bluuebunny/crossref_metadata_2025_split huggingface dataset:
Achieving high-resolution single-cell segmentation in convoluted cancer spheroids via Bayesian optimization and deep-learning
Authors: Mogollon Isabel , Feodoroff Michaela , Neto Pedro , Montedeoca Alba , Pietiänen Vilja , Paavolainen Lassi 
Published: September 2024

ABSTRACTUnderstanding cellular function within 3D multicellular spheroids is essential for advancing cancer research, particularly in studying cell-stromal interactions as potential targets for novel drug therapies. However, accurate single-cell segmentation in 3D cultures is challenging due to dense cell clustering and the impracticality of manual annotations. We present a high-throughput (HT) 3D single-cell analysis pipeline that combines optimized wet-lab conditions, deep learning (DL)-based segmentation models, and Bayesian optimization to address these challenges. By using live-cell nuclear and cytoplasmic dyes, we achieved clear and uniform staining of cell populations in renal cancer and immune T-cell monocultures and cocultures, improving single-cell detection in spheroids. Our pipeline integrates image preprocessing and DL models based on 3DUnet architecture, enabling robust segmentation of densely packed 3D structures. Bayesian optimization, guided by a custom objective function, was employed to refine segmentation parameters and improve quality based on biologically relevant criteria. The pipeline successfully segments cells under various drug treatments, revealing drug-induced changes not detectable by bulk conventional assays. This approach has potential for application to more complex biological samples, including, organoid co-cultures, diverse drug treatments, and integration with additional immunostaining assays, paving the way for detailed HT analyses of single-cell responses.
Exploring the sequence and structural determinants of the energy landscape from thermodynamically stable and kinetically trapped subtilisins: ISP1 and SbtE
Authors: Hood Miriam R. , Marqusee Susan 
Published: September 2024

AbstractA protein’s energy landscape, all accessible conformations, their populations, and dynamics of interconversion, is encoded in its primary sequence. While how a protein’s primary sequence encodes its native state is well understood, how sequence encodes the kinetic barriers such as unfolding and refolding is not. Here we have looked at two subtiliase homologs from theBacillus subtilis, Intracellular Subtilisin Protease 1 (ISP1) and Subtilisin E (SbtE), that are expected to have very different dynamics. As an intracellular protein, ISP1 has a small pro-domain thought to act simply as a zymogen, whereas the extracellular SbtE has a large pro-domain required for folding. The stability and kinetics of ISP1 and ProSbtE have been previously characterized. We now directly compare their energy landscapes with and without the pro-domain, examining global and local energetics of the mature proteases and the effect of each pro-domain. We find that ISP1’s pro-domain has limited impact on the energy landscape of the mature protein while without the pro-domain, SbtE is thermodynamically unstable and kinetically trapped. The pro-domains show opposite effects on the flexibility of the core of the protein: in the absence of its pro-domain, ISP1’s core becomes more flexible while SbtE’s core becomes more rigid. ISP1 contains a unique conserved insertion, which points to a potential source for these differences. These homologs are an example of how changes in the primary sequence can dramatically alter a proteins energy landscape, and highlight the need for large scale, high-throughput studies on the relationship between primary sequence and conformational dynamics.
LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation
Authors: Nalawansha Dhanusha A. , Mazis Georgios , Husemoen Gitte , Ashton Kate S. , Deng Weixian , Wurz Ryan P. , Tran Anh T. , Lanman Brian A. , Xie Jiansong , Guenette Robert G. , Li Shiqian , Smith Christopher E. , Archunan Suresh , Agnihotram Manoj K. , Sadhukhan Arghya , Kapoor Rajiv , Koirala Sajjan , De Sousa E Melo Felipe , Potts Patrick Ryan 
Published: September 2024

AbstractProximity-inducing modalities that co-opt cellular pathways offer new opportunities to regulate oncogenic drivers. Inspired by the success of proximity-based chimeras in both intracellular and extracellular target space, here we describe the development ofLYsosomeMembraneTArgetingChimeras(LYMTACs) as a novel small molecule-based platform that functions intracellularly to modulate the membrane proteome. Conceptually, LYMTACs are heterobifunctional small molecules that co-opt short-lived lysosomal membrane proteins (LMPs) as effectors to deliver targets for lysosomal degradation. We demonstrate that a promiscuous kinase inhibitor-based LYMTAC selectively targets membrane proteins for lysosomal degradation via RNF152, a short-lived LMP. To extend these findings, we show that oncogenic, membrane-associated KRASG12Dprotein can be tethered to RNF152, inducing KRAS relocalization to the lysosomal membrane, inhibiting downstream phospho-ERK signaling, and leading to lysosomal degradation of KRASG12Din a LYMTAC-dependent manner. Notably, potent cell killing could be attributed to the multi-pharmacology displayed by LYMTACs, which differentiates the LYMTAC technology from existing modalities. Thus, LYMTACs represent a proximity-based therapeutic approach that promises to expand the target space for challenging membrane proteins through targeted protein relocalization and degradation.
Modeling the antigenic evolution of seasonal influenza viruses with PREDAC for vaccine strain selection in the season of 2025
Authors: Peng Yousong , Du Xiangjun , Mi Liu , Ding Xiao , Meng Jing , Han Wenjie , Yang Lei , Huang Weijuan , Jiang Taijiao , Wang Dayan 
Published: September 2024

AbstractIntroductionThe antigenic evolution of seasonal influenza viruses have been reported to be discontinuous as clusters. Timely identification of antigenic clusters could help for influenza virus vaccine strain selections.MethodsHere, we revealed the antigenic evolution of seasonal influenza viruses, including A(H3N2), A(H1N1pdm) and B-Victoria, using our previously developed PREDAC methods, and identified antigenic clusters of seasonal influenza viruses based on existing HA protein sequences in GISAID database.ResultsDuring the last six months (2024.2∼2024.7), for the influenza A(H3N2) virus, the antigenic clusters DA21 (A/Darwin/6/2021-like and A/Massachusetts/18/2022-like) continuously dominated and nearly all viruses are antigenic similar to the current vaccine strain A/Massachusetts/18/2022; for the influenza A(H1N1pdm) virus, the antigenic cluster SY21 (A/Sydney/5/2021-like and A/Victoria/4897/2022-like) dominated and nearly all viruses are antigenic similar to the current vaccine strain A/Victoria/4897/2022; for the influenza B-Victoria, antigenic clusters AU21 (B/Austria/1359417/2021-like) dominated and nearly all viruses are antigenic similar to the current vaccine strain B/Austria/1359417/2021.ConclusionThe analysis suggests that there is no need to update the vaccine strains in the flu season of 2025 on the aspect of antigenicity.
Mechanisms of urate transport and uricosuric drugs inhibition in human URAT1
Authors: Guo Wenjun , Wei Miao , Li Yunfeng , Xu Jiaxuan , Zang Jiahe , Chen Yuezhou , Chen Lei 
Published: September 2024

AbstractHigh urate levels in circulation lead to the accumulation of urate crystals in joints and ultimately inflammation and gout1. The reabsorption process of urate in the kidney by the urate transporter URAT1 plays a pivotal role in controlling serum urate levels2. Pharmacological inhibition of URAT1 by uricosuric drugs is a valid strategy for gout management3. Despite the clinical significance of URAT1, its structure and mechanism remain elusive. Here, we report the structures of human URAT1 (hURAT1) in complex with substrate urate or inhibitors benzbromarone and verinurad at resolution ranges from 3.0 to 3.3 Å. Urate-bound hURAT1 adopts the outward-facing conformation. Urate is wrapped in the center of hURAT1 by five phenylalanines and coordinated by two positively charged residues on each side. Uricosuric compounds benzbromarone and verinurad occupy the urate-binding site of hURAT1 in the inward-facing conformation. Structural comparison between different conformations of hURAT1 reveals the rocker-switch-like mechanism for urate transport. Benzbromarone and verinurad exert their inhibitory effect by blocking not only the binding of urate but also the structural isomerization of hURAT1.
Decomposed interaction testing improves detection of genetic modifiers of the relationship of dietary omega-3 fatty acid intake and its plasma biomarkers with hsCRP in the UK Biobank
Authors: Westerman Kenneth E. , Patel Chirag J. , Meigs James B. , Chasman Daniel I. , Manning Alisa K. 
Published: September 2024

AbstractDiscovery and translation of gene-environment interactions (GxEs) influencing clinical outcomes is limited by low statistical power and poor mechanistic understanding. Molecular omics data may help address these limitations, but their incorporation into GxE testing requires principled analytic approaches. We focused on genetic modification of the established mechanistic link between dietary long-chain omega-3 fatty acid (dN3FA) intake, plasma N3FA (pN3FA), and chronic inflammation as measured by high sensitivity CRP (hsCRP). We considered an approach that decomposes the overall genetic effect modification into components upstream and downstream of a molecular mediator to increase the potential to discover gene-N3FA interactions. Simulations demonstrated improved power of the upstream and downstream tests compared to the standard approach when the molecular mediator for many biologically plausible scenarios. The approach was applied in the UK Biobank (N = 188,700) with regression models that used measures of dN3FA (based on fish and fish oil intake), pN3FA (% of total fatty acids measured by nuclear magnetic resonance), and hsCRP. Mediation analysis showed that pN3FA fully mediated the dN3FA-hsCRP main effect relationship. Next, we separately tested modification of the dN3FA-hsCRP (“standard”), dN3FA-pN3FA (“upstream”), and pN3FA-hsCRP (“downstream”) associations. The knownFADS1-3locus variant rs174535 reachedp= 1.6x10-12in the upstream discovery analysis, with no signal in the downstream analysis (p= 0.94). It would not have been prioritized based on a naïve analysis with dN3FA exposure and hsCRP outcome (p= 0.097), indicating the value of the decomposition approach. Gene-level enrichment testing of the genome-wide results further prioritized two genes from the downstream analysis,CBLL1andMICA, with links to immune cell counts and function. In summary, a molecular mediator-focused interaction testing approach enhanced statistical power to identify GxEs while homing in on relevant sub-components of the dN3FA-hsCRP pathway.
The impact of tobacco control policies on illicit tobacco trade: A scoping review
Authors: Meciar Isabel , Gartner Coral , McLay Ariel , Puljević Cheneal 
Published: September 2024

ABSTRACTIntroductionThere are concerns that progressively stringent tobacco control policies will result in increased illicit tobacco trade (ITT). We reviewed the international evidence base to identify studies examining the impact of tobacco control polices on ITT.MethodsGuided by JBI scoping review methodology, we searched six databases (PubMed, CINAHL, EMBASE, PsycINFO, Scopus, and Web of Science) in 2022 and 2024 for original research published in English since 2000. One reviewer screened titles and abstracts, and two reviewers screened full-text articles against inclusion criteria. We report the results according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews Checklist.ResultsWe identified 60 articles examining the impact of eight tobacco control policies or combination of policies on the ITT, including tobacco tax increases or decreases (n=43); menthol bans (n=5); decreasing allowable cigarette filter ventilation levels (n=1); standardised packaging (n=5); tobacco sales bans (n=1); a substantial reduction in the number of tobacco retailers (n=1); a very low nicotine content mandate (VLNC; n=2); and multicomponent policy interventions (n=4).ConclusionsThe available evidence is predominantly focused on the impact of tobacco tax increases on the ITT, with equal numbers of studies describing an increase in the ITT or no impact on the ITT, countering claims that tobacco tax increases are the primary driver of increased ITT. Evidence on the impact of other tobacco control policies on the ITT is limited. Collaborative efforts to develop accurate and consistent methods to assess the ITT are needed.ImplicationsTo our knowledge, this scoping review is the first to synthesise the available evidence on the association between multiple tobacco control policies and ITT. Findings suggest that evidence for the association between tobacco tax increases and ITT is mixed, with an equal number of studies (n=18) describing an increase or no impact of this policy on ITT. Evidence for the impact of other policies was limited; the only policies with some evidence for increasing the ITT include tobacco sales bans (n=1), a concurrent tobacco sales and smoking ban (n=2), and a hypothetical VLNC standard mandate (n=2).
Preschool musicality is associated with school-age communication abilities through genes related to rhythmicity
Authors: de Hoyos Lucía , Verhoef Ellen , Okbay Aysu , Vermeulen Janne R , Figaroa Celeste , Lense Miriam , Fisher Simon E , Gordon Reyna L , Pourcain Beate St 
Published: September 2024

AbstractEarly-life musical engagement is an understudied but developmentally important and heritable precursor of later (social) communication and language abilities. This study aims to uncover the aetiological mechanisms linking musical to communication abilities. We derived polygenic scores (PGS) for self-reported beat synchronisation abilities (PGSrhythmicity) in children (N≤6,737) from the Avon Longitudinal Study of Parents and Children and tested their association with preschool musical (0.5-5 years) and school-age (social) communication and cognition-related abilities (9-12 years). We further assessed whether relationships between preschool musicality and school-age communication are shared through PGSrhythmicity, using structural equation modelling techniques. PGSrhythmicitywere associated with preschool musicality (Nagelkerke-R2=0.70-0.79%), and school-age communication and cognition-related abilities (R2=0.08-0.41%), but not social communication. We identified links between preschool musicality and school-age speech-and syntax-related communication abilities as captured by known genetic influences underlying rhythmicity (shared effect β=0.0065(SE=0.0021),p=0.0016), above and beyond general cognition, strengthening support for early music intervention programmes.
Leveraging CRISPR-Cas13d in an inducible knockdown system to interrogateDrosophilagerm granule mRNAs
Authors: Gauhar Zoya A. , Myhrvold Cameron , Gavis Elizabeth R. 
Published: September 2024

ABSTRACTRibonucleoprotein (RNP) germ granules are hallmarks of germ cells across the animal kingdom and are thought to be hubs for post-transcriptional regulation that promote formation of the germ cell precursors. While numerous RNAs are associated with germ granules inDrosophila, the functions of many in germline development are poorly understood. Current methods for RNA knockdown, such as RNAi, do not allow local depletion of transcripts such as those found in the germ granules. We leveraged CRISPR-Cas13 to create a subcellular RNA knockdown system and tested it on two mRNAs,nanos (nos)andsarah (sra), whose abundance in germ granules differs. Because Cas13 has bothcisandtranscleavage activities, we evaluated the effect of target abundance on off-target RNA depletion. We show on and off-target RNA depletion is coupled when targeting the more abundantnosgerm granule transcripts. Off-target RNA knockdown is less potent when the system is used for less abundantsratranscripts. Whensrais knocked down in germ granules, we observe defective primordial germ cell migration, and an increase in the calcium indicator GCaMP at the posterior, consistent withsraencoding a negative regulator of calcium signaling. In sum, we report anin vivoCas13-based system for subcellular knockdown, evaluate its feasibility, and uncover a novel function forsragerm granule transcripts in promoting germline development.
Comparing newly developed SNP barcode panels with microsatellites to explore population genetics of malaria parasites in the Peruvian Amazon
Authors: Cabrera-Sosa Luis , Safarpour Mahdi , Kattenberg Johanna H. , Ramirez Roberson , Vinetz Joseph , Rosanas-Urgell Anna , Gamboa Dionicia , Delgado-Ratto Christopher 
Published: September 2024

AbstractMalaria molecular surveillance (MMS) can provide insights into transmission dynamics, guiding national control/elimination programs. Considering the genetic differences among parasites from different areas in the Peruvian Amazon, we previously designed SNP barcode panels forPlasmodium vivax(Pv) andP. falciparum(Pf), integrated into AmpliSeq assays, to provide population genetics estimates of malaria parasites. These AmpliSeq assays are ideal for MMS: multiplexing different traits of interest, applicable to many use cases, and high throughput for large numbers of samples. The present study compares the genetic resolution of the SNP barcode panels in the AmpliSeq assays with widely used microsatellite (MS) panels to investigate Amazonian malaria parasites.Malaria samples collected in remote areas of the Peruvian Amazon (51 Pv & 80 Pf samples) were characterized using the Ampliseq assays and MS. Population genetics estimates (complexity of infection, genetic diversity and differentiation, and population structure) were compared using the SNP barcodes (Pv: 40 SNPs & Pf: 28 SNPs) and MS panels (Pv: 16 MS & Pf: 7 MS).The genetic diversity of Pv (expected heterozygosity,He) was similar across the subpopulations for both makers:HeMS= 0.68 - 0.78 (p = 0.23) andHeSNP= 0.36 - 0.38 (p = 0.80). Pairwise genetic differentiation (fixation index, FST) was also comparable: FST-MS= 0.04 - 0.14 and FST-SNP= 0.03 - 0.12 (p = 0.34 – 0.85). No geographic clustering was observed with any panel. In addition, Pf genetic diversity trends (HeMS= 0 – 0.48 p = 0.03 – 1;HeSNP= 0 - 0.09, p = 0.03 – 1) and pairwise FSTcomparisons (FST-MS= 0.14 – 0.65, FST-SNP= 0.19 – 0.61, p = 0.24 – 0.83) were concordant between the panels. Similar population structure clustering was observed with both SNP and MS, highlighting one Pf subpopulation in an indigenous community.The SNP barcodes in the Pv AmpliSeq v2 Peru and Pf AmpliSeq v1 Peru assays offer comparable results to MS panels when investigating population genetics in Pv and Pv populations. Therefore, the AmpliSeq assays can efficiently characterize malaria transmission dynamics and population structure and support malaria elimination efforts in Peru.
Osterix Facilitates Osteocytic Communication by Targeting Connexin43
Authors: Wu Zuping , Chen Qian , Gao Qian , Liang Muchun , Zhou Yumeng , Zhu Li , Wang Jiahe , Shen Yang , Jing Junjun , Xie Jing , Liu Xiaoheng , Zou Shujuan , Zhang Demao , Zhou Chenchen 
Published: September 2024

AbstractOsteocytes, terminal-differentiated cells in bone, are now considered as more pivotal regulators of mature bone homeostasis than other bone cells, since they constitute 90- 95% of the bone cell population. Given their non-migratory nature within the mineralized matrix, their unique dendrites are crucial for cell-to-cell communication in response to both intracellular and extracellular stimuli, such as bone fracture or mechanical load. Here, we showed that Osterix (Osx), usually recognized as a specific doorkeeper for osteoblast differentiation during new bone formation marked by collagen type I α 1 (Col1α1), was unexpectedly co-expressed with Col1α1 in osteocytes within the cortical bone of mice. Deleting Osx in Col1α1-positive osteocytes disrupted cortical bone structure and osteocytic dendrites in mice, thus impairing transcellular fluid flow and intercellular communication. Conversely, overexpression of Osx in osteocytes enhanced these processes. Furthermore, we identified Connexin43, a critical protein of gap junction channel, was a direct transcriptional target of Osx in regulating dendrites of osteocytes. Pharmacological restoration of Connexin43 levels rescued the dysfunction in Osx-deficient osteocytes both in vitro and in vivo. Taken together, this work demonstrated Osx’s distinct role in osteocyte function through maintaining intercellular signaling, which broadened the current understanding of its role in Col1α1-positive bone cells, extending beyond osteoblasts and bone mineralization, offering new insights into bone diseases such as fracture nonunion or disuse osteoporosis.
Long-term protein synthesis with PURE in a mesoscale dialysis system
Authors: Julià Laura Roset , Grasemann Laura , Stellacci Francesco , Maerkl Sebastian J. 
Published: September 2024

AbstractCell-free systems are powerful tools in synthetic biology with versatile and wide-ranging applications. However, a significant bottleneck for these systems, particularly the PURE cell-free system, is their limited reaction lifespan and yield. Dialysis offers a promising approach to prolong reaction lifetimes and increase yields, yet most custom dialysis systems require access to sophisticated equipment like 3D printers or microfabrication tools. In this study, we utilized an easy-to-assemble, medium-scale dialysis system for cell-free reactions using commercially available components. By employing dialysis with periodic exchange of the feeding solution, we achieved a protein yield of 1.16 mg/mL GFP in the PURE system and extended protein synthesis for at least 12.5 consecutive days, demonstrating the system’s excellent stability.
Comparative analysis of uncoupled succinate production by the FeII/2-oxoglutarate-dependent dioxygenases
Authors: Das Susmita , Ranjan Saumya , Keerthana Carmel , Seenivasan Gayathri , Tuti Nikhil , Shaji Unnikrishnan P , Meur Gargi , Anindya Roy 
Published: September 2024

AbstractNon-heme iron (FeII) and 2-oxoglutarate(2OG)-dependent dioxygenases catalyse a diverse array of biological reactions. These enzymes couple the oxidative decarboxylation of 2OG to the hydroxylation of the substrates. However, in the absence of the substrate, oxidative decarboxylation of 2OG generates succinate. We have determined succinate level by using succinyl-CoA synthetase to monitor this uncoupled decarboxylation of FeII/2OG-dependent dioxygenases and measured the uncoupled 2OG turnover of different FeII/2OG-dependent dioxygenases. We also performed comparative analysis and verified the functionality of human dioxygenase ALKBH6.