bluuebunny/crossref_metadata_2025_split huggingface dataset:
Orchestrating Improbable Chemistries: Structural Snapshots of B12-Dependent Methionine Synthase’s Catalytic Choreography
Authors: Mendoza Johnny , Yamada Kazuhiro , Castillo Carmen , Wilhelm Catherine A. , Koutmos Markos 
Published: August 2024

AbstractCobalamin (vitamin B12) and its derivatives play an essential role in biological methylation, with cobalamin-dependent methionine synthase (MS) serving as a canonical example. MS catalyzes multiple methyl transfers within a single, dynamic multi-domain architecture that has proven challenging to study, hampering efforts to elucidate its catalytic mechanism(s). Utilizing a thermostable MS homolog and non-native cobalamin cofactors, we have captured crystal structures of transient conformational states of MS, including those directly involved in folate demethylation and homocysteine methylation. These snapshots reveal the mechanistic significance of five-coordinate, His-off methylcobalamin in homocysteine methylation and highlight the crucial role of the folate-binding domain and interdomain linkers in orchestrating the intricate structural rearrangements required for catalysis. This expanded conformational ensemble, including the unexpected capture of novel ‘Cap-on’ conformations, underscores the remarkable plasticity of MS, exceeding previous estimations. Our findings provide crucial insights into the catalytic mechanism of MS, laying the foundation for harnessing cobalamin’s biocatalytic potential and elucidating how nature exploits protein dynamics to facilitate complex transformations.
Imaging the interactions of chimeric antigen receptor-expressing T-cells with colorectal cancer cells
Authors: Banik Debasis , Ward Christopher , Zhang Ziwei , Heraghty Daniel , Suresh Prasanna , Li Bing , Kedia Shekhar , Davis Simon J. , Roy James P. , Chapman Michael A. , Mahata Bidesh , Klenerman David 
Published: August 2024

ABSTRACTChimeric antigen receptor (CAR) T-cell therapy has shown unprecedented success in haematological cancers but faces challenges in solid tumours. Although carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is differentially expressed in many solid tumours, CEACAM5 CAR T-cells are ineffective. Here, we have studied the interaction of CEACAM5 targeting CAR primary T-cells with colorectal cancer (CRC) cells using fluorescence microscopy. We find that CRC cells’ glycocalyx is much thicker than the CAR T-cell and likely contributes to immune-escape. Oscillating calcium flux, a signature of non-sustained triggering and decreased killing, was observed when CAR T-cells interacted with CRC, which increased with increasing cell-seeding time. This was because CEACAM5 became increasingly unavailable on the CRC cell monolayer, as revealed by fluorescence imaging. Local proteolytic treatment with trypsin to disrupt the CRC cell monolayer, using a micropipette, increased CEACAM5 availability, decreased glycocalyx thickness, and restored sustained CAR T-cell calcium fluxes, increasing the killing of CRC cells. Our results reveal why CAR T-cells targeting CEACAM5 are ineffective and suggest possible routes for improved therapy.One Sentence SummaryCEACAM5 unavailability and the thick CRC cell glycocalyx are major barriers to CAR T-cells, which local proteolysis helps to overcome.
Development of a Robust Gel-Free 3D Culture System for Generating Spheroids from Axolotl Blastema Cells
Authors: Aladağ Zeynep , Vatandaşlar Emre , Vilain Sven , Öztürk Gürkan 
Published: August 2024

AbstractRegenerative biology is a pivotal field aimed at understanding and harnessing the ability of organisms to restore damaged tissues and organs. The axolotl (Ambystoma mexicanum) is a key model organism renowned for its exceptional regenerative capabilities, particularly in limb regeneration. However, currentin vitroresearch is hampered by the lack of well-defined axolotl blastema cell lines and unreliable primary culture protocols. To address these challenges, we present a novel, robust, gel-free 3D culture system for axolotl blastema cells. This system overcomes limitations of 2D culture methods by enabling the formation of spheroid structures that closely mimic thein vivoenvironment. Using this protocol, we observed that spheroids derived from axolotl blastema tissues retained key regenerative markers, including PRRX1, and exhibited stable expression of crucial blastema markers through extended culture periods. Our approach facilitates the study of cellular and molecular mechanisms underlying limb regeneration in axolotls and provides a valuable tool for drug testing and regenerative research. The development of this 3D culture system represents a significant advancement in regenerative biology, offering a more consistent and reliable model for exploring the regenerative potential of axolotl cells.
Enhanced Salt Tolerance inSynechocystissp. PCC 6803 Through Adaptive Evolution: Mechanisms and Applications for Environmental Bioremediation
Authors: Zhu Xiaofei , Zou Rongsong , Liu Dailin , Liu Jing , Wu Xuejing , Chen Lei , Sun Tao , Zhang Weiwen 
Published: August 2024

AbstractSalt stress is common in natural environments, where elevated salt levels in brackish water and saline soil can hinder the growth of organisms, thereby exacerbating environmental challenges. Developing salt-tolerant organisms not only uncovers novel mechanisms of salt tolerance but also lays the groundwork for managing and utilizing saline environments. Cyanobacteria, which are widely distributed in hydrosphere and soil, serve as ideal models for studying salt stress. In this study, the model cyanobacteriumSynechocystissp. PCC 6803 was selected, whose salt (NaCl) tolerance improved from 4.0% to 6.5% (m/v) through adaptive laboratory evolution. Genome re-sequencing and mutant analysis identified six key genes associated with salt tolerance. Notably, the deletion ofslr1670, which encodes glycerol glucoside hydrolase, improved the strain’s salt tolerance. In addition,slr1753encodes a membrane protein that may enhance salt tolerance by facilitating ion transport to the extracellular space. Further analysis revealed that overexpression ofslr1753significantly accumulates Na+on the cell surface, enabling effective seawater treatment using the engineered strain, resulting in a 6.35% reduction of Na+in the seawater. Moreover, the adapted bacteria can be used for the remediation of saline soil samples, leading to a 184.2% and 43.8% increase in the germination rate and average height ofBrassica rapa chinensis, respectively, along with a 25.3% rise in total organic carbon content and reductions in both total salt content by 1.82% and pH by 1.91% in soil. This study provides novel insights into salt tolerance mechanisms and the bioremediation of high-salinity environments.Graphical abstract
Inferring super-resolved spatial metabolomics from microscopy
Authors: Rappez Luca , Haase Kristina 
Published: August 2024

AbstractCurrent spatial metabolomics techniques have transformed our understanding of cellular metabolism, yet accessible methods are limited in spatial resolution due to sensitivity constraints. MetaLens, a deep generative approach, disrupts this trade-off by quantitatively propagating cellular-resolutionin situimaging mass spectrometry readouts to subcellular scales through integration with high-resolution light microscopy. MetaLens identifies subcellular metabolic domains with distinct molecular composition, enabling accessible label-free subcellular metabolomic analysis from microscopy.
A combination of systemic mannitol administration and mannitol-modified polyester nanoparticles facilitate gene delivery to the brain through caveolae-mediated endocytosis
Authors: Betsy Reshma G , Miglani Chirag , Karmakar Arundhati , Gupta Sarika , Pal Asish , Ganguli Munia 
Published: August 2024

AbstractOvercoming the blood-brain barrier (BBB) remains a significant challenge for nucleic acid delivery to the brain. We have explored a combination of mannitol-modified poly (β-amino ester) (PBAE) nanoparticles and systemic mannitol injection for crossing the BBB. We incorporated mannitol in the PBAE polymer for caveolae targeting and also selected monomers that may help avoid delivery to the liver. We also induced caveolae at the BBB through systemic mannitol injection in order to create an opportunity for the caveolae-targeting nanoparticles (M30 D90)containing plasmid DNA to cross the BBB. When a clinically relevant dose was administered intravenously in this caveolae induction model, M30 D90 demonstrated significant transgene expression of a reporter plasmid in the brain, with selective uptake by neuronal cells and minimal liver accumulation. We also demonstrate that both caveolae modulation using systemic mannitol administration and caveolae targeting using designed nanoparticles are necessary for efficient delivery to the brain. This delivery platform offers a simple, scalable, and controlled delivery solution and holds promise for treating central nervous system diseases with functional targets.
RcsF-independent mechanisms of signaling within the Rcs Phosphorelay
Authors: Petchiappan Anushya , Majdalani Nadim , Wall Erin , Gottesman Susan 
Published: August 2024

AbstractThe Rcs (regulator of capsule synthesis) phosphorelay is a conserved cell envelope stress response mechanism in enterobacteria. It responds to perturbations at the cell surface and the peptidoglycan layer from a variety of sources, including antimicrobial peptides, beta-lactams, and changes in osmolarity. RcsF, an outer membrane lipoprotein, is the sensor for this pathway and activates the phosphorelay by interacting with an inner membrane protein IgaA. IgaA is essential; it negatively regulates the signaling by interacting with the phosphotransferase RcsD. We previously showed that RcsF-dependent signaling does not require the periplasmic domain of the histidine kinase RcsC and identified a dominant negative mutant of RcsD that can block signaling via increased interactions with IgaA. However, how the inducting signals are sensed and how signal is transduced to activate the transcription of the Rcs regulon remains unclear. In this study, we investigated how the Rcs cascade functions without its only known sensor, RcsF and characterized the underlying regulatory mechanisms for three distinct RcsF-independent inducers. Previous reports showed that Rcs signaling can be induced in the absence of RcsF by a loss of function mutation in the periplasmic oxidoreductase DsbA or by overexpression of the DnaK cochaperone DjlA. We identified an inner membrane protein, DrpB, as a multicopy RcsF-independent Rcs activator inE. coli. The loss of the periplasmic oxidoreductase DsbA and the overexpression of the DnaK cochaperone DjlA each trigger the Rcs cascade in the absence of RcsF by weakening IgaA-RcsD interactions in different ways. In contrast, the cell-division associated protein DrpB uniquely requires the RcsC periplasmic domain for signaling; this domain is not needed for RcsF-dependent signaling. This suggests the possibility that RcsC acts as a sensor for some Rcs signals. Overall, the results add new understanding to how this complex phosphorelay can be activated by diverse mechanisms.Author summaryThe Rcs phosphorelay signaling cascade regulates the expression of genes related to capsule synthesis, biofilm formation, virulence, and cell division in Enterobacteria and is critical for cell membrane integrity and response to beta-lactam antibiotics and antimicrobial peptides. RcsF is the sole known sensor, but other proteins have been reported to activate this pathway in the absence of RcsF. We have discovered a novel RcsF-independent Rcs activator and found that each of three RcsF-independent proteins activate the system differently. Most significantly, we find that the histidine kinase RcsC can be involved in signal sensing independently of RcsF. Our study sheds light into the complex mechanisms of Rcs activation and adds to our knowledge of non-orthodox signaling systems across organisms.
RNG2 tethers the conoid to the apical polar ring inToxoplasma gondii: a key mechanism in parasite motility and invasion
Authors: Haase Romuald , Ren Bingjian , Santos Pacheco Nicolas Dos , Visentin Rémy , Maco Bohumil , Flores Ricardo Mondragon , Vadas Oscar , Soldati-Favre Dominique 
Published: August 2024

AbstractInToxoplasma gondii, the conoid is a dynamic organelle composed of spiraling tubulin fibers that extrudes during egress, gliding motility, and invasion. This organelle traverses the apical polar ring (APR) in response to calcium waves and plays a critical role in controlling parasite motility. While the actomyosin-dependent extrusion of the conoid is beginning to be understood, the mechanism by which it is anchored apically to the APR remains unclear. RNG2, a protein localized at both the conoid and the APR, has emerged as a key candidate for this function. By combining iterative ultrastructure expansion microscopy and immunoelectron microscopy we discovered that RNG2 forms 22 tethers between the APR and the conoid. The unique biochemical properties of RNG2, including several proteolytic processing events and its ability to form concatenations, enable it to function as a dynamic bridge between these structures. Conditional depletion of RNG2 resulted in the conoid organelle detaching from the APR without compromising the integrity of its structure, thereby confirming RNG2 essential tethering role. Although microneme secretion remains normal, parasites lacking RNG2 were unable to move and impaired in rhoptry discharge, highlighting the conoid’s crucial role in parasite motility and invasion. RNG2 is a pivotal protein that ensures conoid functionality inCoccidia.
Chemical crosslinking extends and complements UV crosslinking in analysis of RNA/DNA nucleic acid–protein interaction sites by mass spectrometry
Authors: Welp Luisa M. , Sachsenberg Timo , Wulf Alexander , Chernev Aleksandar , Horokhovskyi Yehor , Neumann Piotr , Pašen Martin , Siraj Arslan , Raabe Monika , Johannsson Sven , Schmitzova Jana , Netz Eugen , Pfeuffer Julianus , He Yi , Fritzemeier Kai , Delanghe Bernard , Viner Rosa , Vos Seychelle M. , Cramer Patrick , Ficner Ralf , Liepe Juliane , Kohlbacher Oliver , Urlaub Henning 
Published: August 2024

ABSTRACTUV (ultra-violet) crosslinking with mass spectrometry (XL-MS) has been established for identifying RNA-and DNA-binding proteins along with their domains and amino acids involved. Here, we explore chemical XL-MS for RNA-protein, DNA-protein, and nucleotide-protein complexesin vitroandin vivo. We introduce a specialized nucleotide-protein-crosslink search engine, NuXL, for robust and fast identification of such crosslinks at amino acid resolution. Chemical XL-MS complements UV XL-MS by generating different crosslink species, increasing crosslinked protein yields in vivo almost four-fold and thus it expands the structural information accessible via XL-MS. Our workflow facilitates integrative structural modelling of nucleic acid–protein complexes and adds spatial information to the described RNA-binding properties of enzymes, for which crosslinking sites are often observed close to their cofactor-binding domains.In vivoUV and chemical XL-MS data fromE. colicells analysed by NuXL establish a comprehensive nucleic acid–protein crosslink inventory with crosslink sites at amino acid level for more than 1500 proteins. Our new workflow combined with the dedicated NuXL search engine identified RNA crosslinks that cover most RNA-binding proteins, with DNA and RNA crosslinks detected in transcriptional repressors and activators.
Reliability of canopy photography for forest ecology and biodiversity studies
Authors: von Meijenfeldt Anouk , Chianucci Francesco , Rigo Francesca , Ottenburghs Jente , Hilpold Andreas , Mina Marco 
Published: August 2024

AbstractUnderstory is a key component of forest biodiversity. The structure of the forest stand and the horizontal composition of the canopy play a major role on the light regime of the understory, which in turn affects the abundance and the diversity of the understory plant community. Reliable assessments of canopy structural attributes are essential for forest research and biodiversity monitoring programs, as well as to study the relationship between canopy and understory plant communities. Canopy photography is a widely used method but it is still not clear which photographic techniques is better suited to capture canopy attributes at stand-level that can be relevant in forest biodiversity studies. For this purpose, we collected canopy structure and understory plant diversity data on 51 forest sites in the north-eastern Italian Alps, encompassing a diversity of forest types. Canopy images were acquired using both digital cover (DCP) and hemispherical (DHP) photography. Canopy structural attributes were then compared to tree species composition data to evaluate whether they were appropriate to differentiate between forest types. Additionally, we tested what canopy attributes derived from DCP and DHP best explained the species composition of vascular plants growing in the understory. We found that hemispherical canopy photography was most suitable to capture differences in forest types, which was best expressed by variables such as leaf inclination angle and canopy openness. On our sites, DHP-based canopy attributes were also able to better distinguish between different conifer forests. Leaf clumping was the most important attribute for determining plant species distribution of the understory, indicating that diverse gap structures create different microclimate conditions enhancing diverse plant species with different ecological strategies. This study supports the reliability of canopy photography in forest ecology and biodiversity monitoring, but also provide insights for increasing understory diversity in managed forests of high conservation value.Abstract Figure
An active model for the basilar membrane and the outer hair cells
Authors: Berger Jorge , Rubinstein Jacob 
Published: August 2024

AbstractA model for the joint motion of the basilar membrane (BM) and the outer hair cells (OHC) in the cochlea is presented. The model consists of two one-dimensional mass distributions, one along the OHC and outer hair bundle (OHB) interface, and one along the BM. The motion of these masses is driven by the forces exerted on them by the elastic bodies connecting them and by the pressure difference in the fluids separated by the BM. The model includes a nonlinear motility of the OHC and its coupling with the vibrations of the BM. The model implies a Hopf bifurcation for the dynamical system governing the two coupled distributed oscillators. It is shown that when the system operates near the bifurcation point the BM motion is amplified up to a saturation level. The model provides very sharp frequency decomposition of the incident audio signal according to the place principle. It also acts as a powerful filter that distinguishes pure tones even in the presence of louder noisy background. In addition to simulations of the model, the unusual role played by the OHC friction is studied. Energy estimates are derived for the model functions.
Hawkmoth pheromone transduction involves G protein-dependent phospholipase Cβ signaling
Authors: Schneider Anna C. , Schröder Katrin , Chang Yajun , Nolte Andreas , Gawalek Petra , Stengl Monika 
Published: August 2024

1AbstractEvolutionary pressures adapted insect chemosensation to the respective insect’s physiological needs and tasks in their ecological niches. Solitary nocturnal moths rely on their acute olfactory sense to find mates at night. Pheromones are detected with maximized sensitivity and high temporal resolution through mechanisms that are mostly unknown. While the inverse topology of insect olfactory receptors and heteromerization with the coreceptor Orco suggest ionotropic transduction via odorant-gated receptor-ion channel complexes, contradictory data propose amplifying G protein-coupled transduction. Here, we usedin vivotip-recordings of pheromone-sensitive sensilla of maleManduca sextahawkmoths at specific times of day (rest vs. activity). Since the olfactory receptor neurons distinguish signal parameters in three consecutive temporal windows of their pheromone response (phasic; tonic; late, long-lasting), respective response parameters were analyzed separately. Disruption of G protein-coupled transduction and block of phospholipase C decreased and slowed the phasic response component during the activity phase of hawkmoths without affecting any other component of the response during activity and rest. A more targeted disruption of Gαsubunits by blocking Gαoor sustained activation of Gαsusing bacterial toxins affected the phasic pheromone response, while toxins targeting Gαqand Gα12/13were ineffective. Consistent with these data, the expression of phospholipase Cβ4 depended on zeitgeber time, which indicates circadian clock-modulated metabotropic pheromone transduction cascades that maximize sensitivity and temporal resolution of pheromone transduction during the hawkmoth’s activity phase. Thus, discrepancies in the literature on insect olfaction may be resolved by considering circadian timing and the distinct odor response components.2 Significance statementInsect chemosensory transduction is typically thought to be ionotropic, but data from different insect species suggests that metabotropic olfactory signaling may occur, either alongside or instead of ionotropic mechanisms. Nocturnal moths, known for their extraordinarily sensitive pheromone-detecting olfactory receptor neurons, likely use metabotropic signal amplification. To overcome limitations of previousin vitrostudies, we conducted tip-recordings of pheromone-sensitive sensilla in healthy hawkmoths at specific zeitgeber times. Disrupting G protein signaling and phospholipase Cβ reduced sensitivity and altered response kinetics, revealing strict temporal control of transduction. Thus, contradictory findings in insect olfaction may be reconciled by considering diverse evolutionary pressures for distinct chemosensory signals in different species, zeitgeber time, and disparate odor response parameters.
Dance displays in gibbons: Biological and linguistic perspectives on structured, intentional and rhythmic body movement
Authors: Coye C. , Caspar K.R. , Patel-Grosz P. 
Published: August 2024

AbstractFemale crested gibbons perform conspicuous sequences of twitching movements involving the rump and extremities. However, these dances have attracted little scientific attention and their structure and meaning remain largely obscure. Here we analyse close-range video recordings of captive crested gibbons, extracting descriptions of dance in fourNomascusspecies (N. annamensis, N. gabriellae, N. leucogenys,andN. siki). Additionally, we report results from a survey amongst relevant professionals clarifying behavioural contexts of dance in captive and wild crested gibbons. Our results demonstrate that dances inNomascusrepresent a common and intentional form of visual communication restricted to sexually mature females. While primarily used as a proceptive signal to solicit copulation, dances occur in a wide range of contexts related to arousal and/or frustration in captivity. A linguistically informed view of this sequential behaviour demonstrates that dances follow a grouping organisation and isochronous rhythm – patterns not described for visual displays in other non-human primates. We argue that applying the concept of dance to gibbons allows us to expand our understanding of the communicative behaviours of non-human apes and develop hypotheses on the rules and regularities characterizing this behaviour. We propose that gibbons dances likely evolved from less elaborate rhythmic proceptive signals, similar to those found in siamangs. Although dance displays in humans and crested gibbons share a number of key characteristics, they cannot be assumed to be homologous. Nevertheless, gibbon dances represent a model behaviour whose investigation could be extended to the study of complex gestural signals in hominoid primates.