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bioRxiv2025

Boosting Cellular Longevity Through Intracellular ATP Modulation

Mitochondrial dysfunction and declining ATP production are common features of aging, yet whether ATP availability itself directly regulates cellular lifespan. However, the causal relationship between cellular ATP homeostasis and aging has not been established. Here, we developed a synthetic system to manipulate intracellular ATP independently of endogenous energy production by expressing a plasma membrane-targeted nucleotide transporter, NTT1, from the intracellular parasite Encephalitozoon cuniculi in Saccharomyces cerevisiae. NTT1 expression depleted intracellular ATP in the absence of extracellular ATP, whereas ATP supplementation produced robust NTT1-dependent ATP uptake and increased intracellular ATP abundance. ATP availability strongly influenced replicative lifespan: ATP depletion shortened lifespan, whereas ATP supplementation restored and extended lifespan in NTT1-expressing cells. Unexpectedly, extracellular ATP also extended lifespan in wild-type cells that lack ATP import, revealing an NTT1-independent response to extracellular ATP. Transcriptomic analyses showed that NTT1- mediated ATP import suppresses glucose uptake, carbohydrate catabolism, mitochondrial respiration, and autophagy, whereas extracellular ATP elicits a distinct transcriptional response in wild-type cells involving metabolic, mitochondrial, and signaling pathways. Single-cell aging analyses further showed that ATP supplementation extends lifespan across distinct aging trajectories and shifts cells away from the mitochondrial dysfunction-associated aging state. Finally, experiments in cells lacking mitochondrial DNA separated these effects mechanistically: NTT1-associated lifespan phenotypes and high-ATP toxicity required functional mitochondria, whereas extracellular ATP extended lifespan in wild-type cells independently of mitochondrial respiration. Together, these findings demonstrate that ATP availability is a direct regulator of cellular aging and reveal distinct metabolic and extracellular ATP-responsive routes through which cellular energy state influences longevity. SignificanceCellular energy homeostasis is a crucial factor in determining the health and longevity of organisms. While intracellular ATP levels are tightly regulated, the idea that cells can directly take in extracellular ATP to influence metabolism has not been thoroughly explored. In this study, we engineered yeast cells to import external ATP and demonstrated that this approach significantly alters mitochondrial function, metabolic flow, and aging processes. Our findings show that ATP uptake inhibits catabolic pathways and modulates mitochondrial bioenergetics function, thereby extending cellular lifespan through a novel and non-traditional mechanism. This research reveals an unexpected degree of metabolic flexibility and introduces a synthetic biology-based method to reprogram energy metabolism and longevity. The principles established in this study provide a new framework for understanding the role of cellular bioenergetics in aging, highlighting how the modulation of ATP availability can impact metabolic states and lifespan regulation.

molecular biology↗

bioRxiv2017

Transcription activator-coactivator specificity is mediated by a large and dynamic fuzzy protein-protein complex

Transcription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient activation by yeast Gcn4 requires tandem Gcn4 ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous, containing nearly all possible AD-ABD interactions. This complex forms using a dynamic fuzzy protein-protein interface where ADs use hydrophobic residues to bind hydrophobic surfaces of the ABDs in multiple orientations. This combinatorial mechanism allows individual interactions of low affinity and specificity to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators and allows great flexibility in combinations of activators that synergize to regulate genes with variable coactivator requirements.

molecular biology↗

bioRxiv2020

Potential Functions of Histone H3.3K56 Acetylation in Mammals

H3K56 acetylation (H3K56Ac) was first identified in yeast and has recently been reported to play important roles in maintaining genomic stability, chromatin assembly, DNA replication, cell cycle progression and DNA repair. Although H3.1K56Ac has been relatively well studied, the function of H3.3K56Ac remains mostly unknown in mammals. In this study, we used H3.3K56Q and H3.3K56R mutants to study the possible function of H3.3K56 acetylation. The K-to-Q substitution mimics a constitutively acetylated lysine, while the K-to-R replacement mimics a constitutively unmodified lysine. We report that cell lines harboring mutation of H3.3K56R exhibit dramatic morphology changes and cell death. Using Tet-Off inducible system, we show an increased population of polyploid/aneuploid cells and a decreased cell viability in H3.3K56R mutant cells. In consistence with these results, H3.3K56R mutant compromised H3.3 incorporation into several pericentric and centric heterochromatin regions we tested. Moreover, mass spectrometry analysis coupled with label free quantification reveal that biological processes regulated by the H3.3-associating proteins, whose interaction with H3.3 is markedly increased by H3.3K56Q mutation but decreased by H3.3K56Q mutation, include sister chromatid cohesion, mitotic nuclear division, and mitotic nuclear envelope disassembly. These results suggest that H3.3K56 acetylation is crucial for chromosome segregation and cell division in mammals.

molecular biology↗

bioRxiv2025

Functional Characterization of a Novel Long Non-Coding RNA in Leishmania braziliensis Identified Through Computational Screening for Conserved RNA Structures

Leishmania parasites alternate between hosts, facing environmental changes that demand rapid gene expression adaptation. Lacking canonical RNA polymerase II promoters, transcription in these eukaryotes is polycistronic, with gene regulation occurring post-transcriptionally. Although non-coding RNAs (ncRNAs) have been identified in Leishmania transcriptomes, their functions remain unclear. Recognizing RNA structures importance, we performed a genome-wide alignment of L. braziliensis and related species, identifying conserved RNA structures, 38 of which overlap with known ncRNAs. One such ncRNA, lncRNA45, was functionally characterized. Using a knockout cell line, we demonstrated that lncRNA45 is crucial for parasite fitness. Reintroducing the wild-type lncRNA45 restored fitness, while a version with a single nucleotide substitution in the structured region did not. This mutation also altered RNA-protein interactions. These findings suggest that lncRNA45s regulatory role and protein interactions rely on its secondary structure. This study highlights the significance of structured lncRNAs in Leishmania biology and their potential as therapeutic targets. Further research into these ncRNAs could uncover new parasite regulation mechanisms and inspire novel treatment strategies.

molecular biology↗

bioRxiv2022

Optimizing Cryo-FIB Lamellas for sub-5 Angstrom in situ Structural Biology

We here present a method based on metallic platinum sputtering that can substantially enhance the quality of subtomogram averages from lamellas and thereby reduce the number of particles needed for high-resolution subtomogram averaging. We provide evidence for the physical background of this improvement and demonstrate its usefulness by producing sub-5[A] ribosome averages from yeast.

molecular biology↗

bioRxiv2022

Transcriptomic analysis reveals a role for the nervous system in regulating growth and development of Fasciola hepatica juveniles

Fasciola spp. liver fluke have significant impacts in veterinary and human medicine. The absence of a vaccine and increasing anthelmintic resistance threaten sustainable control and underscore the need for novel flukicides. Functional genomic approaches underpinned by in vitro culture of juvenile Fasciola hepatica facilitate control target validation in the most pathogenic life stage. Comparative transcriptomics of in vitro and in vivo maintained 21 day old F. hepatica finds that 86% of genes are expressed at similar levels across maintenance treatments suggesting commonality in core biological functioning within these juveniles. Phenotypic comparisons revealed higher cell proliferation and growth rates in the in vivo juveniles compared to their in vitro counterparts. These phenotypic differences were consistent with the upregulation of neoblast-like stem cell and cell-cycle associated genes in in vivo maintained worms. The more rapid growth/development of in vivo juveniles was further evidenced by a switch in cathepsin protease expression profiles, dominated by cathepsin B in in vitro juveniles and then by cathepsin L in in vivo juveniles. Coincident with more rapid growth/development was the marked downregulation of both classical and peptidergic neuronal signalling components in in vivo maintained juveniles, supporting a role for the nervous system in regulating liver fluke growth and development. Differences in the miRNA complements of in vivo and in vitro juveniles identified 31 differentially expressed miRNAs, notably fhe-let-7a-5p, fhe-mir-124-3p and, miRNAs predicted to target Wnt-signalling, supporting a key role for miRNAs in driving the growth/developmental differences in the in vitro and in vivo maintained juvenile liver fluke. Widespread differences in the expression of neuronal genes in juvenile fluke grown in vitro and in vivo expose significant interplay between neuronal signalling and the rate of growth/development, encouraging consideration of neuronal targets in efforts to dysregulate growth/development for parasite control. Author SummaryParasitic worms are notoriously difficult to study outside of a host organism. However, recent developments in culture methods for Fasciola hepatica liver fluke juveniles support growth and development of these parasites in the laboratory (in vitro) towards adult parasites. Having the ability to grow pathogenic juvenile stages in vitro enables functional studies to validate potential drug and vaccine targets. However, comparison of in vitro grown juveniles to juveniles retrieved from infected hosts (in vivo) shows considerable size differences suggesting at least some differences in biology that could undermine the relevance of data generated from in vitro maintained parasites. This study examines gene expression differences between in vitro and in vivo maintained F. hepatica juveniles via transcriptomic analysis to identify similarities and differences in their biology which may help explain differences in the rate of growth and development. 86% of genes were shown to be expressed at similar levels across treatment groups suggesting a high level of biological similarity between in vitro and in vivo juveniles. However, the genes that are expressed differently between these juveniles will help improve current culture methods and provide a new group of potential drug targets that impact on juvenile growth and development.

molecular biology↗

bioRxiv2024

Urinary Proteome Characterization of Stroke-Prone Spontaneously Hypertensive Rats

BackgroundHypertension is a multifactorial, complex disease related to genetic and environmental factors and has become the most serious public health problem. This study aimed to explore the changes of hypertension based on urinary proteome. MethodsThe stroke-prone spontaneously hypertensive rats (SHRSPs) model was used to examined urinary proteome changes during hypertension development. Urine proteome at months 1, 4, 8, 10, 12, and 14 was profiled using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Considered the degree of disease progression may differ, each rat was compared before and after hypertension developed to screen for differential proteins. ResultsThe differential proteins in each rat can be enriched into some important biological processes and pathways associated with hypertension, such as regulation of systemic arterial blood pressure by renin-angiotensin, renin-angiotensin signaling, response to glucocorticoid and glucocorticoid receptor signaling, calcium transport I, aldosterone adipocyte signaling pathway, apelin adipocyte signaling pathway and oxidative stress response. The biological processes and pathways enriched at the same time point in the progression of hypertension differed significantly among different rat individuals. ConclusionsThis study showed that the changes of hypertension can be reflected in urine proteins. The urine proteomics has the potential to be used to study the mechanisms of hypertension, discovery new drug targets, and provide personalized antihypertensive treatment strategies.

molecular biology↗

bioRxiv2017

Computational docking reveals evolutionary conservation of a specific interaction between 15d-Prostaglandin-J2 and eIF4A.

15-deoxy-delta 12,14-prostaglandin J2 (15d-PGJ2) is anti-inflammatory/antineoplastic prostaglandin which functions through covalent binding to cysteine residues of various target proteins. We previously showed that 15d-PGJ2 mediated anti-inflammatory responses are dependent on the translational inhibition through its interaction with eIF4A. Binding of 15d-PGJ2 to eIF4A specifically blocks the interaction between eIF4G and eIF4A leads to the formation of stress granules (SGs), which cluster mRNAs with inhibited translation. Here we show that the binding between 15d-PGJ2 and eIF4A specifically blocks the interaction between the MIF4G domain of eIF4G and eIF4A. To reveal the mechanism of this interaction, we used computational simulation-based docking studies and identified that the carboxyl tail of 15d-PGJ2 could stabilize the binding of 15d-PGJ2 to eIF4A through arginine 295 of eIF4A, which is the first suggestion that the 15d-PGJ2 tail play a physiological role. Interestingly, the putative 15d-PGJ2 binding site on eiF4A is conserved across many species, suggesting a biological role. Our data propose that studying 15d-PGJ2 and its targets will may uncover new therapeutic approaches in anti-inflammatory drug discovery.

molecular biology↗

bioRxiv2023

Establishing CRISPR-Cas9 in the sexually dimorphic moss, Ceratodon purpureus.

The development of CRISPR technologies provides a powerful tool for understanding the evolution and functionality of essential biological processes. Here we demonstrate successful CRISPR-Cas9 genome editing in the dioecious moss species, Ceratodon purpureus. Using an existing selection system from the distantly related hermaphroditic moss, Physcomitrium patens, we generated knock-outs of the APT reporter gene by employing CRISPR targeted mutagenesis under expression of native U6 snRNA promoters. Next, we used the native homology-directed repair (HDR) pathway, combined with CRISPR-Cas9, to knock-in two reporter genes under expression of an endogenous RPS5A promoter in a newly developed landing site in C. purpureus. Our results show that the molecular tools developed in P. patens can be extended to other mosses across this ecologically important and developmentally variable group. These findings pave the way for precise and powerful experiments aimed at identifying the genetic basis of key functional variation within the bryophytes and between the bryophytes and other land plants. Significance StatementWe have developed CRISPR-Cas9 genome editing tools and protocols for the sexually dimorphic moss, Ceratodon purpureus to generate gene knock-outs and knock-ins within targeted loci. This work facilitates future functional genomic experiments in this fast growing, haploid, dioecious system and suggests these tools will function across much of moss diversity.

molecular biology↗

bioRxiv2024

Long-read sequencing to detect full-length protein-protein interactions

Given the increased predictions on interactome size and demand for protein function information, methods for detecting protein-protein interactions remain a significant development area. The all-vs.-all sequencing (AVA-Seq) method utilizes a convergent fusion plasmid design to make two-hybrid technology amenable to next-generation sequencing. Here, we further innovate to take advantage of synthetic DNA technologies and Oxford Nanopore Technologies long-read sequencing improvements to allow us to determine full-length protein-protein interactions. Here, using this approach we recovered 159 protein-protein interactions from a set of 57 human proteins using multiple forms of validation. Further, when referencing a human gold standard set of interactions, eight full-length protein-protein interactions were recovered from an expected 28 interaction pairs (28.6%), a typical recovery rate for two-hybrid technologies. The AVA-Seq, in combination with the ease of synthetic DNA production and the MinION platform, offers a low-cost, high-throughput alternative for determining protein-protein interactions, which can be utilized in research labs at all stages. 3 Key PointsO_LIFirst application of long-read sequencing for full-length protein-protein interaction studies. C_LIO_LIThe recovery rate of the AVA-Seq method using full-length proteins is on par with other leading methods. C_LIO_LIAdvances in synthetic biology and sequencing technologies make full-length protein interactomes affordable and accessible. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/586447v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1d81412org.highwire.dtl.DTLVardef@10dbb19org.highwire.dtl.DTLVardef@76a04borg.highwire.dtl.DTLVardef@15baf0_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Role of the osaA transcription factor gene in development, secondary metabolism and virulence in the mycotoxigenic fungus Aspergillus flavus

Aspergillus flavus colonizes oil-seed crops contaminating them with aflatoxins, highly carcinogenic mycotoxins that cause severe health and economic losses. Genetic studies may reveal new targets for effective control strategies. Here we characterized a putative WOPR transcription factor gene, osaA, in A. flavus. Our results revealed that osaA regulates conidiation and sclerotial formation. Importantly, deletion of osaA reduces aflatoxin B1 production, while, unexpectedly, transcriptome analysis indicated upregulation of aflatoxin biosynthetic genes, suggesting post-transcriptional or cofactor-mediated regulation. Cyclopiazonic acid production also decreased in absence of osaA. In addition, the osaA mutant exhibited upregulation of genes in the imizoquin and aspirochlorine clusters. Moreover, osaA is indispensable for normal seed colonization; deletion of osaA significantly reduced fungal burden in corn kernels. Aflatoxin content in seeds also decreased in the absence of osaA. Furthermore, deletion of osaA caused a reduction in cell-wall chitin content, as well as alterations in oxidative stress sensitivity, which could in part contribute to the observed reduction in pathogenicity. Additionally, promoter analysis of osaA-dependent genes indicated potential interactions with stress-responsive regulators, indicated by an enrichment in Sko1 and Cst6 binding motifs. Understanding the osaA regulatory scope provides insight into fungal biology and identifies potential targets for controlling aflatoxin contamination and pathogenicity. Key ContributionAspergillus flavus osaA controls morphological and chemical development, as well as phytopathogenicity, and could be a promising target for a control strategy against A. flavus to reduce health risks and economic losses associated with aflatoxin contamination.

molecular biology↗

bioRxiv2019

A tertiary structure protein model for the ab-initio interpretation of small angle X-ray scattering data

Small angle X-ray scattering (SAXS) has become an important tool to investigate the structure of proteins in solution. In this paper we present a novel ab-initio method to represent polypeptide chains as discrete curves that can be used to derive a meaningful three-dimensional model from only the primary sequence and experimental SAXS data. High resolution crystal structures were used to generate probability density functions for each of the common secondary structural elements found in proteins. These are used to place realistic restraints on the model curves geometry. To evaluate the quality of potential models and demonstrate the efficacy of this novel technique we developed a new statistic to compare the entangled geometry of two open curves, based on mathematical techniques from knot theory. The chain model is coupled with a novel explicit hydration shell model in order derive physically meaningful 3D models by optimizing configurations against experimental SAXS data using a monte-caro based algorithm. We show that the combination of our ab-initio method with spatial restraints based on contact predictions successfully derives a biologically plausible model of the coiled-coil component of the human synaptonemal complex central element protein.\n\nSIGNIFICANCESmall-angle X-ray scattering allows for structure determination of biological macromolecules and their complexes in aqueous solution. Using a discrete curve representation of the polypeptide chain and combining it with empirically determined constraints and a realistic solvent model we are now able to derive realistic ab-initio 3-dimensional models from BioSAXS data. The method only require a primary sequence and the scattering data form the user.

molecular biology↗

bioRxiv2019

A genetic pathway encoding double-stranded RNA transporters and interactors regulates growth and plasticity in Caenorhabditis elegans

The environment and genes shape the development, physiology and behaviour of organisms. Many animal species can take-up double-stranded RNA (dsRNA) from the environment. Environmental dsRNA changes gene expression through RNA interference (RNAi). While environmental RNAi is used as a laboratory tool, e.g. in nematodes, planaria and insects, its biological role remains enigmatic. Here we characterise the environmental dsRNA receptor SID-2 to understand the biological function of dsRNA uptake in Caenorhabditis elegans. First we determine that SID-2 localises to the apical membrane and the trans-Golgi-network (TGN) in the intestine, implicating the TGN as a central cellular compartment for environmental dsRNA uptake. We demonstrate that SID-2 is irrelevant for nucleotide uptake from the environment as a nutritional (nitrogen) source. Instead RNA profiling and high-resolution live imaging revealed a new biological function for sid-2 in growth and phenotypic plasticity. Surprisingly, lack of the ability to uptake environmental RNA reduces plasticity of gene expression. Furthermore, using genetic analyses we show that the dsRNA pathway genes sid-2, sid-1 and rde-4 together regulate growth. This work suggest that environmental RNA affects morphology and plasticity through gene regulation.

molecular biology↗

bioRxiv2022

Single-nucleotide Differences and Cell Type Decide the Subcellular Localization of miRNA Isoforms (isomiRs), tRNA-derived Fragments (tRFs) and rRNA-derived Fragments (rRFs)

BackgroundMicroRNAs (miRNAs) and their isoforms (isomiRs), tRNA-derived fragments (tRFs), and rRNA-derived fragments (rRFs) represent ~95% of all short RNAs found in cells. All three types modulate mRNA and protein abundance and are dysregulated in diseases. Experimental studies to date assumed that the subcellular localization of these molecules is well understood and constant across cell types. ResultsWe investigated the localization of isomiRs, tRFs, and rRFs in biological replicates from three frequently-used model cell lines. In each case, we analyzed the contents of the nucleus, cytoplasm, whole mitochondrion, mitoplast, and the whole cell. We used a rigorous mathematical model to account for cross-fraction contamination and technical errors and adjusted abundances accordingly. We found that isomiRs, tRFs, and rRFs exhibit complex and unexpected patterns of subcellular localization. These patterns depend on the type of the RNA molecule, its exact sequence, and the cell type. Even for "sibling" RNAs from the same parental RNA whose sequences differ by only a few nucleotides, their subcellular localization depends on each siblings exact sequence and the cell type. ConclusionsPrevious studies of isomiRs, tRFs, and rRFs that used ectopic expression without accounting for isoforms may need to be re-evaluated. Future experiments with these molecules will need to distinguish among the multiple isoforms and account for the fact that each isoforms abundance and destination depend on its exact sequence and cell type. The findings additionally suggest the existence of an intracellular trafficking program for isomiRs, tRFs, and rRFs and, by extension, expanded roles for these molecules - both dimensions await characterization. To help design future experiments, we compiled a first-of-its-kind Atlas to catalogue the subcellular localization and abundance of 5,898 isomiRs, tRFs, and rRFs across three model cell lines. Results SummaryO_LIWe analyzed the distribution of microRNA isoforms (isomiRs), tRNA-derived fragments (tRFs), and rRNA-derived fragments (rRFs) in the{whitebullet} nucleus {whitebullet}cytoplasm {whitebullet}mitochondrion, and {whitebullet}mitoplast of biological replicates from three cell lines from the same tissue. C_LIO_LIWe corrected the measured abundances by accounting for cross-fraction contamination and technical errors through a rigorous mathematical model. C_LIO_LIOur analysis revealed complex localization patterns involving numerous isomiRs, tRFs, and rRFs. C_LIO_LIThe subcellular localization of these RNAs depends on their exact sequence and differs even for molecules that arise from the same parental miRNA, tRNA, or rRNA. C_LIO_LIFor a given RNA, its subcellular localization additionally depends on cell type. C_LIO_LIThe findings have implications for previous and future molecular studies of the function of isomiRs, tRFs, and rRFs. C_LIO_LIThe findings suggest the existence of a complex subcellular trafficking program, and hint at expanded functions for these RNA molecules that differ by compartment. C_LIO_LITo assist with the design of future experiments, we created a first-of-its-kind Atlas that catalogues the subcellular distribution and abundance of 5,898 isomiRs, tRFs, and rRFs across three cell lines. C_LI

molecular biology↗

bioRxiv2023

Identification roles of NFE2L3 in Digestive System Cancers

(1) BackgroundThe morbidity and mortality rate of Digestive System Cancers (DSC) continue to threaten human lives and health. Nuclear factor erythroid 2-like protein 3 (NFE2L3) is associated with the development, growth, and progression of multiple cancers. Nevertheless, the clinical value and underlying mechanisms of NFE2L3 in DSC remains unknown. This study aimed to clarify the possibilities of NFE2L3 as a novel biomarker in DSC. (2) MethodsWe obtained the data of NFE2L3 in cancers from database to evaluate the expression level and clinical value of NFE2L3 in DSC, and to explore underlying mechanism and biological functions of NFE2L3 in DSC. (3) ResultsNFE2L3 expression is up-regulated in DSC and have both prognostic and diagnostic value. NFE2L3 contributes to oncogenesis through a variety of mechanisms. In vitro experiment showed that NFE2L3 stimulated the proliferation and migration ability of gastric cancer cells. (4) ConclusionOur study confirms the clinical applicability of NFE2L3 as a promising biomarker for DSC.

molecular biology↗

bioRxiv2025

The coordinated regulatory roles of two LysR-Type Transcriptional Regulators balance chorismate and protocatechuate partition in Listeria organisms

Listeria monocytogenes is an economically deleterious foodborne pathogens that continually challenges the global food supply chain. Listeria species in general, synthesize protocatechuate from saprophytically-derived quinate and shikimate utilizing a novel class of bacterial dehydroshikimate dehydratase. Paradoxically, Listeria species are unable to metabolically utilize protocatechuate, as such, it was proposed that this compound is used as a currency to Listeria interactions with other microorganisms to improve their environmental proliferation. Therefore, an understanding of the regulatory mechanism for the metabolic pathway for protocatechuate biosynthesis is of great importance. Two LysR Type Transcriptional Regulators (LTTR), annotated QuiR and in this study QuiR2, are found upstream of genomic operons, qui1 and qui2, which transcribe genes for protocatechuate synthesis. QuiR, has been shown to activate the expression of genes from both operons with shikimate as a coinducer. However, the role of QuiR2, Lmo2233, is not clear. In this study, we conducted structural, biochemical and bioinformatics analyses of QuiR2 and demonstrated that it functions as a negative regulator of protocatechuate biosynthesis in Listeria species. Moreover, we determined that protocatechuate functions in modulating QuiR2 repressive properties through our mobility shift assay and LacZ reporter activity studies. Furthermore, phylogenetic analyses reveal that QuiR2 clusters closely but independently from QuiR thus supporting their distinct regulatory roles. We propose that QuiR2 prevents metabolic commitment of dehydroshikimate to protocatechuate when elevated and in limiting shikimate condition. In this study we revisited the biological role of the shikimate pathway in microbes and demonstrated that in addition to it producing chorismite for aromatic compound metabolism it is also important in allowing organisms to shuttle shikimate and quinate to produce protocatechuate which can be used as an energy source and more importantly in Listeria it is used to facilitate microbial interactions.

molecular biology↗

bioRxiv2025

Polysaccharides in cryopreservation: multidimensional systematic review of extremophilic traits and the role of selective pressure in structure-function relationships

Cryopreservation of biological matter has accumulated apex biomedical interest for its potential in elongating the shelf-life of biological matter in a state of suspended animation. In Nature, extremophilic microorganisms have an outstanding ability of surviving in habitats where extreme cold, heat, salinity and acidity defy the established boundaries for life. Through Darwinian selective adaptation, they have developed biochemical defense strategies to counteract lethal stimuli. Here, we have compiled an extremophilic EPS structure-function relationship database (XPOL-DB) which aggregates reports on 145 extremophilic and mesophilic EPS, for a total of 128 biochemical and establishes the psychrophilic chemical profile for cold adaptation. Psychrophilic EPS are highly-branched, polyanionic, elongated structures of increased flexibility and molecular weight (16-300 MDa), with predominant expression of polar monomers (GalNAc, GalA, GlcNAc, GlcA) - compared to the linear, rigid, neutral thermophilic EPS. This critical analysis revealed the significant EPS similarity between psychrophiles and halophiles suggests ice growth and extreme salinity are rooted in a shared mechanism of physical membrane destabilization, for which similar chemical traits can dualistically imbue freeze and salt tolerance. Psychrophiles are exciting testbeds for the mapping of how extreme cold selects for cryobiological EPS adaptation; and can fuel reverse engineering efforts to design optimal bio-based, non-cytotoxic cryoprotectant polysaccharides.

molecular biology↗

bioRxiv2026

Integrated serum proteomics and autoantibody analyses reveal a biomarker signature predictive of flare during biologic tapering in rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by a heterogeneous clinical course with periods of remission and flare. Although biologic DMARDs (bDMARDs) have revolutionized RA treatment by enabling sustained disease control, their long-term use is associated with adverse effects and high costs, making dose tapering an attractive but clinically challenging strategy. The lack of reliable biomarkers to predict flare risk limits safe implementation of treatment de-escalation. This study aimed to identify novel circulating protein biomarkers associated with flare risk in RA patients undergoing bDMARDs tapering, useful to enable biomarker-guided treatment optimization strategies. MethodsA discovery proteomic analysis using mass spectrometry was performed on baseline serum samples from a subset of the OPTIBIO clinical trial (n=44), followed by validation in the full cohort (n=194) using ELISA. Functional pathway analysis explored biological processes associated with candidate biomarkers. In parallel, anti-cytokine autoantibodies were profiled using multiplex immunoassays. Logistic and Cox regression models were used to assess associations with flare risk. Predictive models integrating biomarkers and clinical variables were evaluated using receiver operating characteristic (ROC) analysis, sensitivity and specificity metrics, and decision curve analysis to assess clinical utility. ResultsMass spectrometry identified 806 proteins, of which 87 were differentially expressed at baseline between patients who flared and those who maintained remission during follow-up within the intervention (tapering) arm. Functional enrichment analysis highlighted immune-regulatory and innate immune pathways. Among the candidates, V-set immunoglobulin-domain-containing 4 (VSIG4) was validated as a biomarker associated with increased flare risk. Anti-interferon-{gamma} (anti-IFN{gamma}) autoantibodies were also associated with flare. A combined model including VSIG4, anti-IFN{gamma}, and the clinical variable DAS28-CRP improved predictive performance compared with clinical variables alone (AUC 0.76 vs 0.66), achieving significantly higher sensitivity. Decision curve analysis demonstrated higher net benefit of the combined model, indicating improved clinical decision-making. In a secondary analysis focused on patients with prolonged remission, representing the most suitable candidates for safe treatment tapering, the model performance further improved (AUC 0.84). ConclusionIntegration of novel serum proteomic and autoantibody biomarkers with clinical parameters improves prediction of flare during biologic tapering in RA and provides clinically relevant benefit for patient stratification. These findings support further development of biomarker-driven approaches for personalized treatment optimization strategies.

molecular biology↗