GC content of 66.6% with 7 protein encoding genes, 346 sub-systems with 6165 coding sequences and 112 RNAs. The genome revealed five functional secondary metabolite biosynthetic gene clusters viz. terpene, resorcinol, NRP+PKS, T2PKS, and RiPPS related to antimicrobial, anticancer and antimalarial functionality.In vitro, screening studies revealed the uricase potential of the strainD. tsuruhatensisIICT-RSP4. This is the first report on the whole genome sequence of an uricase-producingD. tsuruhatensisIICT-RSP4.
A New Species ofPachytritonfrom Anhui, China (Amphibia: Caudata: Salamandridae)
Authors: He Zhirong , Li Jiaqi , Wu Siyu , Wang Shanqing , Zhao Na , Ma Li , Jiang Jianping , Wu Xiaobing , Wang Supen
Published: May 2024

A new species belonging to the genusPachytritonis delineated based on four specimens obtained from the Qingliangfeng Nature Reserve, Huangshan, Anhui, China. The identification of this distinct species within the Southeast Chinese Hilly Area enhances the recognized biodiversity ofPachytritonand signifies unresolved interrelations among species in southeastern China. This new species can be distinguished fromP. granulosusandP. feiithrough meticulous morphological and molecular examinations. The phylogenetic correlation was established utilizing the mitochondrial NADH dehydrogenase subunit 2 gene (ND2). The four recent specimens constitute a cohesive monophyletic clade with robust affirmation. The acknowledgment of this new species elevates the tally of documentedPachytritonspecies to 11.
Bayesian Estimation of Muscle Mechanisms and Therapeutic Targets Using Variational Autoencoders
Authors: Tune Travis , Kooiker Kristina B , Davis Jennifer , Daniel Thomas , Moussavi-Harami Farid
Published: May 2024

Cardiomyopathies, often caused by mutations in genes encoding muscle proteins, are traditionally treated by phenotyping hearts and addressing symptoms post irreversible damage. With advancements in genotyping, early diagnosis is now possible, potentially introducing earlier treatment. However, the intricate structure of muscle and its myriad proteins make treatment predictions challenging. Here we approach the problem of estimating therapeutic targets for a mutation in mouse muscle using a spatially explicit half sarcomere muscle model. We selected 9 rate parameters in our model linked to both small molecules and cardiomyopathy-causing mutations. We then randomly varied these rate parameters and simulated an isometric twitch for each combination to generate a large training dataset. We used this dataset to train a Conditional Variational Autoencoder (CVAE), a technique used in Bayesian parameter estimation. Given simulated or experimental isometric twitches, this machine learning model is able to then predict the set of rate parameters which are most likely to yield that result. We then predict the set of rate parameters associated with twitches from control mice with the cardiac Troponin C (cTnC) I61Q variant and control twitches treated with the myosin activator Danicamtiv, as well as model parameters that recover the abnormal I61Q cTnC twitches.

SIGNIFICANCE

Machine learning techniques have potential to accelerate discoveries in biologically complex systems. However, they require large data sets and can be challenging in high dimensional systems such as cardiac muscle. In this study, we combined experimental measures of cardiac muscle twitch forces with mechanistic simulations and a newly developed mixture of Bayesian inference with neural networks (in autoencoders) to solve the inverse problem of determining the underlying kinetics for observed force generation by cardiac muscle. The autoencoders are trained on millions of simulations spanning parameter spaces that correspond to the mechanochemistry of cardiac sarcomeres. We apply the trained model to experimental data in order to infer parameters that can explain a diseased twitch and ways to recover it.
Plasmodesmal closure elicits stress responses
Authors: Tee Estee E. , Breakspear Andrew , Papp Diana , Thomas Hannah R. , Walker Catherine , Bellandi Annalisa , Faulkner Christine
Published: May 2024

Plant cells are connected to their neighbors via plasmodesmata facilitating the exchange of nutrients and signaling molecules. During immune responses, plasmodesmata close, but how this contributes towards a full immune response is unknown. To investigate this, we developed two transgenic lines with which we could induce plasmodesmal closure independently of immune elicitors, using the over-active CALLOSE SYNTHASE3 alleleicals3mand the C-terminus of PDLP1 to drive callose deposition at plasmodesmata. Induction of plasmodesmal closure increased the expression of stress responsive genes, salicylic acid accumulation and resistance toPseudomonas syringaeDC3000. More homogeneous plasmodesmal closure usingicals3malso led to the accumulation of starch and sugars, decreased leaf growth, as well as hypersusceptibility toBotrytis cinerea. Based on the profile of responses, we conclude that plasmodesmal closure itself activates stress signaling, raising questions of what signals mediate this response and whether these responses occur in all circumstances when plasmodesmata close.
EMT-dependent cell-matrix interactions are linked to unjamming transitions in cancer spheroid invasion
Authors: van der Net Anouk , Rahman Zaid , Bordoloi Ankur D. , Muntz Iain , ten Dijke Peter , Boukany Pouyan E. , Koenderink Gijsje H.
Published: May 2024

The plasticity of cancer cells allows them to switch between different migration modes, promoting their invasion into the extracellular matrix (ECM) and hence increasing the risks of metastasis. Epithelial-to-mesenchymal transitions (EMT) and unjamming transitions provide two distinct pathways for cancer cells to become invasive, but it is still unclear to what extent these pathways are connected. Here we addressed this question by performing 3D spheroid invasion assays of lung adenocarcinoma (A549, epithelial) and melanoma (MV3, mesenchymal-like) cancer cell lines in collagen-based hydrogels, where we varied both the invasive character of the cells (using Transforming Growth Factor (TGF)-βto promote EMT and matrix metalloprotease (MMP) inhibition to block cell-mediated matrix degradation) and the porosity of the matrix. Using a quantitative image analysis method to track spheroid invasion, we discovered that the onset time of invasion mostly depended on the matrix porosity and corresponded with vimentin levels, while the subsequent spheroid expansion rate mostly depended on metalloprotease MMP1 levels and thus cell-matrix interaction. Morphological analysis revealed that spheroids displayed solid-like (non-invasive) behavior in small-pore hydrogels and switched to fluid-like (strand-based) or gas-like (disseminating cells) phases in large-pore hydrogels and when cells were more mesenchymal-like. Our findings are consistent with unjamming transitions as a function of cell motility and matrix confinement predicted in recent models for cancer invasion, but show that cell motility and matrix confinement are coupled via EMT-dependent matrix degradation.
Oncogenic Ras-driven Dorsal/NF-κB signaling contributes to tumorigenesis in aDrosophilacarcinoma model
Authors: Dillard Caroline , Reis Jose Teles , Jain Ashish , Le Borgne Roland , Jasper Heinrich , Rusten Tor Erik
Published: May 2024

Cancer-driving mutations synergize with inflammatory stress signaling pathways during carcinogenesis.Drosophila melanogastertumour models are increasingly recognized as models to inform conserved molecular mechanisms of tumorigenesis with both local and systemic effects of cancer. Although initial discoveries of the Toll-NFκB signaling pathway in development and immunity was pioneered inDrosophila, limited information is available for its role in cancer progression. Using a well-studied cooperative RasV12-driven epithelial-derived tumour model, we here describe functions of Toll-NF-κB signaling in malignantRasV12, scrib-tumors. The extracellular Toll pathway components ModSP and PGRP-SA and intracellular signaling Kinase, Pelle/IRAK, are rate-limiting for tumor growth. The Toll pathway NFκB protein Dorsal, as well ascactus/IκB show elevated expression in tumors with highest expression in invasive cell populations. Oncogenic RasV12, and not loss ofscribble,confers increased expression and heterogenous distribution of two Dorsal isoforms, DorsalA and DorsalB in different tumour cell populations. Mechanistic analyses demonstrates that Dorsal drives growth and malignancy by suppressing differentiation, counteracting apoptosis and promoting invasion ofRasV12, scrib-tumors genetically dependent ontwistandsnail.
B cell-mediated antigen presentation promotes adverse cardiac remodeling in chronic heart failure
Authors: Lovell Jana P. , Duque Carolina , Rousseau Sylvie , Bhalodia Aashik , Bermea Kevin , Cohen Charles D. , Adamo Luigi
Published: May 2024

Cardiovascular disease remains the leading cause of death worldwide. A primary driver of cardiovascular mortality is ischemic heart failure, a form of cardiac dysfunction that can develop in patients who survive myocardial infarction. Acute cardiac damage triggers robust changes in the spleen with rapid migration of immune cells from the spleen to the heart. Activating this “cardio-splenic” axis contributes to progressive cardiac dysfunction. The cardio-splenic axis has, therefore, been identified as a promising therapeutic target to prevent or treat heart failure.

However, our understanding of the precise mechanisms by which specific immune cells contribute to adverse cardiac remodeling within the cardio-splenic axis remains limited. Here, we show that splenic B cells contribute to the development of heart failure via MHC II-mediated antigen presentation. We found that the adoptive transfer of splenic B cells from mice with ischemic heart failure promoted adverse cardiac remodeling and splenic inflammatory changes in naïve recipient mice. Based on single-cell RNA sequencing analysis of splenic B cells from mice with ischemic heart failure, we hypothesized that B cells contributed to adverse cardiac remodeling through antigen presentation by MHC II molecules. This mechanism was confirmed using transgenic mice with B cell-specific MHC II deletion, and by analyzing circulating B cells from humans who experienced myocardial infarction. Our results broaden our understanding of B lymphocyte biology, reshape current models of immune activation in response to myocardial injury, and point towards MHC II-mediated signaling in B cells as a novel and specific therapeutic target in chronic heart failure.
PCIF1 is partly cytoplasmic, dynamically localizes to stress granules and binds mRNA coding regions upon oxidative stress
Authors: Tat Trinh T. , Raza Sabeen , Khan Shaheerah , Watson Tiara L. , Jung Sung Yun , Kiss Daniel L.
Published: May 2024

PCIF1 (PhosphorylatedCTD-InteractingFactor1) is the mRNA (2’-O-methyladenosine-N(6)-)-methyltransferase that catalyzes the formation of cap-adjacent N6,2’-O-dimethyladenosine (m6Am) by methylating adenosines at the first transcribed position of capped mRNAs. While previous studies assumed that PCIF1 was nuclear, cell fractionation and immunofluorescence both show that a population of PCIF1 is localized to the cytoplasm. Further, PCIF1 redistributes to stress granules upon oxidative stress. Immunoprecipitation studies with stressed cells show that PCIF1 also physically interacts with G3BP and other stress granule components. In addition, PCIF1 behaves as a stress granule component as it disassociates from stress granules upon recovery from stress. Overexpressing full-length PCIF1 also inhibits stress granule formation, while knocking out PCIF1 slows stress granule disassembly. Next, our enhanced crosslinking and immunoprecipitation (eCLIP) data show that PCIF1 binds mRNAs in their coding sequences rather than cap-proximal regions. Further PCIF1’s association with mRNAs increased upon NaAsO2stress. In contrast to eCLIP data, ChIP-Seq experiments show that PCIF1 is predominantly associated with transcription start sites rather than gene bodies, indicating that PCIF1’s association with mature mRNA is not co-transcriptional. Collectively, our data suggest that PCIF1 has cytoplasmic RNA surveillance role(s) independent of transcription-associated cap-adjacent mRNA modification, particularly during the stress response.
SCO-pH: Microfluidic dynamic phenotyping platform for high-throughput screening of single cell acidification
Authors: Jeong Hyejoong , Porello Emilia A. Leyes , Rosario Jean Gabriel , Kuang Da , Han Syung Hun , Sul Jai-Yoon , Lim Bomyi , Lee Daeyeon , Kim Junhyong
Published: May 2024

Studies on the dynamics of single cell phenotyping have been hampered by the lack of quantitative high-throughput metabolism assays. Extracellular acidification, a prominent phenotype, yields significant insights into cellular metabolism, including tumorigenicity. Here, we develop a versatile microfluidic system for single cell optical pH analysis (SCO-pH), which compartmentalizes single cells in 140-pL droplets and immobilizes approximately 40,000 droplets in a two-dimensional array for temporal extracellular pH analysis. SCO-pH distinguishes cells undergoing hyperglycolysis induced by oligomycin A from untreated cells by monitoring their extracellular acidification. To facilitate pH sensing in each droplet, we encapsulate a cell-impermeable pH probe whose fluorescence intensities are quantified. Using this approach, we can differentiate hyperglycolytic cells and concurrently observe single cell heterogeneity in extracellular acidification dynamics. This high-throughput system will be useful in applications that require dynamic phenotyping of single cells with significant heterogeneity.
eIF4A controls translation of estrogen receptor alpha and is a therapeutic target in advanced breast cancer
Authors: Boyer Jacob A. , Sharma Malvika , Dorso Madeline A. , Mai Nicholas , Amor Corina , Reiter Jason M. , Kannan Ram , Gadal Sunyana , Xu Jianing , Miele Matthew , Li Zhuoning , Chen Xiaoping , Chang Qing , Pareja Fresia , Worland Stephan , Warner Douglas , Sperry Sam , Chiang Gary G. , Thompson Peggy A. , Yang Guangli , Ouerfelli Ouathek , de Stanchina Elisa , Wendel Hans-Guido , Rosen Ezra Y. , Chandarlapaty Sarat , Rosen Neal
Published: May 2024

The majority of human breast cancers are dependent on hormone-stimulated estrogen receptor alpha (ER) and are sensitive to its inhibition. Treatment resistance arises in most advanced cancers due to genetic alterations that promote ligand independent activation of ER itself or ER target genes. Whereas re-targeting of the ER ligand binding domain (LBD) with newer ER antagonists can work in some cases, these drugs are largely ineffective in many genetic backgrounds including ER fusions that lose the LBD or in cancers that hyperactivate ER targets. By identifying the mechanism of ER translation, we herein present an alternative strategy to target ER and difficult to treat ER variants. We find that ER translation is cap-independent and mTOR inhibitor insensitive, but dependent on 5’ UTR elements and sensitive to pharmacologic inhibition of the translation initiation factor eIF4A, an mRNA helicase. EIF4A inhibition rapidly reduces expression of ER and short-lived targets of ER such as cyclin D1 and other components of the cyclin D-CDK complex in breast cancer cells. These effects translate into suppression of growth of a variety of ligand-independent breast cancer models including those driven by ER fusion proteins that lack the ligand binding site. The efficacy of eIF4A inhibition is enhanced when it is combined with fulvestrant—an ER degrader. Concomitant inhibition of ER synthesis and induction of its degradation causes synergistic and durable inhibition of ER expression and tumor growth. The clinical importance of these findings is confirmed by results of an early clinical trial (NCT04092673) of the selective eIF4A inhibitor zotatifin in patients with estrogen receptor positive metastatic breast cancer. Multiple clinical responses have been observed on combination therapy including durable regressions. These data suggest that eIF4A inhibition could be a useful new strategy for treating advanced ER+ breast cancer.
Identifying Candidate Genes for Sugar Accumulation in Sugarcane Cultivars: From a Syntenic Genomic Region to a Gene Coexpression Network
Authors: Martins Mônica Letícia Turibio , Sforça Danilo Augusto , dos Santos Luís Paulo , Pimenta Ricardo José Gonzaga , Mancini Melina Cristina , Aono Alexandre Hild , da Silva Cláudio Benício Cardoso , Vautrin Sonia , Bellec Arnaud , Vicentini Renato , Bérgès Helene , da Silva Carla Cristina , de Souza Anete Pereira
Published: May 2024

Elucidating the intricacies of the sugarcane genome is essential for breeding superior cultivars. This economically important crop originates from hybridizations of highly polyploidSaccharumspecies. However, the large size (10 Gb), high polyploidy, and aneuploidy of the sugarcane genome pose significant challenges to complete genome sequencing, assembly, and annotation. One successful strategy for identifying candidate genes linked to agronomic traits, particularly those associated with sugar accumulation, leverages synteny and potential collinearity with related species. In this study, we explored synteny between sorghum and sugarcane. Genes from a sorghum Brix QTL were used to screen bacterial artificial chromosome (BAC) libraries from two Brazilian sugarcane varieties (IACSP93-3046 and SP80-3280). The entire region was successfully recovered, confirming synteny and collinearity between the species. Manual annotation identified 51 genes in the hybrid varieties that were subsequently confirmed to be present inSaccharum spontaneum. To identify candidate genes for sugar accumulation, this study employed a multifaceted approach, including retrieving the genomic region of interest, performing gene-by-gene analysis, analyzing RNA-seq data of internodes fromSaccharum officinarumandS. spontaneumaccessions, constructing a coexpression network to examine the expression patterns of genes within the studied region and their neighbors, and finally identifying differentially expressed genes (DEGs). This comprehensive approach led to the discovery of three candidate genes potentially involved in sugar accumulation: an ethylene-responsive transcription factor (ERF), an ABA 8’-hydroxylase, and a prolyl oligopeptidase (POP). These findings could be valuable for identifying additional candidate genes for other important agricultural traits and directly targeting candidate genes for further work in molecular breeding.
Gradual labeling with fluorogenic probes for MINFLUX nanoscopy of densely packed structures
Authors: Yao Longfang , Si Dongjuan , Chen Liwen , Li Shu , Guan Jiaxin , Ma Jiong , Zhang Qiming , Wang Jing , Wang Lu , Gu Min
Published: May 2024

Single-molecule localization in densely packed structures represents a formidable challenge in nanoscopy. Here we develop a gradual labeling method with fluorogenic probes for MINFLUX nanoscopy (GLF-MINFLUX), with which individual proteins in packed structures can be sequential illuminated and quantitatively localized at the nanoscale solely by adjusting probe concentration. With a 1.7-fold localization precision improvement and 2.2-fold faster acquisition, GLF-MINFLUX enables dual-channel nanoscale imaging of densely neuronal microtubules and microfilaments, and three-dimensional quantitative mapping of individual TOM20 proteins in mitochondrial clusters.
Context dependent activity of p63-bound gene regulatory elements
Authors: McCann Abby A , Baniulyte Gabriele , Woodstock Dana , Sammons Morgan A
Published: May 2024

The p53 family of transcription factors regulate numerous organismal processes including the development of skin and limbs, ciliogenesis, and preservation of genetic integrity and tumor suppression. p53 family members control these processes and gene expression networks through engagement with DNA sequences within gene regulatory elements. Whereas p53 binding to its cognate recognition sequence is strongly associated with transcriptional activation, p63 can mediate both activation and repression. How the DNA sequence of p63-bound gene regulatory elements is linked to these varied activities is not yet understood. Here, we use massively parallel reporter assays (MPRA) in a range of cellular and genetic contexts to investigate the influence of DNA sequence on p63-mediated transcription. Most regulatory elements with a p63 response element motif (p63RE) activate transcription, with those sites bound by p63 more frequently or adhering closer to canonical p53 family response element sequences driving higher transcriptional output. The most active regulatory elements are those also capable of binding p53. Elements uniquely bound by p63 have varied activity, with p63RE-mediated repression associated with lower overall GC content in flanking sequences. Comparison of activity across cell lines suggests differential activity of elements may be regulated by a combination of p63 abundance or context-specific cofactors. Finally, changes in p63 isoform expression dramatically alters regulatory element activity, primarily shifting inactive elements towards a strong p63-dependent activity. Our analysis of p63-bound gene regulatory elements provides new insight into how sequence, cellular context, and other transcription factors influence p63-dependent transcription. These studies provide a framework for understanding how p63 genomic binding locally regulates transcription. Additionally, these results can be extended to investigate the influence of sequence content, genomic context, chromatin structure on the interplay between p63 isoforms and p53 family paralogs.
Modeling Riboswitches: The impact of SAM concentration on the folding of the SAM-II riboswitch
Authors: Alaidi Osama
Published: May 2024

Several mechanistic (thermodynamic) models have been developed for the folding of SAM-II riboswitch as a function of SAM and magnesium concentrations. For each model, the model parameters (equilibrium constants) have been determined from experimental (apparent) binding data, based on the underlying assumptions of the model. The predicted titration curves computed from the different models were calculated and compared with actual experimental observation of the fraction of the RNA forming a pseudoknot at specific concentration of the ligands. Strikingly, only one of the six models correctly predicts the experimental findings, confirming the dominant mechanism of the riboswitch function. More interestingly, the latter mechanism is found to be the most efficient compared to the other possible mechanisms. The study sheds light on the cognate ligand conformational capture mechanism of the SAM-II riboswitch in the presence of specific concentrations of magnesium ions. The presented mathematical and thermodynamic framework, as well as the inferred equilibrium constants, provide foundations for making accurate quantitative prediction of the SAM-II riboswitch ensemble populations as a function of SAM and magnesium concentrations. The mechanistic linked equilibria model can be generalized to describe other thermodynamically driven riboswitches and hence facilitate identifying RNA intermediates that can be leveraged for small molecule drug design.
In vitro spatiotemporal reconstruction of human skeletal muscle organogenesis
Authors: Mavrommatis Lampros , Daya Nassam , Volke Leon , Lu I-Na , Zhuge Heidi , Stehling Martin , Zeuschner Dagmar , Jeong Hyun-Woo , Yang Ji-Hun , Meyer zu Hörste Gerd , Brand-Saberi Beate , Schöler Hans R. , Vorgerd Matthias , Zaehres Holm
Published: May 2024

Spatiotemporal recapitulation of long-range trajectories for lineages that influence body patterning along the medio-lateral and proximal-distal axes during embryogenesis in anin vitrosystem remains elusive. Here we introduce a three-dimensional organoid approach, termed Gastruloids-Lateraloid-Musculoids (GLMs), to model human neural crest, lateral plate mesoderm and skeletal muscle lineage development at the forelimb level following gastrulation and during limb patterning. GLMs harvest neuro-mesodermal progenitors with the potential to establish neural and paraxial mesodermal populations, while single cell analyses and spatial transcriptomics demonstrate promotion of mesodermal lineage segregation during gastrulation and spatial recapitulation of migration events along the medio-lateral axis for vagal neural crest, hypaxial myogenesis and lateral plate mesodermal lineages. Comparative analyses to developmental atlases and adult muscle stem cell data confirm a pool of hypaxial migrating myogenic progenitors that in a niche dependent manner change their embryonic anatomical developmental program to a fetal myogenic program, thus enabling them to resist specification in a cell autonomous manner and facilitate long termin vitroexpansion. GLMs model human myogenesis at the forelimb level, establish fetal muscle stem cells equivalent to those that sustain the growth phase of the embryo and provide a 3Din vitrosystem for investigating neural crest, early fore-gut and lateral plate mesoderm development.
An MST-based assay reveals new binding preferences of IFIT1 for canonically and non-canonically capped RNAs
Authors: Spiewla Tomasz , Grab Katarzyna , Depaix Anais , Ziemkiewicz Kamil , Warminski Marcin , Jemielity Jacek , Kowalska Joanna
Published: May 2024

IFIT proteins (interferon-induced proteins with tetratricopeptide repeats) are key components of the innate immune response that bind to viral and cellular RNA targets to inhibit viral translation and replication. The RNA target recognition is guided by molecular patterns, particularly at the RNA 5’ ends. IFIT1 preferably binds RNAs modified with the 7-methylguanosine (m7G) cap-0 structure, while RNAs with cap-1 structure are recognized with lower affinity. Less is known about the propensity of IFIT1 to recognize non-canonical RNA 5’ ends, including hypermethylated and non-canonical RNA caps. Deciphering the structure-function relationship for IFIT1-RNA interaction may improve understanding of cellular selection of IFIT targets and guide the design of exogenously delivered therapeutic RNAs, but requires high-throughput and robust analytical methods. Here, we report a biophysical assay for quick, direct, in-solution affinity assessment of differently capped RNAs with IFIT1. The procedure, which relies on measuring microscale thermophoresis (MST) of fluorescently labelled protein as a function of increasing ligand concentration, is applicable to various RNA lengths and sequences without the need for labelling or affinity tagging. Using the assay, we examined thirteen canonically and non-canonically 5’-capped RNAs, revealing new binding preferences of IFIT1. The 5’ terminal m6A mark in the m7G cap had a protective function against IFIT1, which was additive with the effect observed for the 2’-O position (m6Amcap-1). In contrast, an increased affinity for IFIT1 was observed for several non-canonical caps, including trimethylguanosine (TMG), unmethylated (G), and flavin-adenine dinucleotide (FAD) caps. The results suggest new potential cellular targets of IFIT1 and may contribute to broadening the knowledge on the mechanisms of the innate immune response as well as the more effective design of chemically modified mRNAs.
Oncogenic signaling in the adultDrosophilaprostate-like accessory gland leads to activation of a conserved pro-tumorigenic program, in the absence of proliferation
Authors: Church S. Jaimian , Pulianmackal Ajai J. , Dixon Joseph A. , Loftus Luke V. , Amend Sarah R. , Pienta Kenneth , Cackowski Frank C. , Buttitta Laura A.
Published: May 2024

Drosophilamodels for tumorigenesis and metastasis have revealed conserved mechanisms of signaling that are also involved in mammalian cancer. Many of these models use the proliferating tissues of the larval stages ofDrosophiladevelopment, when tissues are highly mitotically active, or stem cells are abundant. FewerDrosophilatumorigenesis models use adult animals to initiate tumor formation when many tissues are largely terminally differentiated and postmitotic. TheDrosophilaaccessory glands are prostate-like tissues and a model for some aspects of prostate tumorigenesis using this tissue has been explored. In this model, oncogenic signaling was induced during the proliferative stage of accessory gland development, raising the question of how oncogenic activity would impact the terminally differentiated and postmitotic adult tissue. Here, we show that oncogenic signaling in the adultDrosophilaaccessory gland leads to activation of a conserved pro-tumorigenic program, similar to that observed in mitotic larval tissues, but in the absence of proliferation. Oncogenic signaling in the adult postmitotic gland leads to tissue hyperplasia with nuclear anaplasia and aneuploidy through endoreduplication, which increases polyploidy and occasionally results in non-mitotic neoplastic-like extrusions. We compare gene expression changes in ourDrosophilamodel with that of endocycling prostate cancer cells induced by chemotherapy, which potentially mediate tumor recurrence after treatment. Similar signaling pathways are activated in theDrosophilagland and endocycling cancer cells, suggesting the adult accessory glands provide a useful model for aspects of prostate cancer progression that do not involve cellular proliferation.
Spike-wave discharges during low-current thalamic deep brain stimulation in mice
Authors: Flores Francisco J , Dalla Betta Isabella , Tauber John , Schreier David , Stephen Emily P , Wilson Matthew A , Brown Emery N
Published: May 2024

Background: Deep brain stimulation of central thalamus (CT-DBS) has potential for modulating states of consciousness, but it can also trigger spike-wave discharges (SWDs). Objectives: To report the probability of inducing SWDs during CT-DBS in awake mice. Methods: Mice were implanted with electrodes to deliver unilateral and bilateral CT-DBS at different frequencies while recording EEG. We titrated stimulation current by gradually increasing it at each frequency until an SWD appeared. Subsequent stimulations to test arousal modulation were performed at the current one step below the current that caused an SWD during titration. Results: In 2.21% of the test stimulations (10 out of 12 mice), CT-DBS caused SWDs at currents lower than the titrated current, at currents as low as 20 uA. Conclusion: Our study found a small but significant probability of inducing SWDs even after titration and at relatively low currents. EEG should be closely monitored for SWDs when performing CT-DBS in both research and clinical settings.
Associations Between Prenatal Vitamin D and Placental Gene Expression
Authors: Parenti Mariana , Melough Melissa M. , Lapehn Samantha , MacDonald James , Bammler Theo , Firsick Evan J. , Choi Hyo Young , Derefinko Karen J. , Enquobahrie Daniel A. , Carroll Kecia N. , LeWinn Kaja Z. , Bush Nicole R. , Zhao Qi , Sathyanarayana Sheela , Paquette Alison G.
Published: May 2024

Background

Vitamin D is a hormone regulating gene transcription. Prenatal vitamin D has been linked to immune and vascular function in the placenta, a key organ of pregnancy. To date, studies of vitamin D and placental gene expression have focused on a limited number of candidate genes. Transcriptome-wide RNA sequencing can provide a more complete representation of the placental effects of vitamin D.

Objective

We investigated the association between prenatal vitamin D levels and placental gene expression in a large, prospective pregnancy cohort.

Methods

Participants were recruited in Shelby County, Tennessee in the Conditions Affecting Neurocognitive Development and Learning in Early childhood (CANDLE) study. Vitamin D level (plasma total 25-hydroxyvitatmin D, [25(OH)D]) was measured at mid-pregnancy (16-28 weeks’ gestation) and delivery. Placenta samples were collected at birth. RNA was isolated and sequenced. We identified differentially expressed genes (DEGs) using adjusted linear regression models. We also conducted weighted gene co-expression network analysis (WGCNA).

Results

The median 25(OH)D of participants was 21.8 ng/mL at mid-pregnancy (N=774, IQR: 15.4-26.5 ng/mL) and 23.6 ng/mL at delivery (N=753, IQR: 16.8-29.1 ng/mL). Placental expression of 25 DEGs was associated with 25(OH)D at mid-pregnancy, but no DEG was associated with 25(OH)D at delivery. DEGs were related to energy metabolism, cytoskeletal function, and RNA transcription. Using WGCNA, we identified 2 gene modules whose expression was associated with 25(OH)D at mid-pregnancy and 1 module associated with 25(OH)D at delivery. These modules were enriched for genes related to mitochondrial and cytoskeletal function, and were regulated by transcription factors includingARNT2,BHLHE40,FOSL2,JUND, andNFKB1.

Conclusions

Our results indicate that 25(OH)D during mid-pregnancy, but not at delivery, is associated with placental gene expression at birth. Future research is needed to investigate a potential role of vitamin D in programming placental mitochondrial metabolism, intracellular transport, and transcriptional regulation during pregnancy.
A phosphorylation code coordinating transcription condensate dynamics with DNA replication
Authors: Marmolejo Carlos Origel , Sanchez Celina , Lee Juyoung , Werner Marcel , Roberts Paige , Hamperl Stephan , Saldivar Joshua C.
Published: May 2024

Chromatin is organized into compartments enriched with functionally-related proteins driving non-linear biochemical activities. Some compartments,e.g.transcription foci, behave as liquid condensates. While the principles governing the enrichment of proteins within condensates are being elucidated, mechanisms that coordinate condensate dynamics with other nuclear processes like DNA replication have not been identified. We show that at the G1/S cell cycle transition, large transcription condensates form at histone locus bodies (HLBs) in a cyclin-dependent kinase 1 and 2 (CDK1/2)-dependent manner. As cells progress through S phase, ataxia-telangiectasia and Rad3-related (ATR) accumulates within HLBs and dissolves the associated transcription condensates. Integration of CDK1/2 and ATR signaling creates a phosphorylation code within the intrinsically-disordered region of mediator subunit 1 (MED1) coordinating condensate dynamics with DNA replication. Disruption of this code results in imbalanced histone biosynthesis, and consequently, global DNA damage. We propose the spatiotemporal dynamics of transcription condensates are actively controlled via phosphorylation and essential for viability of proliferating cells.
pH-Controlled chemoselective rapid azo-coupling reaction (CRACR) enables global profiling of serotonylation proteome in cancer cells
Authors: Zhang Nan , Wu Jinghua , Gao Shuaixin , Peng Haidong , Li Huapeng , Gibson Connor , Wu Sophia , Zhu Jiangjiang , Zheng Qingfei
Published: May 2024

Serotonylation has been identified as a novel protein post-translational modification for decades, where an isopeptide bond is formed between the glutamine residue and serotonin through transamination. Transglutaminase 2 (also known as TGM2 or TGase2) was proven to act as the main writer enzyme for this PTM and a number of key regulatory proteins (including small GTPases, fibronectin, fibrinogen, serotonin transporter, and histone H3) have been characterized as the substrates of serotonylation. However, due to the lack of pan-specific antibodies for serotonylated glutamine, the precise enrichment and proteomic profiling of serotonylation still remain challenging. In our previous research, we developed an aryldiazonium probe to specifically label protein serotonylation in a bioorthogonal manner, which depended on a pH-controlled chemoselective rapid azo-coupling reaction (CRACR). Here, we report the application of a photoactive aryldiazonium-biotin probe for the global profiling of serotonylation proteome in cancer cells. Thus, over 1,000 serotonylated proteins were identified from HCT 116 cells, many of which are highly related to carcinogenesis. Moreover, a number of modification sites of these serotonylated proteins were determined, attributed to the successful application of our chemical proteomic approach. Overall, these findings provided new insights into the significant association between cellular protein serotonylation and cancer development, further suggesting that to target TGM2-mediated monoaminylation may serve as a promising strategy for cancer therapeutics.
Highly accurate metagenome-assembled genomes from human gut microbiota using long-read assembly, binning, and consolidation methods
Authors: Portik Daniel M , Feng Xiaowen , Benoit Gaetan , Nasko Daniel J , Auch Benjamin , Bryson Samuel J , Cano Raul , Carlin Martha , Damerum Anabelle , Farthing Brett , Grove Jonas R , Islam Moutusee , Langford Kyle W , Liachko Ivan , Locken Kristopher , Mangelson Hayley , Tang Shuiquan , Zhang Siyuan , Quince Christopher , Wilkinson Jeremy E
Published: May 2024

Long-read metagenomic sequencing is a powerful approach for cataloging the microbial diversity present in complex microbiomes, including the human gut microbiome. We performed a deep-sequencing experiment using PacBio HiFi reads to obtain metagenome-assembled genomes (MAGs) from a pooled human gut microbiome. We performed long-read metagenome assembly using two methods (hifiasm-meta, metMDBG), used improved bioinformatic and proximity ligation binning strategies to cluster contigs and identify MAGs, and developed a novel framework to compare and consolidate MAGs (pb-MAG-mirror). We found proximity ligation binning yielded more MAGs than bioinformatic binning, but our novel comparison framework resulted in higher MAG yields than either binning strategy individually. In total, from 255 Gbp of total HiFi data we produced 595 total MAGs (including 175 high-quality MAGs) using hifiasm-meta, and 547 total MAGs (including 277 high-quality MAGs) with metaMDBG. Hifiasm-meta assembled almost twice as many strain-level MAGs as metaMDBG (246 vs. 156), but both assembly methods produced up to five strains for a species. Approximately 85% of the MAGs were assigned to known species, but we recovered >35 high-quality MAGs that represent uncultured diversity. Based on strict similarity scores, we found 125 MAGs were unequivocally shared across the assembly methods at the strain level, representing ~22% of the total MAGs recovered per method. Finally, we detected more total viral sequences in the metaMDBG assembly versus the hifiasm-meta assembly (~6,700 vs. ~4,500). Overall, we find the use of HiFi sequencing, improved metagenome assembly methods, and complementary binning strategies is highly effective for rapidly cataloging microbial genomes in complex microbiomes.