bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of Machine Learning-Assisted Directed Evolution Across Diverse Combinatorial Landscapes
Authors: Li Francesca-Zhoufan , Yang Jason , Johnston Kadina E. , Gürsoy Emre , Yue Yisong , Arnold Frances H. 
Published: October 2024

SummaryVarious machine learning-assisted directed evolution (MLDE) strategies have been shown to identify high-fitness protein variants more efficiently than typical wet-lab directed evolution approaches. However, limited understanding of the factors influencing MLDE performance across diverse proteins has hindered optimal strategy selection for wet-lab campaigns. To address this, we systematically analyzed multiple MLDE strategies, including active learning and focused training using six distinct zero-shot predictors, across 16 diverse protein fitness landscapes. By quantifying landscape navigability with six attributes, we found that MLDE offers a greater advantage on landscapes which are more challenging for directed evolution, especially when focused training is combined with active learning. Despite varying levels of advantage across landscapes, focused training with zero-shot predictors leveraging distinct evolutionary, structural, and stability knowledge sources consistently outperforms random sampling for both binding interactions and enzyme activities. Our findings provide practical guidelines for selecting MLDE strategies for protein engineering.
A new screening assay reveals that aminoglycoside antibiotics interfere with the Tau/MDM2 interaction
Authors: Sola Martina , Ciccaldo Matteo , Paganetti Paolo , Papin Stéphanie 
Published: October 2024

AbstractMAPTgene mutations cause some neurodegenerative tauopathies and theMAPT-encoded protein Tau is deposited in neurofibrillary tangles, hallmarks of this disease family. In addition to its canonical function in regulating microtubule dynamics, Tau modulates chromatin compaction, gene expression, and the cellular response to DNA damage. During the DNA damage response, Tau positively modulates P53 by binding to MDM2 thereby preventing P53 inactivation and degradation. The aberrant presence of both MDM2 associated to neurofibrillary tangles and of P53 misfolding in brains affected by neurodegenerative diseases suggests that the sequestration of MDM2 may prevent P53 clearance in tauopathies and so contribute to progressive neuronal dysfunction and death. Following this evidence, a pharmacological inhibition of the Tau/MDM2 interaction may represent a viable strategy to reduce P53-dependent cell damage in brain disorders. With the screening FDA-approved drugs and natural compounds, we discovered that members of the aminoglycoside antibiotic family antagonize the Tau/MDM2 interaction. The use of these reagents may advance the understanding of the implication of the Tau/MDM2/P53 axis in neurodegeneration models. However, their unfavorable pharmacokinetic properties may limit their systemic use when targeting the brain.
GABAergic neurons can facilitate the propagation of cortical spreading depolarization: experiments in mouse neocortical slices and a novel neural field computational model
Authors: Baspinar Emre , Simonti Martina , Srour Hadi , Desroches Mathieu , Avitabile Daniele , Mantegazza Massimo 
Published: October 2024

AbstractCortical spreading depolarization (CSD) is a wave of depolarization with local onset and extended propagation implicated in several pathological conditions. Its mechanisms have been extensively investigated, including our recent studies showing with experimental and computational approaches that the hyperactivity of GABAergic neurons can initiate migraine-related CSD because of spiking-generated extracellular potassium (K+) build-up. However, less is known about the role played by GABAergic neurons in CSD propagation. Here we studied mechanisms of CSD propagation, focusing on the role of GABAergic neurons, with experiments performed in mouse brain slices and with a new spatially extended neural field computational model. Experimentally, we induced CSD by applying brief puffs of potassium chloride (KCl) in somatosensory cortex slices from wild type and VGAT-ChR2-tdtomato mice, which specifically express the excitatory opsin channelrhodopsin (ChR2) in GABAergic neurons. We evaluated the role of GABAergic neurons in CSD propagation by modulating their activity with optogenetic illumination and their synaptic connections with pharmacological tools. We have developed the computational model to obtain realistic simulations of both initiation and propagation of CSD. It includes large populations of interconnected excitatory and inhibitory neurons, as well as the effect of extracellular ion concentrations on their features. We found that the decrease of the synaptic activity of GABAergic neurons can enhance CSD propagation, because of the reduction of the inhibitory synaptic weight, whereas their spiking activity can enhance CSD propagation because of extracellular K+upload. However, differently from for CSD initiation, the latter effect is normally hidden by the action of GABAergic synaptic transmission. A reduction of GABAergic synaptic transmission, which can be observed in pathological states, can reveal the potentiating effect of the K+upload induced by GABAergic activation. The neural field model that we implemented can generate accurate simulations of CSD, providing testable hypotheses on mechanisms, and can also be used for modeling other (patho)-physiological activities of neuronal networks.
Nanoparticles with Curcumin and Piperine Modulate Steroid Biosynthesis in Prostate Cancer
Authors: Yakubu Jibira , Natsaridis Evangelos , du Toit Therina , Barata Isabel Sousa , Tagit Oya , Pandey Amit V. 
Published: October 2024

AbstractEndogenous androgens are pivotal in the development and progression of prostate cancer (PC). We investigated nanoparticle formulations of curcumin and piperine in modulating steroidogenesis within PC cells. Using multiple PC cell lines (LNCaP, VCaP, DU145 and PC3) we studied the effects of curcumin, piperine, and their nanoparticle formulations—curcumin nanoparticles, piperine nanoparticles, and curcumin-piperine nanoparticles (CPN)—on cell viability, migration, and steroid biosynthesis. Curcumin and its nanoparticle formulations significantly reduced cell viability in PC cells, with curcumin-piperine nanoparticles showing the highest efficacy. These treatments also inhibited cell migration, with CPN exhibiting the most pronounced effect. In assays for steroid biosynthesis, curcumin and its nanoparticle formulations, as well as piperine and its nanoparticles, selectively inhibited 17α-hydroxylase and 17,20-lyase activities of cytochrome P450 17A1 (CYP17A1). Abiraterone, a CYP17A1 inhibitor, displayed a broader inhibition of steroid metabolism including cytochrome P450 21-hydroxylase activity, whereas curcumin and piperine provided a more targeted inhibition profile. Analysis of steroid metabolites by liquid chromatography-mass spectrometry revealed that CPN caused significant reduction of androstenedione and cortisol, suggesting potential synergistic effects. In conclusion, nanoformulations co-loaded with curcumin and piperine offer an effective approach to targeting steroidogenesis and could be promising candidates for therapies aimed at managing androgen-dependent PC.
FtsK is Critical for the Assembly of the Unique Divisome Complex of the FtsZ-lessChlamydia trachomatis
Authors: Harpring McKenna , Lee Junghoon , Zhong Guangming , Ouellette Scot P. , Cox John V. 
Published: October 2024

AbstractChlamydia trachomatisserovar L2(Ct),an obligate intracellular bacterium that does not encode FtsZ, divides by a polarized budding process. In the absence of FtsZ, we show that FtsK, a chromosomal translocase, is critical for divisome assembly inCt. Chlamydial FtsK forms discrete foci at the septum and at the base of the progenitor mother cell, and our data indicate that FtsK foci at the base of the mother cell mark the location of nascent divisome complexes that form at the site where a daughter cell will emerge in the next round of division. The divisome inCthas a hybrid composition, containing elements of the divisome and elongasome from other bacteria, and FtsK is recruited to nascent divisomes prior to the other chlamydial divisome proteins assayed, including the PBP2 and PBP3 transpeptidases, and MreB and MreC. Knocking down FtsK prevents divisome assembly inCtand inhibits cell division and septal peptidoglycan synthesis. We further show that MreB does not function like FtsZ and serve as a scaffold for the assembly of theCtdivisome. Rather, MreB is one of the last proteins recruited to the chlamydial divisome, and it is necessary for the formation of septal peptidoglycan rings. Our studies illustrate the critical role of chlamydial FtsK in coordinating divisome assembly and peptidoglycan synthesis in this obligate intracellular bacterial pathogen.
TIR signaling activates caspase-like immunity in bacteria
Authors: Rousset François , Osterman Ilya , Scherf Tali , Falkovich Alla H. , Leavitt Azita , Amitai Gil , Shir Sapir , Malitsky Sergey , Itkin Maxim , Savidor Alon , Sorek Rotem 
Published: October 2024

AbstractProteases of the caspase family, as well as Toll/Interleukin-1 Receptor (TIR)-domain proteins, have central roles in innate immunity and regulated cell death in humans. In this study we describe a bacterial immune system comprising both a caspase-like protease and a TIR-domain protein. We found that the TIR protein, once it recognizes phage invasion, produces the previously unknown immune signaling molecule ADP-cyclo[N7:1′′]-ribose (N7-cADPR). This molecule specifically activates the bacterial caspase-like protease which then indiscriminately degrades cellular proteins to halt phage replication. The TIR-caspase defense system, which we denote as type IV Thoeris, is abundant in bacteria and efficiently protects against phage propagation. Our study highlights the diversity of TIR-produced immune signaling molecules and demonstrates that cell death regulated by proteases of the caspase family is an ancient mechanism of innate immunity.
Virulence regulates and boosts CRISPR-Cas9 immunity in Group BStreptococcus
Authors: Pastuszka Adeline , Mazzuoli Maria-Vittoria , Crestani Chiara , Deborde Léonie , Sismeiro Odile , Lemaire Coralie , Rong Vanessa , Gominet Myriam , Jacquemet Elise , Legendre Rachel , Lanotte Philippe , Firon Arnaud 
Published: October 2024

AbstractCRISPR-Cas9 immune systems protect bacteria from foreign DNA. However, immune efficiency is constrained by Cas9 off-target cleavages and toxicity. How bacteria regulate Cas9 to maximize protection while preventing autoimmunity is not understood. Here, we show that the master regulator of virulence, CovR, regulates CRISPR-Cas9 immunity against mobile genetic elements inStreptococcus agalactiae, a pathobiont responsible for invasive neonatal infections. We show that CovR binds to and represses a distal promoter of thecasoperon, integrating immunity within the virulence regulatory network. The CovR-regulated promoter provides a controlled increase in off-target cleavages to counteract mutations in the target DNA, restores the potency of old immune memory, and stimulates the acquisition of new memory in response to recent infections. Regulation of Cas9 by CovR is conserved at the species level, with lineage specificities suggesting different adaptive trajectories. Altogether, we describe the coordinated regulation of immunity and virulence that enhances the bacterial immune repertoire during host-pathogen interaction.
Oncogenic Alterations, Race, and Survival in US Veterans with Metastatic Prostate Cancer Undergoing Somatic Tumor Next Generation Sequencing
Authors: Valle Luca F. , Li Jiannong , Desai Heena , Hausler Ryan , Haroldsen Candace , Chatwal Monica , Ojo Matthias , Kelley Michael J. , Rebbeck Timothy , Rose Brent S. , Rettig Matthew B. , Nickols Nicholas G. , Garraway Isla P. , Yamoah Kosj , Maxwell Kara N. 
Published: October 2024

ABSTRACTPurposeNational guidelines recommend next generation sequencing (NGS) of tumors in patients diagnosed with metastatic prostate cancer (mPCa) to identify potential actionable alterations. We sought to describe the spectrum and frequency of alterations in PCa-related genes and pathways, as well as associations with self-identified race/ethnicity, and overall survival in US Veterans.Patients and MethodsThis retrospective cohort study included Non-Hispanic Black (NHB) and Non-Hispanic white (NHW) Veterans with mPCa who obtained NGS through the Veterans Affairs National Precision Oncology Program. 45 genes in seven canonical or targetable mPCa pathways were evaluated in addition to TMB and MSI status. Multivariable logistic regression evaluated associations between race/ethnicity and genomic alteration frequencies. Cox proportional hazards models were used to determine associations between race/ethnicity, specific gene/pathway alteration, and overall survival.Results5,015 Veterans with mPCa who had NGS conducted were included (1,784 NHB, 3,231 NHW). NHB Veterans were younger, had higher PSA at diagnosis, were less likely to report Agent Orange exposure, and resided in more deprived neighborhoods compared to NHW Veterans. Nine of the top ten most commonly altered genes were the same in NHB v NHW Veterans; however, the frequencies of alterations varied by race/ethnicity. NHB race/ethnicity was associated with higher odds of genomic alterations inSPOP(OR 1.7 [1.2-2.6]) as well as immunotherapy targets (OR 1.7 [1.1-2.7]) including MSI high status (OR 3.1 [1.1-9.4]).Furthermore, NHB race/ethnicity was significantly associated with lower odds of genomic alterations in the AKT/PI3K pathway (OR 0.6 [0.4-0.7]), AR axis (OR 0.7 [0.5-0.9]), and tumor suppressor genes (OR 0.7 [0.5-0.8]). Cox proportional hazards modelling stratified by race/ethnicity demonstrated alterations in tumor suppressor genes includingTP53were associated with shorter OS in both NHB (HR 1.54 [1.13-2.11] and NHW individuals (HR 1.52 [1.25-1.85]).ConclusionIn the equal access VA healthcare setting, Veterans undergoing NGS for mPCa exhibited differences in alteration frequencies in both actionable and non-actionable pathways that may be associated with survival. This analysis affirms the utility of genomic testing for identifying candidates irrespective of race/ethnicity for precision oncology treatments, which could contribute to equitable outcomes in patients with mPCa.
A framework for target discovery in rare cancers
Authors: Li Bingchen , Sadagopan Ananthan , Li Jiao , Wu Yuqianxun , Cui Yantong , Konda Prathyusha , Weiss Cary N. , Choueiri Toni K. , Doench John G. , Viswanathan Srinivas R. 
Published: October 2024

AbstractWhile large-scale functional genetic screens have uncovered numerous cancer dependencies, rare cancers are poorly represented in such efforts and the landscape of dependencies in many rare cancers remains obscure. We performed genome-scale CRISPR knockout screens in an exemplar rare cancer,TFE3-translocation renal cell carcinoma (tRCC), revealing previously unknown tRCC-selective dependencies in pathways related to mitochondrial biogenesis, oxidative metabolism, and kidney lineage specification. To generalize to other rare cancers in which experimental models may not be readily available, we employed machine learning to infer gene dependencies in a tumor or cell line based on its transcriptional profile. By applying dependency prediction to alveolar soft part sarcoma (ASPS), a distinct rare cancer also driven byTFE3translocations, we discovered and validated thatMCL1represents a dependency in ASPS but not tRCC. Finally, we applied our model to predict gene dependencies in tumors from the TCGA (11,373 tumors; 28 lineages) and multiple additional rare cancers (958 tumors across 16 types, including 13 distinct subtypes of kidney cancer), nominating potentially actionable vulnerabilities in several poorly-characterized cancer types. Our results couple unbiased functional genetic screening with a predictive model to establish a landscape of candidate vulnerabilities across cancers, including several rare cancers currently lacking in potential targets.
Substrate tunnel regulates substrate specificity switching in hyaluronan synthase
Authors: Zhu Kaiyi , Han Yilei , Jian Yupei , Jiang Guoqiang , Lu Diannan , Wu Jianzhong , Liu Zheng 
Published: October 2024

AbstractHyaluronic acid (HA), a ubiquitous linear polysaccharide in the extracellular matrix of vertebrates and the capsule of certain pathogenic bacteria, is characterized by its unique structure of alternating β-1,3-N-acetylglucosamine and β-1,4-glucuronic acid units. HA is synthesized by Class I hyaluronan synthase (HAS) in the majority of organisms, but the molecular mechanisms determining the alternating sequence of HA remain unclear. Here, we demonstrated that HA synthesizedin vitrousingStreptococcus equisimilisHAS maintained its alternating sequence, regardless of substrate ratios and concentrations. While the active pocket lacks intrinsic selectivity, a distal allosteric regulation from the transmembrane tunnel is revealed. The lingering HA terminals in the active pocket ensure lower binding energies for alternating substrates through Coulombic repulsion and competitive binding. Moreover, the dynamic C-terminal loop at the substrate tunnel entrance reduces transport distance for alternating substrates. Our findings indicate that HA-substrate interactions and enzyme dynamics in the substrate tunnel collectively govern the alternating substrate specificity of HAS. This study provides the first molecular insight into precise substrate specificity control within a single active pocket during template-independent polymer synthesis.
A high throughput single molecule platform to study DNA supercoiling effect on protein-DNA interactions
Authors: Lee Huijin , Hwang Jihee , Rashid Fahad , London James A. , Fishel Richard , Berger James M. , Myong Sua , Ha Taekjip 
Published: October 2024

ABSTRACTDNA supercoiling significantly influences DNA metabolic pathways. To examine its impact on DNA-protein interactions at the single-molecule level, we developed a highly efficient and reliable protocol to modify plasmid DNA at specific sites, allowing us to label plasmids with fluorophores and biotin. We then induced negative and positive supercoiling in these plasmids using gyrase and reverse gyrase, respectively. Comparing supercoiled DNA with relaxed circular DNA, we assessed the effects of supercoiling on CRISPR-Cas9 and mismatch repair protein MutS. We found that negative DNA supercoiling exacerbates off-target effects in DNA unwinding by Cas9. For MutS, we observed both negative and positive DNA supercoiling enhances the binding interaction between MutS and a mismatched base pair but does not affect the rate of ATP-induced sliding clamp formation. These findings not only underscore the versatility of our protocol but also opens new avenues for exploring the intricate dynamics of protein-DNA interactions under the influences of supercoiling.
Alanine Scanning to Define Membrane Protein-Lipid Interaction Sites Using Native Mass Spectrometry
Authors: Jayasekera Hiruni S. , Mohona Farhana Afrin , De Jesus Madison J. , Miller Katherine M. , Marty Michael T. 
Published: October 2024

AbstractLipids surrounding membrane proteins interact with different sites on the protein at varying specificities, ranging from highly specific to weak interactions. These interactions can modulate the structure, function, and stability of membrane proteins. Thus, to better understand membrane protein structure and function, it is important to identify the locations of lipid binding and the relative specificities of lipid binding at these sites. In our previous native mass spectrometry (MS) study, we developed a single and double mutant analysis approach to profile the contribution of specific residues toward lipid binding. Here, we extend this method by screening a broad range of mutants of AqpZ to identify specific lipid binding sites and by measuring binding of different lipid types to measure the selectivity of different lipids at selected binding sites. We complemented these native MS studies with molecular dynamics (MD) simulations to visualize lipid interactions at selected sites. We discovered that AqpZ is selective towards cardiolipins (CL) but only at specific sites. Specifically, CL orients with its headgroup facing the cytoplasmic side, and its acyl chains interact with a hydrophobic pocket located at the monomeric interface within the lipid bilayer. Overall, this integrative approach provides unique insights into lipid binding sites and the selectivity of various lipids towards AqpZ, enabling us to map the AqpZ protein structure based on the lipid affinity.
Elucidating the metabolic roles of isoflavone synthase-mediated protein-protein interactions in yeast
Authors: Liu Chang , Han Jianing , Li Sijin 
Published: October 2024

AbstractTransient plant enzyme complexes formed via protein-protein interactions (PPIs) play crucial regulatory roles in secondary metabolism. Complexes assembled on cytochrome P450s (CYPs) are challenging to characterize metabolically due to difficulties in decoupling the PPIs’ metabolic impacts from the CYPs’ catalytic activities. Here, we developed a yeast-based synthetic biology approach to elucidate the metabolic roles of PPIs between a soybean-derived CYP, isoflavone synthase (GmIFS2), and other enzymes in isoflavonoid metabolism. By reconstructing multiple complex variants with an inactive GmIFS2 in yeast, we found that GmIFS2-mediated PPIs can regulate metabolic flux between two competing pathways producing deoxyisoflavonoids and isoflavonoids. Specifically, GmIFS2 can recruit chalcone synthase (GmCHS7) and chalcone reductase (GmCHR5) to enhance deoxyisoflavonoid production or GmCHS7 and chalcone isomerase (GmCHI1B1) to enhance isoflavonoid production. Additionally, we identified and characterized two novel isoflavoneO-methyltransferases interacting with GmIFS2. This study highlights the potential of yeast synthetic biology for characterizing CYP-mediated complexes.
scMoE: single-cell mixture of experts for learning hierarchical, cell-type-specific, and interpretable representations from heterogeneous scRNA-seq data
Authors: Huang Michael , Li Yue 
Published: October 2024

AbstractAdvancements in single-cell transcriptomics methods have resulted in a wealth of single-cell RNA sequencing (scRNA-seq) data. Methods to learn cell representation from atlas-level scRNA-seq data across diverse tissues can shed light into cell functions implicated in diseases such as cancer. However, integrating large-scale and heterogeneous scRNA-seq data is challenging due to the disparity of cell-types and batch effects. We present single-cell Mixture of Expert (scMoE), a hierarchical mixture of experts single-cell topic model. Our key contributions are the cell-type specific experts, which explicitly aligns topics with cell-types, and the integration of hierarchical cell-type lineages and domain knowledge. scMoE is both transferable and highly interpretable. We benchmarked our scMoE’s performance on 9 single-cell RNA-seq datasets for clustering and 3 simulated spatial datasets for spatial deconvolution. We additionally show that our model, using single-cell references, yields meaningful biological results by deconvolving 3 cancer bulk RNA-seq datasets and 2 spatial transcriptomics datasets. scMoE is able to identify cell-types of survival importance, find cancer subtype specific deconvolutional patterns, and capture meaningful spatially distinct cell-type distributions.