bluuebunny/crossref_metadata_2025_split huggingface dataset:
Prolific release of S-layer units and associated proteins by the methanotrophic bacteriumMethylomicrobium albumBG8
Authors: Hermary Mariah K. , Rodriguez Gallo Maria C. , Grigonyte Aurelija M. , Uhrig R. Glen , Sauvageau Dominic , Stein Lisa Y. 
Published: January 2025

AbstractSome gammaproteobacterial methanotrophs are encapsulated by S-layers that overlay their outer membrane. In this study, TEM imaging revealed thatMethylomicrobium albumBG8 constitutively releases abundant cup-shaped S-layer units – a phenotype that was not observed in 7 other methanotroph strains, even inMethylotuvimicrobium buryatense5GB1 that produces a similar S-layer structure. Release of S-layer units occurred regardless of carbon (methane or methanol) or nitrogen (ammonium or nitrate) source, with 50X trace metal availability or under copper deprivation, and at all growth phases. The released S-layer units were isolated from the culture medium ofM. albumBG8 for proteome analysis. The proteome revealed an abundance of transporters related to calcium uptake including TolC and Repeats-in-Toxin (RTX) proteins, transporters for uptake for cobalamin and siderophores, proteins with Type I secretion system (T1SS) target domains, cell wall biogenesis proteins, and the S- layer protein units themselves. The gene encoding the S-layer protein is co-located in theM. albumBG8 genome with genes encoding the T1SS. Together, this information provides clues regardingM. albumBG8 S-layer biogenesis and function, including a role for the calcium and RTX protein domains, involvement of the T1SS in selecting associated proteins, and reasons behind prolific release of S-layer units. This S-layer release and involvement of the T1SS has biological implications and could be applied towards selective secretion of proteins byM. albumBG8 in bioindustrial applications.ImportanceThe methanotrophic bacteriumM. albumBG8 produces and releases large quantities of its S-layer units into the culture medium regardless of carbon or nitrogen source, metal availability or phase of growth. Of the 8 methanotrophic bacteria screened, onlyM. albumBG8 possesses this phenotype. Proteomics analysis of purified S-layer units identified the S-layer protein itself along with associated proteins involved metal uptake and S-layer biogenesis, including involvement of the T1SS. Better understanding ofM. albumBG8 S-layer biogenesis and the release of its S-layer units into culture medium could be harnessed for exporting expressed proteins of bioindustrial interest for ease of collection and purification.
Histone methyltransferase PRDM9 promotes survival of drug-tolerant persister cells in glioblastoma
Authors: Joun George L. , Kempe Emma G. , Chen Brianna , Sterling Jayden R. , Abbassi Ramzi H. , Daniel du Preez W. , Recasens Ariadna , Clark Teleri , Du Tian Y. , Low Jason K.K. , Kim Hani , Yang Pengyi , Khor Jasmine , Hoque Monira , Indurthi Dinesh C. , Kuchibhotla Mani , Palanisamy Ranjith , Jorgensen William T. , Montgomery Andrew P. , Baker Jennifer R. , Higginbottom Sarah L. , Tomaskovic-Crook Eva , Crook Jeremy M. , Loo Lipin , Day Bryan W. , Neely G. Gregory , Guccione Ernesto , Johns Terrance G. , Kassiou Michael , Don Anthony S. , Munoz Lenka 
Published: January 2025

ABSTRACTChemotherapy often kills a large fraction of cancer cells but leaves behind a small population of drug- tolerant persister cells. These persister cells survive drug treatments through reversible, non-genetic mechanisms and cause tumour recurrence upon cessation of therapy. Here, we report a drug tolerance mechanism regulated by the germ-cell-specific H3K4 methyltransferase PRDM9. Through histone proteomic, transcriptomic, lipidomic, and ChIP-sequencing studies combined with CRISPR knockout and phenotypic drug screen, we identified that chemotherapy-induced PRDM9 upregulation promotes metabolic rewiring in glioblastoma stem cells, leading to chemotherapy tolerance. Mechanistically, PRDM9-dependent H3K4me3 at cholesterol biosynthesis genes enhances cholesterol biosynthesis, which persister cells rely on to maintain homeostasis under chemotherapy- induced oxidative stress and lipid peroxidation. PRDM9 inhibition, combined with chemotherapy, resulted in strong anti-cancer efficacy in preclinical glioblastoma models, significantly enhancing the magnitude and duration of the antitumor response by eliminating persisters. These findings demonstrate a previously unknown role of PRDM9 in promoting metabolic reprogramming that enables the survival of drug-tolerant persister cells.
Proximity based proteomics reveals Git1 as a regulator of Smoothened signaling
Authors: Zhang Jingyi , Kaur Gurleen , Cai Eva , Gutierrez Oscar Torres , Liu Xiaoliang , Baboo Sabyasachi , Diedrich Jolene K , Zhu Ju-Fen , Myers Benjamin R. , Yates John R , Ge Xuecai 
Published: January 2025

ABSTRACTThe GPCR-like protein Smoothened (Smo) plays a pivotal role in the Hedgehog (Hh) pathway. To initiate Hh signaling, active Smo binds to and inhibits the catalytic subunit of PKA in the primary cilium, a process facilitated by G protein-coupled receptor kinase 2 (Grk2). However, the precise regulatory mechanisms underlying this process, as well as the events preceding and following Smo activation, remain poorly understood. To address this question, we leveraged the proximity labeling tool TurboID and conducted a time-resolved proteomic study of Smo-associated proteins over the course of Hh signaling activation. Our results not only confirmed previously reported Smo interactors but also uncovered new Smo-associated proteins. We characterized one of these new Smo interactors, Grk-interacting protein 1 (Git1), previously known to modulate GPCR signaling. We found that Git1 localizes to the base of the primary cilium, where it controls the cilium transport of Grk2, an early event in Hh signaling. Loss of Git1 impairs Smo phosphorylation by Grk2, a critical step for Smo-PKA interaction, leading to attenuated Hh signaling and reduced cell proliferation in granule neuron precursors. These results revealed a critical regulatory mechanism of Grk2 phosphorylation on Smo in the primary cilium. Our Smo-TurboID proteomic dataset provides a unique resource for investigating Smo regulations across different stages of Hh pathway activation.
Polymerase Eta Recruits FANCD2 to Common Fragile Sites to Maintain Genome Stability
Authors: Niljikar Mrunmai , Barreto-Galvez Angelica , Patel Saloni , Gagliardi Julia Elizabeth , Kumar Vasudha , Pradeep Archana , Juwarwala Aastha , Gerhardt Jeannine , Chang Yokechen , Montagna Cristina , Madireddy Advaitha 
Published: January 2025

AbstractThe replicative polymerase delta is inefficient copying repetitive DNA sequences. Error-prone translesion polymerases have been shown to switch with high-fidelity replicative polymerases to help navigate repetitive DNA. We and others have demonstrated the importance of one such translesion polymerase, polymerase Eta (pol eta), in facilitating replication at genomic regions called common fragile sites (CFS), which are difficult-to-replicate genomic regions that are hypersensitive to replication stress. However, the mechanistic basis for pol eta’s role in facilitating DNA replication at CFS and(or) at other genomic regions is currently unclear. Importantly, the functional importance of three non-catalytic domains of pol eta, the Ubiquitin-binding Zinc finger (UBZ), PCNA interacting protein (PIP) domain, and the F1 domain which mediates its switch with replicative DNA polymerases in mediating replication stress, especially at CFS loci is not clear. Here, we report that the PIP and UBZ domains of Pol Eta are both critical for its role in mediating cellular replication stress, especially at CFS. The absence of either domain induced elevated replication stress, replication stalling and DNA damage accumulation genome wide. This effect was even more pronounced at CFS loci leading to the accumulation of under replication DNA in G2/M. Importantly, while the inactivation of the UBZ domain resulted in a robust FANCD2 monoubiquitylation (a prominent marker of FANCD2 activation), FANCD2 recruitment genome wide was significantly impacted, especially at CFSs such as FRA16D. These S-phase phenotypes result in ssDNA gap formation and the persistence of under-replicated genomic regions upon transition to G2/M. While post-replicative gap filing/ repair by Mitotic DNA synthesis is activated in the mutants, it only effectively resolves UFBs in the F1* cells. The PIP*, UBZ* and pol eta−/−cells unfortunately manifest excessive toxic cytosolic DNA that instigates a strong innate immune response. These results collectively show that translesion polymerase Eta functions in a common pathway with FANCD2 to prevent replication perturbation and instability at CFS loci.
Structural insights into heterohexameric assembly of epilepsy-related ligand–receptor complex LGI1–ADAM22
Authors: Yamaguchi Takayuki , Okatsu Kei , Kubota Masato , Mitsumori Ayuka , Yamagata Atsushi , Fukata Yuko , Fukata Masaki , Shibata Mikihiro , Fukai Shuya 
Published: January 2025

AbstractLeucine-rich glioma-inactivated 1 protein (LGI1) is a secreted neuronal protein consisting of the N-terminal leucine-rich repeat (LRR) and C-terminal epitempin repeat (EPTP) domains. LGI1 is linked to epilepsy, a neurological disorder caused by genetic mutations. ADAM22 is a membrane receptor that binds to LGI1 extracellularly and interacts with AMPA-type glutamate receptors via PSD-95 intracellularly to maintain normal synaptic signal transmission. Structural analysis of the LGI1–ADAM22 complex is important for understanding the molecular mechanism of epileptogenesis and developing new therapies against epilepsy. We previously reported the crystal structure of a 2:2 complex consisting of two molecules of LGI1 and two molecules of the ADAM22 ectodomain (ECD), which is suggested to bridge neurons across the synaptic cleft. On the other hand, multiangle light scattering, small-angle X-ray scattering, and cryo-EM analyses have suggested the existence of a 3:3 complex consisting of three molecules of LGI1 and three molecules of ADAM22. In the previous cryo-EM analysis, many observed particles were in a dissociated state, making it difficult to determine the three-dimensional (3D) structure of the 3:3 complex. In this study, we stabilized the 3:3 LGI1–ADAM22ECDcomplex using chemical crosslinking and determined the cryo-EM structures of the LGI1LRR–LGI1EPTP–ADAM22ECDand 3:3 LGI1–ADAM22ECDcomplexes at 2.78 Å and 3.79 Å resolutions, respectively. Furthermore, high-speed atomic force microscopy (HS-AFM) visualized the structural features and flexibility of the 3:3 LGI1–ADAM22ECDcomplex in solution. We discuss new insights into the interaction modes of the LGI1–ADAM22 higher-order complex and the structural properties of the 3:3 LGI1–ADAM22 complex.SignificanceThe neuronal secretory protein Leucine-rich glioma-inactivated 1 (LGI1) and its receptor protein ADAM22 play a critical role in maintaining normal synaptic signal transmission. Genetic mutations in LGI1 are linked to epilepsy. Structural analysis of the LGI1–ADAM22 complex is crucial for understanding the molecular mechanisms of epileptogenesis and for developing targeted treatments. In this study, we determined the cryo-EM structure of the heterohexameric LGI1–ADAM22 complex and visualized the dynamics of this complex by high-speed atomic force microscopy.
MLC1alteration in iPSCs give rise to disease-like cellular vacuolation phenotype in the astrocyte lineage
Authors: Sharma Saumya , Bharti Vishal , Das Prosad Kumar , Rahman Abdul , Sharma Harshita , Rauthan Riya , RC Madhumita , Gupta Neerja , Shukla Rashmi , Mohanty Sujata , Kabra Madhulika , Francis Kevin R , Chakraborty Debojyoti 
Published: January 2025

ABSTRACTBackgroundMegalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare and progressive neurodegenerative disorder involving the white matter, is not adequately recapitulated by current disease models. Somatic cell reprogramming, along with advancements in genome engineering, may allow the establishment ofin-vitrohuman models of MLC for disease modeling and drug screening. In this study, we utilized cellular reprogramming and gene-editing techniques to develop induced pluripotent stem cell (iPSC) models of MLC to recapitulate the cellular context of the classical MLC-impacted nervous system.MethodsSomatic cell reprogramming of peripheral patient-derived blood mononuclear cells (PBMCs) was used to develop iPSC models of MLC. CRISPR-Cas9 system-based genome engineering was also utilized to create theMLC1knockout model of the disease. Directed differentiation of iPSCs to neural stem cells (NSCs) and astrocytes was performed in a 2D cell culture format, followed by various cellular and molecular biology approaches, to characterize the disease model.ResultsMLC iPSCs established by somatic cell reprogramming and genome engineering were well characterized for pluripotency. iPSCs were subsequently differentiated to disease-relevant cell types: neural stem cells (NSCs) and astrocytes. RNA sequencing profiling of MLC NSCs revealed a set of differentially expressed genes related to neurological disorders and epilepsy, a common clinical finding within MLC disease. This gene set can serve as a target for drug screening for the development of a potential therapeutic for this disease. Upon differentiation to the more disease relevant cell type-astrocytes, MLC-characteristic vacuoles were clearly observed, which were distinctly absent from controls. This emergence recapitulated a distinguishing phenotypic marker of the disease.ConclusionThrough the creation and analyses of iPSC models of MLC, our work addresses a critical need for relevant cellular models of MLC for use in both disease modeling and drug screening assays. Further investigation can utilize MLC iPSC models, as well as generated transcriptomic data sets and analyses, to identify potential therapeutic interventions for this debilitating disease.
Diverse ssRNA viruses associated withKarenia brevisharmful algal blooms in Southwest Florida
Authors: Lim Shen Jean , Rogers Alexandra , Rosario Karyna , Kerr Makenzie , Garrett Matt , Koester Julie , Hubbard Katherine , Breitbart Mya 
Published: January 2025

AbstractHarmful algal blooms (HABs) caused by the dinoflagellateKarenia brevisfrequently occur in the eastern Gulf of Mexico, where they negatively impact the environment, human health, and economy. Very little is known about viruses associated withK. brevisblooms, although viral infection of other HAB-forming phytoplankton species can play an important role in bloom dynamics. We used viral metagenomics to identify viruses in 11 pooled seawater samples collected from Southwest Florida, USA in 2021 during a severe, spatiotemporally dynamicK. brevisbloom. Assembled viral genomes were similar to published genomes from the orderPicornavirales, familyMarnaviridae, and generaSogarnavirus,Bacillarnavirus, andMarnarnavirus. Viruses from these groups infect bloom-forming diatoms (Chaetocerossp. andRhizosolenia setigera) and the raphidophyteHeterosigma akashiwo. We also recovered unclassifiedRiboviriagenomes related to aSymbiodiniumpositive-sense ssRNA virus sequenced from coral dinoflagellate symbionts. Reverse-transcriptase PCR assays were performed to monitor the occurrence of seven representative virus genomes in these 2021 samples and 43 seawater samples collected during a subsequent, typical bloom between November 2022 and May 2023. Over half of the samples contained multiple viruses, and at least one viral genome was detected in 44 of 54 samples, collected across seasons and years, highlighting the ubiquity of these viruses in this region. Alpha diversity was highest in the summer months and positively correlated withK. breviscell counts. Multiple regression revealed month and the presence of unclassifiedRiboviriasequences most similar to dinoflagellate viruses as significant predictors ofK. breviscellular abundance.ImportanceHarmful algal blooms caused by the dinoflagellateKarenia brevisnegatively impact the tourism, fisheries, and public health sectors. Anticipated impacts of climate change, nutrient pollution, and ocean acidification may sustain and/or exacerbateK. brevisblooms in the future, underscoring the need for proactive monitoring, communication, and mitigation strategies. This study represents a pioneering effort in monitoring viruses associated withK. brevisblooms. The findings lay the groundwork for studying the effects of environmental drivers onK. brevisblooms and their associated viruses, as well as for exploring the roles of viruses in bloom dynamics and potential applications of viruses as biocontrol agents forK. brevisblooms. Furthermore, the comparison of viral dynamics relative to local and regional bloom dynamics in this study helps inform future monitoring and modeling needs.
Fine-tuning molecular mechanics force fields to experimental free energy measurements
Authors: Rufa Dominic , Fass Joshua , Chodera John D. 
Published: January 2025

AbstractAlchemical free energy methods using molecular mechanics (MM) force fields are essential tools for predicting thermodynamic properties of small molecules, especially via free energy calculations that can estimate quantities relevant for drug discovery such as affinities, selectivities, the impact of target mutations, and ADMET properties. While traditional MM forcefields rely on hand-crafted, discrete atom types and parameters, modern approaches based on graph neural networks (GNNs) learn continuous embedding vectors that represent chemical environments from which MM parameters can be generated. Excitingly, GNN parameterization approaches provide a fully end-to-end differentiable model that offers the possibility of systematically improving these models using experimental data. In this study, we treat a pretrained GNN force field—here, espaloma-0.3.2—as afoundation simulation modelandfine-tuneits charge model using limited quantities of experimental hydration free energy data, with the goal of assessing the degree to which this can systematically improve the prediction of other related free energies. We demonstrate that a highly efficient “one-shot fine-tuning” method using an exponential (Zwanzig) reweighting free energy estimator can improve prediction accuracy without the need to resimulate molecular configurations. To achieve this “one-shot” improvement, we demonstrate the importance of using effective sample size (ESS) regularization strategies to retain good overlap between initial and fine-tuned force fields. Moreover, we show that leveraging low-rank projections of embedding vectors can achieve comparable accuracy improvements as higher-dimensional approaches in a variety of data-size regimes. Our results demonstrate that linearly-perturbative fine-tuning of foundation model electrostatic parameters to limited experimental data offers a cost-effective strategy that achieves state-of-the-art performance in predicting hydration free energies on the FreeSolv dataset.
Cleavage cascade of the sigma regulator FecR orchestrates TonB-dependent signal transduction
Authors: Yokoyama Tatsuhiko , Miyazaki Ryoji , Suzuki Takehiro , Dohmae Naoshi , Nagai Hiroki , Tsukazaki Tomoya , Kubori Tomoko , Akiyama Yoshinori 
Published: January 2025

AbstractTonB-dependent signal transduction is a versatile mechanism observed in gram-negative bacteria, integrating energy-dependent substrate transport with signal relay. InEscherichia coli, the TonB-ExbBD motor complex energizes the TonB-dependent transporter FecA, facilitating ferric citrate import. FecA also functions as a sensor, transmitting signals to the cytoplasmic membrane protein FecR. We previously demonstrated that FecR undergoes a three-step cleavage process, culminating in the activation of the cytoplasmic sigma factor FecI, which drivesfecgene transcription. Here, we describe the complete mechanism of FecR cleavage-mediated ferric citrate signaling involving FecA and TonB. The cleavage cascade begins with FecR autoproteolysis prior to membrane integration. The soluble C-terminal domain (CTD) fragment of FecR is co-translocated with the N-terminal domain (NTD) fragment through a Tat system-mediated process. In the periplasm, the interaction between the CTD and NTD fragments prevents further cleavage. This inhibition is lifted by TonB-mediated motor function, which releases the CTD, allowing the cleavage cascade to proceed. This process is essential for ferric citrate signal-induced activation offecgene expression. Our findings reveal that the regulation of FecR cleavage, relying on the TonB-FecA axis, plays a central role in bacterial response to ferric citrate signals.Significance StatementScarcity of iron, an essential element for life, has driven bacteria to evolve intricate acquisition systems, yet the molecular basis of their signal transduction mechanisms remains elusive. Unlike conventional pathways, iron transport systems employ outer membrane receptors that mediate signal transduction across both the outer and cytoplasmic membranes, powered by TonB. Using theEscherichia coliFec system, we uncovered the mechanism by which FecR’s cleavage cascade orchestrates ferric citrate signaling through TonB-mediated energy transfer. These findings shed light on similar mechanisms in gram-negative bacteria and have significant implications for understanding bacterial adaptation and pathogenesis.
DNA data (genome skims and metabarcodes) paired with chemical data demonstrate utility for retrospective analysis of forage linked to fatal poisoning of cattle
Authors: Cook Daniel , Kocurek Brandon , Stonecipher Clint , Welch Kevin D. , Gardner Dale R. , Mammel Mark , Reed Elizabeth , Ramachandran Padmini , Erickson David , Commichaux Seth , Ottesen Andrea 
Published: January 2025

AbstractPrepared and stored feeds, fodder, silage, and hay may be contaminated by toxic plants resulting in the loss of livestock. Several poisonous plants have played significant roles in livestock deaths from forage consumption in recent years in the Western United States includingSalvia reflexa. Metagenomic data, genome skims and metabarcodes, have been used for identification and characterization of plants in complex matrices including diet composition of animals, mixed forages, and herbal products. Here, chemistry, genome skims, and metabarcoding were used to retrospectively describe the composition of contaminated alfalfa hay from a case ofSalvia reflexapoisoning that killed 165 cattle. Genome skims and metabarcoding provided similar estimates of the relative abundance of theSalviain the hay samples when compared to chemical methods. Additionally, genome skims and metabarcoding provided similar estimates of species composition in the contaminated hay and rumen contents of poisoned animals. The data demonstrate that genome skims and DNA metabarcoding may provide useful tools for plant poisoning investigations.