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bioRxiv2025

Internal phase separation in synthetic DNA condensates

Biomolecular condensates regulate cellular biochemistry by organizing enzymes, substrates and metabolites, and often acquire partially de-mixed states whereby distinct internal domains play functional roles. Despite their physiological relevance, questions remain about the principles underpinning the emergence of multi-phase condensates. Here, we present a model system of synthetic DNA nanostructures able to form monophasic or biphasic condensates. Key condensate features, including the degree of interphase mixing and the relative size and spatial arrangement of domains, can be controlled by altering nanostructure stoichiometries. The modular nature of the system facilitates an intuitive understanding of phase behavior, and enables mapping of the experimental phenomenology onto a predictive Flory-Huggins model. The experimental and theoretical framework we introduce will help address open questions on multiphase condensation in biology and aid the design of functional biomolecular condensates in vitro, in synthetic cells, and in living cells.

biophysics↗

bioRxiv2025

Differential cell survival outcomes in response to diverseamino acid stress

Amino acid (AA) detection is fundamental for cellular function, balancing translation demands, biochemical pathways, and signaling networks. While the GCN2 and mTORC1 pathways are known to regulate AA sensing, the global cellular response to AA deprivation remains poorly understood, particularly in non-transformed cells, which may exhibit distinct adaptive strategies compared to cancer cells. Here we employed murine pluripotent embryonic stem (ES) cells as a model system to dissect responses to AA stress. Using multi-omics analyses over an extended time course, we examined the effects of arginine (Arg) and leucine (Leu) deprivation and uncovered a broad array of proteomic, phosphoproteomic, transcriptomic, and metabolomic adaptations, including an increase in lysosome production, all occurring without lethality. We found that Arg or Leu starvation induces reversible cell cycle exit, promoting a quiescent state that enhances resistance to cytotoxic stressors. By contrast, cysteine (Cys) and threonine (Thr) deprivation led to cell death via distinct pathways: ferroptosis for Cys starvation, while Thr deprivation triggered a previously uncharacterized form of cell death, which could be entirely suppressed by methionine (Met) co-starvation, mTOR or translational inhibition. These findings suggest that ES cells implement specialized survival strategies in response to different AA limitations, highlighting their ability to reprogram cellular biochemistry under nutrient stress.

cell biology↗

bioRxiv2025

Sub-optimal temperature leads to tighter coupling between photosynthetic electron transport and CO2 assimilation under fluctuating light in maize.

The C4 carbon concentrating pathway promotes high CO2 assimilation rates. To keep C4 photosynthesis energetically efficient, electron transport reactions and downstream biochemistry need to be carefully balanced. Here we use a combination of non-invasive measurements and metabolic profiling to study the efficiency of C4 photosynthesis in maize under two conditions that can lead to decoupling between electron transport and carbon assimilation: fluctuating light and suboptimal temperature. Measurements were performed for three fluctuating light regimes and three temperatures, providing the most detailed study to date of the interaction between fluctuating light and suboptimal temperature on the photosynthetic performance of maize, an important global crop. At room temperature, CO2 assimilation rates were decoupled from photosynthetic electron transport under fluctuating light regimes, in contrast to tight coordination observed under constant light. This decoupling was underpinned by metabolic flexibility and buffering by large pools of C4 transfer metabolites. Surprisingly, at sub-optimal temperatures, CO2 assimilation rates became more tightly coupled to photosynthetic electron transport rates under fluctuating light regimes. This appeared to be caused by strong feedback downregulation of electron transport and a stronger degree of light-saturation of CO2 assimilation at low temperature. Low temperature impacted carbon assimilation rates more strongly than metabolite pools or intercellular metabolite distribution, which could reflect negative effects on diffusional metabolite transfer through plasmodesmata. Altogether, these results show that maize is able to maintain energetic efficiency by buffering light transitions under room temperature, as well as avoid oxidative damage by strongly downregulating electron transfer under short-term exposure to low temperature. One-sentence summaryAnalysis of maize CO2 assimilation under fluctuating light shows significant decoupling from photosynthetic electron transport at room temperature, supported by metabolic flexibility and buffering by large pools of C4 transfer metabolites, but tight coordination is restored under suboptimal temperature due to enhanced feedback regulation of electron transport and a stronger degree of light saturation of CO2 assimilation.

plant biology↗

bioRxiv2025

Tomato Omics Research: Mapping Bibliometric Footprints of Global Research Trends, Collaboration Networks, and Impact Trajectories

Tomato is a globally vital crop and model system for fleshy fruit development, with profound agricultural and economic significance. Omics-based tomato research has revolutionized understanding of fruit quality traits, disease resistance mechanisms, and stress tolerance pathways reflecting insights for breeding innovations to address food security challenges. This bibliometric analysis on tomato omics delivered crucial insights into the evolution and impact of tomato omics research over two decades. Examining 1,702 Scopus-indexed publications, we have shown a remarkable acceleration in research output after 2017, signifying the fields growing significance. Our findings revealed that Biochemistry, Genetics and Molecular Biology dominated the landscape with 1,054 publications, establishing the molecular foundation for tomato research. It identified key global research hubs, with the United States (450 publications) leading, followed by China (310) and Italy (171), while mapping eight distinct International collaboration networks that drive innovation. Through citation analysis, we have pinpointed transformative contributions in microRNA research and genome sequencing, with the fields most influential paper garnering 2,570 citations. Co-authorship network visualization exposed 25 research clusters among 8,103 authors, highlighting prolific contributors including Fernie A.R. (39 publications) and Lucini L. (26). The keyword co-occurrence mapping revealed critical research priorities centered on stress responses, fruit quality, and plant immunity, with "Frontiers in Plant Science" emerging as the fields premier publication venue (92 articles). Our analysis provided a roadmap of tomato omics research progression, offering strategic value for prioritization, collaboration development, and funding allocation to address pressing challenges in global food security and crop improvement.

plant biology↗

bioRxiv2025

A genome-guided atlas to the composition, activity, and β-bungarotoxin dimerization in many-banded krait venom inferred by functional venomics

1Snakebite is a neglected tropical disease claiming [~]140,000 lives every year. One of the most medically relevant snakes of Asia is the many-banded krait (Bungarus multicinctus). Approximately 8% of the global human population is at risk of being envenomated by this species, able to cause fatal neurotoxicity. Here, we present a proteogenomic and functional assessment of the B. multicinctus venom via genome-guided bottom-up and top-down proteomics, combined with traditional protein profiling and bioassays. We report its venom profile alongside the primary structures of its toxins, revealing a relatively simple venom containing 55 components from 16 protein families. It is largely composed by three-finger toxins and phospholipase A2, besides acetylcholinesterase and snake venom metalloprotease. Top-down data unveiled the diversity of the highly lethal {beta}-bungarotoxins and allowed us to infer the complex dimerization patterning of these multi-domain neurotoxins. Our functional analysis revealed that B. multicinctus venom exerts potent phospholipase A2 and acetylcholinesterase activities, but protease activity as well as effects on cell viability and release of second messengers were virtually absent. This suggests, that B. multicinctus venom causes its devastating neurotoxic symptoms due to a heavy reliance on phospholipase A2 and acetylcholinesterase, but without impairing viability of neurons nor via interference of second messenger release. Antibacterial and antiviral screens further revealed activity against some pathogenic microbes that warrant further translational investigations. A comparison to previously published venom proteomes of B. multicinctus and its congeners suggests, that intraspecific venom variation occurs more widely in kraits than previously acknowledged and deserves higher attention. Overall, our investigation provides pivotal new insights into the biochemistry and pathophysiology of one of earths most lethal snakes and represents an important resource to inform future proteogenomic and functional studies in krait venom and beyond.

molecular biology↗

bioRxiv2025

Structural modeling reveals the allosteric switch controlling the chitin utilization program of Vibrio cholerae

Signal transduction by histidine kinases (HKs) is nearly ubiquitous in bacterial species. HKs can either sense ligands directly or indirectly via a cognate solute binding protein (SBP). The molecular basis for SBP-dependent signal reception, however, remains poorly understood in most cases. CBP and ChiS are the SBP-HK pair that activate the chitin utilization program of Vibrio cholerae. Here, we elucidate the molecular basis for allosteric regulation of CBP-ChiS by generating structural models of this complex in the unliganded and liganded states, which we support with extensive genetic, biochemical, and cell biological analysis. Our results reveal that ligand-binding induces a large conformational interface switch that is distinct from previously described SBP-HKs. Structural modeling suggests that similar interface switches may also regulate other uncharacterized SBP-HKs. Together, these results extend our understanding of signal transduction in bacterial species and highlight a new approach for uncovering the molecular basis of allostery in protein complexes. SIGNIFICANCE STATEMENTAll living things use protein receptors to sense and respond to environmental changes via a process termed signal transduction. However, how these proteins sense environmental stimuli remains poorly understood in many cases. In this study, we study allosteric activation of the chitin sensor, ChiS, by chitin-binding protein, CBP, in Vibrio cholerae as a model system. Using a combination of structural modeling, genetics, and biochemistry we uncovered the molecular basis underlying CBP-ChiS allosteric regulation, which we find is distinct from previously described systems. This work expands our understanding of bacterial signal transduction and highlights an approach for uncovering new modes of allosteric regulation.

microbiology↗

bioRxiv2025

Genetic modification of Clostridium kluyveri for heterologous n-butanol and n-hexanol production

The mesophilic microbe Clostridium kluyveri serves as the most commonly used model microbe to elucidate the physiology and biochemistry of ethanol-based chain elongation via reverse {beta}-oxidation. In this pathway, ethanol and acetate are converted into short- and medium-chain carboxylates. However, to date, no genetic system has been published in a peer-reviewed publication. Here, we report the development of versatile genetic tools for C. kluyveri, utilizing the pMTL Clostridia shuttle vector system and thiamphenicol as a selective marker. We identified the native restriction-modification system of C. kluyveri as a critical barrier to DNA transfer and overcame it by identifying and characterizing the crucial methyltransferase. To mimic the native DNA methylation pattern of C. kluyveri, we performed in-vivo methylation of the shuttle vector plasmid by expressing the methyltransferase in Escherichia coli, followed by DNA transfer via conjugation. After validating the genetic system, we demonstrated heterologous expression of different combinations of both NADH and NADPH-dependent alcohol dehydrogenases from Clostridium acetobutylicum. The expression of these genes was controlled by the Pthl promoter, which is commonly used in Clostridia, and the PadhE2 promoter, leading to n-butanol and n-hexanol production of the mutant strains. This genetic system for C. kluyveri will not only enable further research on the metabolism of this microbe but also enable more profound insights into ethanol-based chain elongation in general. IMPORTANCEMedium-chain carboxylates are required in various everyday products, including cosmetics, pharmaceuticals, and fragrances, and show a natural antimicrobial property. Further, they represent food additives and serve as chemical building blocks for several other compounds. Traditionally, these carboxylates are produced from fossil resources, contributing to increased greenhouse gas emissions. Alternatively, they are derived from animal- or plant-based fat (e.g., coconut oil), which competes with agricultural land that is needed for food production. However, microbial chain elongation, which is a biotechnological approach relying on microbes, such as Clostridium kluyveri, is sustainable and a promising alternative to the conventional production of medium-chain carboxylates. Notably, it enables the use of industrial waste streams (e.g., off-gases, carbohydrate-rich industrial waste) as substrates, making the process more environmentally friendly. By applying our genetic system for C. kluyveri, a better understanding of microbial chain elongation can be achieved, and potentially even an extension of its product portfolio.

microbiology↗

bioRxiv2025

FastConformation: A Standalone ML-Based Toolkit for Modeling and Analyzing Protein Conformational Ensembles at Scale

Deep learning approaches like AlphaFold 2 (AF2) have revolutionized structural biology by accurately predicting the ground state structures of proteins. Recently, clustering and subsampling techniques that manipulate multiple sequence alignment (MSA) inputs into AlphaFold to generate conformational ensembles of proteins have also been proposed. Although many of these techniques have been made open source, they often require integrating multiple packages and can be challenging for researchers who have a limited programming background to employ. This is especially true when researchers are interested in subsampling to produce predictions of protein conformational ensembles, which require multiple computational steps. This manuscript introduces FastConformation, a Python-based application that integrates MSA generation, structure prediction via AF2, and interactive analysis of protein conformations and their distributions, all in one place. FastConformation is accessible through a user-friendly GUI suitable for non-programmers, allowing users to iteratively refine subsampling parameters based on their analyses to achieve diverse conformational ensembles. Starting from an amino acid sequence, users can make protein conformation predictions and analyze results in just a few hours on their local machines, which is significantly faster than traditional molecular dynamics (MD) simulations. Uniquely, by leveraging the subsampling of MSAs, our tool enables the generation of alternative protein conformations. We demonstrate the utility of FastConformation on proteins including the Abl1 kinase, LAT1 transporter, and CCR5 receptor, showcasing its ability to predict and analyze the protein conformational ensembles and effects of mutations on a variety of proteins. This tool enables a wide range of high-throughput applications in protein biochemistry, drug discovery, and protein engineering.

biophysics↗

bioRxiv2025

De Novo Protocell Membrane Formation Fueled by Primitive Metabolites

Lipid membranes define cell boundaries, acting as gatekeepers for transport and signaling. A central principle in biology is that all cellular membranes descend from a common ancestral membrane, as they cannot be generated in the absence of preexisting lipid structures. It is thus unclear how the first protocell membranes originated from membrane-less precursors. Here we demonstrate the de novo generation of lipid bilayers in the absence of any preexisting membranes, membrane-bound proteins, or lipid nanostructure templates. Using acetate as a two-carbon precursor, lipid tails are constructed by soluble enzymes and spontaneously conjugate to cysteine backbones, forming diacyl lipids that assemble into vesicles. Pore-forming peptides facilitate precursor transport into vesicles, allowing the continuous generation of new lipids. Formation of glycolipid membranes creates compartments that can maintain proton gradients. The de novo formation of membrane bilayers using primitive chemical building blocks may have been an intermediary step between primitive prebiotic biochemistry and the emergence of cellular life.

synthetic biology↗

bioRxiv2025

Allosteric Modulation of MIF-2 Structure, Catalysis, and Biological Signaling via Cysteine Residues and a Small Molecule, Ebselen

The macrophage migration inhibitory factor (MIF) family of cytokines comprised of the MIF and D-dopachrome tautomerase (or MIF-2) paralogs share identical tertiary and quaternary structures that contribute to their overlapping enzymatic and signaling activities. Recent investigations of MIF and MIF-2 have shown them to possess N-to-C-terminal allosteric crosstalk, but despite the similarity of this "allosteric pathway," its regulation of MIF and MIF-2 is not identical. Thus, structure alone does not preserve the precise allosteric mechanism and additional residues that modulate MIF and MIF-2 allosteric function must be characterized. Cysteines have been identified as allosteric switches for the same biochemical functions of MIF and small molecules targeting its N-terminal enzymatic site have affected the structure of three proximal cysteines. Ebselen is a compound that forms covalent selenylsulfide bonds with MIF cysteines and is hypothesized to destabilize and dissociate the MIF trimer into monomers. Ebselen-bound MIF also displays little-to-no catalysis or biological signaling. However, it is unclear whether Ebselen similarly affects the MIF-2 paralog, despite MIF-2 containing two related cysteines (MIF contains three). We used mutagenesis, nuclear magnetic resonance (NMR), molecular dynamics (MD) simulations, in vitro and in vivo biochemistry to investigate the mechanism of Ebselen as an allosteric modulator of MIF-2 via its cysteines. Our findings suggest that Ebselen partially disrupts the MIF-2 homotrimer, though the overall population of such a structure is <35%, even on the timescale of many hours. Ebselen does attenuate the biological functions of MIF-2 and solution structural biology captures the conformational transitions preceding the destabilized MIF-2 trimer. Significance StatementThe nearly identical MIF and MIF-2 structures have been recently shown to contain an N-to-C-terminal crosstalk between non-overlapping functional sites in vitro and in vivo. Small molecule inhibitors designed as therapeutics for the MIF superfamily have primarily targeted the N-terminal active site, while very few allosteric molecules have been reported. Ebselen, however, destabilizes MIF by covalently modifying its distal Cys80 residue and disrupting its obligate trimeric assembly. This unique mechanism has never been evaluated in the MIF-2 paralog, and this study reports the structural, dynamic, and functional impact of Ebselen on MIF-2. We define the essential structural hallmarks preceding trimer dissociation and reveal that Ebselen inhibits MIF-2 function before trimer disruption, leading to destabilized monomer-monomer interfaces that slowly degrade the quaternary assembly.

biophysics↗

bioRxiv2025

Phosphoribosyl ubiquitination of SNARE proteins regulate autophagy in Legionella infection

Legionella pneumophila is an intracellular pathogen that causes Legionnaires disease. The bacteria releases effector proteins some of which remodel the host autophagic-lysosomal pathways. One effector is RavZ, which delipidates ATG8 proteins, making Legionella-infected cells deficient in autophagy. Here we show that SidE effectors mediate the phosphoribosyl ubiquitination (PR-Ub) of autophagic SNARE proteins STX17 and SNAP29. STX17 modification induces recruitment of STX17+ membranes from the endoplasmic reticulum to Legionella-containing phagosomes to form replicative vacuoles. Using proximity labeling, biochemistry and Legionella infection studies, we have discovered a mechanism by which autophagy is hijacked by bacteria to recruit ER membranes to the bacterial vacuole which has autophagy markers but do not fuse with lysosomes. Mass spectrometric identification of PR-Ub sites and mutational studies show that PR-Ub modification of STX17 alters its interaction with ATG14L, which drives the recruitment of ER membranes to the bacterial vacuole in a PI3K dependent manner. On the other hand, PR-Ub of SNAP29 inhibits the formation of the autophagosomal SNARE complex (STX17-SNAP29-VAMP8) by steric hindrance, thereby preventing the fusion of bacterial vacuoles with lysosomes.

cell biology↗

bioRxiv2025

TRiC-assisted folding of class I HDAC family proteins regulated by distinct co-chaperone and cofactor networks

Class I histone deacetylases (HDACs), including HDAC1, HDAC2, HDAC3, and HDAC8, are essential for diverse cellular processes. Although the chaperonin TRiC is implicated in the activation of class I HDACs, the underlying mechanisms remain elusive. Using cryo-electron microscopy (cryo-EM), cross-linking mass spectrometry (XL-MS), and biochemistry analyses, we established class I HDACs as novel TRiC substrates and elucidate TRiC-assisted folding of HDAC1 and HDAC3 during its ATPase cycle, orchestrated by distinct co-chaperone and cofactor networks. In the closed TRiC chamber, both HDAC1 and HDAC3 adopt near-native states with shared binding interfaces. However, their open-state configurations diverge: Hsp70 and PDCD5 engage atop and within TRiC, respectively, for HDAC3, whereas prefoldin (PFD) binds atop TRiC for HDAC1, suggesting roles in substrate delivery and folding modulation. Furthermore, an unexpected bent conformation of CCT4, detected in TRiC-HDAC1 complex, may facilitate co-chaperone dissociation from TRiC. In contrast, HDAC8 folds independently of TRiC. Our study reveals the mechanism governing TRiC-assisted folding of class I HDACs in orchestration of dynamic co-chaperone/cofactor network, shielding new lights on the sophisticated regulatory landscape of TRiC, and open promising avenues for designing peptides or small molecules to selectively modulate TRiC-assisted folding of class I HDACs and other substrates.

molecular biology↗

bioRxiv2025

Quantification of small molecule partitioning in a biomolecular condensate with 2D NMR spectroscopy

Several intrinsically disordered proteins have been shown to undergo phase separation into a dense and dilute phase and this process is intimately linked with the regulation of cellular processes. It is therefore highly relevant to know how metabolites partition between these phases. We show here that the partitioning of components in a complex mixture can be robustly obtained from a single set of 2D NMR spectra recorded on the dilute and dense phases separately using a so-called "time zero extrapolated approach" known as HSQC0. The spectral separation power of 2D NMR spectroscopy circumvents the need for physical isolation or workup of the mixture components in the two samples. Using quantitative 1D 1H NMR, it is validated that the HSQC0 approach effectively removes all the undermining effects that plague quantification in common 2D NMR experiments, including differential attenuation due to relaxation in the two phases, pulse imperfections, partial decoupling, off-resonance effects, and incomplete coherence transfer in the case of scalar coupling variation. These results should be of widespread interest as partitioning into biomolecular condensates is crucial for the calibration of computational physicochemical models of phase separation and key to the further understanding of cellular biochemistry involving membraneless organelles.

biophysics↗

bioRxiv2025

Did Iron Suppress Eukaryote Emergence and Early Radiation?

The last eukaryotic common ancestor (LECA) is widely thought to have been an oxygen-respiring organism, arising through endosymbiosis when a free-living bacterium became the mitochondrion. Owing to the mitochondrions central metabolic task of oxidative phosphorylation, oxygen availability has long been a hypothesized driver of eukaryogenesis. However, this hypothesis is challenged by a temporal disconnect, spanning several hundred million years, between the earliest geochemical evidence for oxygen in the environment ([~]3.2-2.5 Ga) and the oldest widely accepted eukaryotic fossils ([~]1.7 Ga). Notably, the earliest candidate eukaryotes appear contemporaneous with the cessation of major iron deposits and rise of sulfide- and sulfate-rich marine sediments in coastal environments. Here, we integrate Proterozoic surface geochemical records with the microbial biochemistry of iron to examine potential environmental constraints on early eukaryotic evolution. Iron bioavailability exerts complex and often antagonistic effects on both aerobic and anaerobic microbial lineages that contributed to LECA. Elevated iron levels likely disrupted cellular homeostasis, particularly by destabilizing labile iron pools and promoting oxidative damage to bacterial lipids. The programmed cell death pathways known as ferroptosis, which is widespread among eukaryotic lineages, may trace its origins to iron-rich conditions in Archaean and Paleoproterozoic seawater and LECA. Our findings challenge oxygen-centered paradigms of eukaryogenesis and reframes the long-recognized temporal gap as a consequence of iron-mediated physiological constraints.

evolutionary biology↗

bioRxiv2025

GNC is a regulator of metabolic and productivity responses to elevated CO2 in Arabidopsis thaliana

Despite established understanding of plant physiological responses to elevated [CO2], the underlying genes are poorly understood. Soybean transcriptomics previously identified a GATA transcription factor, involved in carbon and nitrogen metabolism, as responsive to elevated [CO2]. Supported by in silico modeling, we therefore hypothesized that this gene plays a previously unrecognized role in responding to elevated [CO2]. Wildtype and a T-DNA insertion line of Arabidopsis thaliana for GNC (GATA, Nitrate Inducible, Carbon Metabolism Involved) were grown under three treatments: sustained ambient [CO2], sustained elevated [CO2], and transfer from ambient to elevated [CO2], to assess changes in their physiology, biochemistry, and transcriptome. Photosynthetic and biomass responses to elevated [CO2] and transfer [CO2] in plants lacking GNC were significantly weaker than WT. A lag of 25-73 hrs in transcriptomic responses after transfer to elevated [CO2] was consistent with indirect sensing, presumably via sugar signals. The breakdown of the gene expression network around GNC was most pronounced in the transfer treatment and suggests targets for further study of interactions between elevated [CO2] and sulfur and nitrogen metabolism. This work provides a case study of a CO2-responsive transcription factor that may be a compelling target for adapting crops to future growing conditions after further characterization. Summary StatementA GATA transcription factor modulates plant metabolic and productivity responses to elevated CO2.

plant biology↗

bioRxiv2025

Juvenile hormone degradation enzymes have shared and unique requirements in Drosophila development

Precise control of hormones is essential for animal development. Hormone bioavailability is regulated by their synthesis, transport, sequestration, natural turnover, and programmed degradation. Here, we use Drosophila melanogaster to investigate developmentally programmed degradation of the retinoid-like juvenile hormones (JHs). JHs promote juvenile growth and timely development by functionally opposing the steroid hormone, ecdysone. Despite well-characterized biochemistry, the developmental requirements for programmed JH degradation are poorly understood due to paralogue expansion. To close this knowledge gap, we generated double and triple knockout animals lacking one of two classes of JH degradation enzymes: JH esterases (JHEs) and JH epoxide hydrolases (JHEHs). We found that while both JHEs and JHEHs restrain JH signaling during Drosophila juvenile development, each has separate requirements in the regulation of developmental timing and growth. Strikingly, loss of all three JHEHs doubled the length of juvenile development, and the resulting pupae were smaller. Through targeted and genome-wide transcriptome analysis, measurements of hormone-producing glands, and rescue experiments, we uncovered both shared and unique dysregulated gene networks, some of which have established roles in the regulation of both body size and developmental timing. Our comparative analysis of both JH degradation pathways demonstrated that loss of JHEH, but not JHE, activates multiple levels of compensatory feedback to maintain homeostasis within JH and ecdysone axes. These data not only suggest that JHEH-mediated degradation is the dominant programmed JH degradation pathway in D. melanogaster development, but they also revealed new JH homeostatic mechanisms more generally. Together, this study provides new genetic tools and insights into programmed hormone degradation. Article summaryHormones control the timing of developmental transitions and growth. As such, multiple mechanisms ensure precise amounts of a hormone are available at the right time and place. Here, we investigated the poorly understood process of developmentally programmed hormone degradation using juvenile hormones (JHs) in Drosophila melanogaster as a model. We generated double and triple knockout strains to disrupt JH degradation and found an unexpected separation of requirements in regulating developmental growth and timing. Through phenotypic and transcriptomic analysis, this study provides insights into the mechanisms and complexities of programmed hormone degradation in animal development.

genetics↗

bioRxiv2025

Amyloid Folding: an Origami-Based Approach

Amyloid is an ordered folding pattern that involves a cross-beta arrangement, with hydrogen-bonding between adjacent chains in the fiber elongation axis, and side-chain interactions perpendicular to it, forming steric and/or polar zippers intra- and/or inter-chains. The arrangement of the polypeptide backbone and the side-chain interactions can be arranged with different morphologies that are repeated along the fiber growth axis as stacking units. A simple representation of amyloid folding of proteins using paper origami is reported here, which can be used to visualize the {beta}-sheet along fiber axis and the perpendicular variability in topological arrangement. The model uses regular office paper, in the form of multiple linearized polypeptide chains, arranged in parallel or anti-parallel forms, connected by hydrogen bonds. Alternating mountain/valley creases along the growth axis of the fibrils and, over the front/back side-chains, results in a pleated sheet that can be used to study the topological arrangement of amyloid fibrils and the polar/steric zippers. The present models can be used as teaching tools for understanding the structural and molecular basis of amyloid folding, chain growth, homo- and cross-seeding, and other features of amyloid function in health, disease and biotechnology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/659339v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@888d0eorg.highwire.dtl.DTLVardef@e4b027org.highwire.dtl.DTLVardef@1e8ff0forg.highwire.dtl.DTLVardef@1c160ed_HPS_FORMAT_FIGEXP M_FIG C_FIG Key featuresO_LIIntroduces a physical origami model of amyloid {beta}-sheet structure using paper folding. C_LIO_LIDemonstrates how hydrogen bonding and side-chain interactions can be conceptualized in 3D. C_LIO_LIModels morphological polymorphism and parallel/antiparallel alignment. C_LIO_LIUseful for teaching complex biophysical concepts like cross-seeding, fibril growth, and molecular stability in amyloids. C_LIO_LIHighlights the educational value for biochemistry, structural biology, biomedical and material sciences. C_LI

biophysics↗

bioRxiv2025

Cultivation of Methanonezhaarchaeia, the third class of methanogens within the phylum Thermoproteota

Methane is a potent greenhouse gas that is largely produced through the activity of methanogenic archaea, which contribute to Earths dynamic climate and biogeochemical cycles. In the past decade, metagenomics revealed that lineages outside of the traditional Euryarchaeota superphylum encode genes for methanogenesis. This was recently confirmed through the cultivation of two classes of methanogenic Thermoproteota. Thus far, all methanogens within the Thermoproteota are predicted or were shown to be methylotrophic. The only exception to this is the Nezhaarchaea, which, based on metagenomics, are predicted to be CO2-reducing methanogens. Here, we demonstrate methanogenic activity in a third class of Thermoproteota, the Methanonezhaarchaeia. We expand the metabolic diversity of this class by cultivating a methylotrophic species, Candidatus Methanonezhaarchaeum fastidiosum YNP3N. We describe novel genes involved in methanogenesis that are not found in other methanogens. We investigate the metabolic diversity of Methanonezhaarchaeia, including metabolic modifications accompanying frequent loss of methanogenesis in this class. This highlights gaps in our understanding of the biochemistry, diversity, and evolution of non-traditional methanogens and their contributions to carbon cycling. TeaserThe cultivation of a new group of methanogens illuminates their metabolic diversity and evolution of archaea.

microbiology↗