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Results for “molecular biology”

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bioRxiv2025

Developing terrestrial environmental DNA sampling methods for detecting arboreal invasive reptiles: a case study of the green anole in the Ogasawara Island, Japan

Early detection of invasive alien species is essential for preventing establishment and mitigating ecological impacts, particularly in island ecosystems harbouring evolutionarily isolated endemic species. Recently, despite increasing reptile introductions and their suggested widespread impacts, methods for monitoring arboreal invasive reptiles remain limited. This study addressed the need for practical detection tools by developing terrestrial environmental DNA (eDNA) sampling methods that collect DNA from leaf surfaces to detect the arboreal invasive green anole (Anolis carolinensis) in the Ogasawara Islands, Japan. Two methods, wiping leaf surfaces with gauze and rinsing with sprayed water, were tested. At an invaded site, green anoles were successfully detected in eight out of 10 samples in both methods. Although no significant difference in eDNA concentration was observed, the wiping method was selected for its greater simplicity. Subsequently, the relationship between green anole population density and eDNA concentrations detected using the wiping method was investigated, suggesting a significant positive relationship. This is the first report demonstrating that terrestrial eDNA concentrations can reflect arboreal reptile population density, suggesting potential applications in quantitative terrestrial biodiversity assessments. Furthermore, the successful detection of eDNA even in the extremely low-density habitat of the green anole demonstrates the usefulness of eDNA-based surveys for early detection of invasions. The method developed here may be broadly applicable to terrestrial biodiversity monitoring, especially in tree-dwelling taxa. Given accelerating biological invasions and biodiversity loss, this approach is expected to benefit managers, conservationists, and researchers concerned with terrestrial ecosystems.

molecular biology↗

bioRxiv2017

Antigen B from Echinococcus granulosus enters mammalian cells by endocytic pathways

Cystic hydatid disease is a zoonosis caused by the larval stage (hydatid cyst) of Echinococcus granulosus (Cestoda, Taeniidae). The hydatid cyst develops in the viscera of intermediate host as a unilocular structure filled by the hydatid fluid, which contains parasitic excretory/secretory products. Antigen B (AgB) is the major component of E. granulosus metacestode hydatid fluid. Functionally, AgB has been implicated in immunomodulation and lipid transport. However, the mechanisms underlying AgB functions are not completely known. In this study, we investigated AgB interactions with different mammalian cell types and the pathways involved in its internalization. AgB uptake was observed in four different cell lines, NIH-3T3, A549, J774 and RH. Inhibition of raft-mediated endocytosis causes about 50 and 69% decrease in AgB internalization by RH and A549 cells, respectively. Interestingly, AgB colocalized with the raft endocytic marker, but also showed a partial colocalization with the clathrin endocytic marker. The results indicate that raft-mediated endocytosis is the main route to AgB internalization, and that a clathrin-mediated entry may also occur at a lower frequency. Cellular internalization could be a requirement for AgB functions as a lipid carrier and/or immunomodulatory molecule, contributing to create a more permissive microenvironment to metacestode development and survival.\n\nAuthor summaryAntigen B (AgB) is an oligomeric lipoprotein highly abundant in Echinococcus granulosus hydatid fluid. AgB has already been characterized as an immunomodulatory protein, capable of inducing a permissive immune response to parasite development. Also, an important role in lipid acquisition is attributed to AgB, because it has been found associated to different classes of host lipids. However, the mechanisms of interaction employed by AgB to perform its functions remain undetermined. In this study, we demonstrate that mammalian cells are able to internalize E. granulosus AgB in culture and found that specific mechanisms of endocytosis are involved. Our results extend the understanding of AgB biological role indicating cellular internalization as a mechanism of interaction, which in turn, may represent a target to intervention.

molecular biology↗

bioRxiv2018

Association of oxidative stress, telomere length, and frailty in an elder population

BackgroundA global aging population requires focusing on the risk factors for unhealthy aging, preventive medicine, and chronic disease management. The identification of adverse health outcomes in older adults has been addressed by the characterization of frailty as a biological syndrome. On the other hand, oxidative stress and telomere length have been suggested as biomarkers of aging.\n\nObjectiveTo study the association of oxidative stress, telomere length, and frailty in an old age population.\n\nMethodsThis was a cross-sectional study based on 2015 data from 202 members from a cohort of older adults (n=202; gender F/M ratio: 133/69; mean age: 69.89 {+/-} 7.39 years). Reactive oxygen species (ROS) were measured by dichlorofluorescin diacetate, and lipid peroxidation by malondialdehyde. Telomere length was determined using qPCR with SYBR Green Master Mix.\n\nResultsStatistical analysis showed an association between telomere length and frailty but no association between oxidative stress on telomere length or frailty.\n\nConclusionsTelomere length could eventually be used as a marker to discriminate between healthy and unhealthy aging as expressed by frailty phenotype. However, oxidative stress seems as just a biological process of aging.

molecular biology↗

bioRxiv2019

A smart polymer for sequence-selective binding, pulldown and release of DNA targets

Selective isolation of DNA is crucial for applications in biology, bionanotechnology, clinical diagnostics and forensics. We herein report a smart methanol-responsive polymer (MeRPy) that can be programmed to bind and separate single- as well as double-stranded DNA targets. Captured targets are quickly isolated and released back into solution by denaturation (sequence-agnostic) or toehold-mediated strand displacement (sequence-selective). The latter mode allows 99.8% efficient removal of unwanted sequences and 79% recovery of highly pure target sequences. We applied MeRPy for the depletion of insulin, glucagon, and transthyretin cDNA from clinical next-generation sequencing (NGS) libraries. This step improved data quality for low-abundance transcripts in expression profiles of pancreatic tissues. Its low cost, scalability, high stability and ease of use make MeRPy suitable for diverse applications in research and clinical laboratories, including enhancement of NGS libraries, extraction of DNA from biological samples, preparative-scale DNA isolations, and sorting of DNA-labeled non-nucleic acid targets.

molecular biology↗

bioRxiv2026

Airspace miR-146a levels in ventilated patients decrease with age and correlate with mortality

The acute respiratory distress syndrome is a heterogenous syndrome characterized by the rapid development of respiratory failure. Nearly 40% of patients who develop ARDS will die, and there is growing interest in identification of biomarkers to identify patients at risk of death and/or inform treatment decisions. Most prior work on biomarkers in ARDS has focused on the plasma compartment, but there is concern that circulating biomarkers may not reflect alveolar pathobiology. The anti-inflammatory microRNA-146a has been shown to be upregulated in inflammatory cells in human bronchoalveolar lavage fluid, but it is not known if these levels correspond with outcomes. We measured miR-146a expression by digital droplet PCR in human biospecimens from four different cohorts of patients with respiratory failure requiring mechanical ventilation - two plasma cohorts, one bronchoalveolar lavage cohort, and one heat moisture exchange (HME) filter fluid cohort. We found that miR-146a was detectible in plasma, bronchoalveolar lavage fluid, and HME fluid. However, only when measured in the alveolar space, was miR-146a expression significantly lower in older adults and those who died. It did not correlate with outcomes when measured in plasma. To our knowledge, this is the first report that nucleotides can be measured in HME fluid and builds upon expanding literature that circulating biomarkers may not reflect complex biology of the alveolar microenvironment during ARDS.

molecular biology↗

bioRxiv2026

Cardiomyocytes possess an intrinsic catecholaminergic machinery that regulates cellular homeostasis and electrophysiological stability

BackgroundCatecholamines play a central role in cardiac performance, coordinating myocardial contractility, conduction, metabolism, and electrophysiological stability. In the heart, their actions have been attributed primarily to sympathetic nerve terminals and circulating adrenal catecholamines. The discovery of an intrinsic non-neuronal cholinergic system within cardiomyocytes challenges this neurocentric paradigm and raises the possibility that cardiomyocytes also possess an intrinsic catecholaminergic programme. Here, we investigated whether cardiomyocytes possess an intrinsic catecholaminergic programme and its contribution to cardiomyocyte homeostasis and stress responses. MethodsWe investigated catecholamine biosynthesis and handling in human induced pluripotent stem cell-derived cardiomyocytes, adult mouse cardiomyocytes, H9C2 cells, rat ventricular tissue, and Langendorff-perfused mouse hearts. Protein expression of catecholamine biosynthetic enzymes and vesicular monoamine transporters was assessed by immunoblotting and immunohistochemistry, while vesicular monoamine uptake was evaluated using fluorescent false neurotransmitters. Functional consequences of catecholamine biosynthesis inhibition were examined using pharmacological approaches, assessing cell viability, apoptosis, organelle homeostasis, metabolic signalling, and cardiac electrophysiology. ResultsTyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine {beta}-hydroxylase, and vesicular monoamine transporters were detected in cardiomyocytes across models. Expression of catecholamine biosynthetic enzymes increased following ischaemia-reperfusion injury in rat heart tissue (TH p=0.008, AADC p=0.031, DBH p=0.008). Pharmacological inhibition of catecholamine biosynthesis caused dose-dependent reductions in cardiomyocyte viability (p<0.0001), increased apoptosis, organelle stress, and mitochondrial dysfunction, with greater effects under oxidative stress. Mechanistically, catecholamine depletion suppressed mTORC1 signalling and activated LKB1-AMPK-ULK1 pathways. In Langendorff-perfused hearts, tyrosine hydroxylase inhibition induced ventricular arrhythmias in 5 of 6 hearts, including sustained ventricular tachycardia, polymorphic ventricular tachycardia, and ventricular fibrillation. ConclusionsThese findings identify cardiomyocytes as previously unrecognised catecholamine-competent cells expressing intrinsic machinery for catecholamine biosynthesis and vesicular handling. Disruption of this pathway compromises metabolic and organelle homeostasis, activates energy-stress and autophagy-related signalling, and promotes malignant ventricular arrhythmias. Intrinsic cardiomyocyte catecholamine biology therefore represents a non-neuronal regulatory axis essential for myocardial resilience and electrical stability, with potential relevance to ischaemic injury and stress-induced dysfunction.

molecular biology↗

bioRxiv2025

BoltzDesign1: Inverting All-Atom Structure Prediction Model for Generalized Biomolecular Binder Design

Deep learning in structure prediction has revolutionized protein research, enabling large-scale screening, novel hypothesis generation, and accelerated experimental design across biological domains. Recent advances, including RoseTTAFold-AA and AlphaFold3, have extended structure prediction models to work with small molecules, nucleic acids, ions, and covalent modifications. We present BoltzDesign1, which inverts the Boltz-1 model, an open source reproduction of AlphaFold3, to enable the design of protein binders for diverse molecular targets without requiring model finetuning. By utilizing only the Pairformer and Confidence modules, our method significantly reduces computational costs while achieving outstanding in silico success rates and diversity in binder generation. Optimizing directly on the distogram allows us to shape the probability distribution of atomic distances, rather than adjusting a single structure, steering the design toward sequences that yield robust structures with well-defined energy minima. By leveraging a fully atomic model trained on a wide variety of macromolecules, we can generate diverse heterocomplexes with flexible ligand conformations--a capability not currently matched by existing methods. This approach enables the design of novel protein interactions with potential applications in biosensors, enzyme engineering, therapeutic development, and biotechnological innovations.

molecular biology↗

bioRxiv2022

Programmable RNA Sensing for Cell Monitoring and Manipulation

RNAs are the central and universal mediator of genetic information underlying the diversity of cell types and cell states, which together shape tissue organization and organismal function across species and life spans. Despite advances in RNA sequencing and massive accumulation of transcriptome datasets across life sciences, the dearth of technologies that leverage RNAs to observe and manipulate cell types remains a prohibitive bottleneck in biology and medicine. Here, we describe CellREADR (Cell access through RNA sensing by Endogenous ADAR), a programmable RNA sensing technology that leverages RNA editing mediated by ADAR (adenosine deaminase acting on RNA) for coupling the detection of cell-defining RNAs with translation of effector proteins. Viral delivery of CellREADR conferred specific cell type access in mouse and rat brains and in ex vivo human brain tissues. Furthermore, CellREADR enabled recording and control of neuron types in behaving mice. CellREADR thus highlights the potential for RNA-based monitoring and editing of animal cells in ways that are specific, versatile, easy, and generalizable across organ systems and species, with broad applications in biology, biotechnology, and programmable RNA medicine.

molecular biology↗

bioRxiv2022

Unique and shared proteome responses of rice plants (Oryza sativa) to individual abiotic stresses

Food safety of staple crops such as rice is of global concern and is at the top of the policy agenda worldwide. Abiotic stresses are one of the main limitations to optimizing yields for sustainability, food security and food safety. We analyzed proteome changes in Oryza sativa ssp. Nipponbare in response to three adverse abiotic treatments, including three levels of drought (mild, moderate, and severe), soil salinization, and non-optimal temperatures. All treatments had modest, negative effects on plant growth, enabling us to identify proteins that were common to all stresses, or unique to one. More than 75% of the total of differentially abundant proteins in response to abiotic stresses were specific to individual stresses, while fewer than 5% of stress-induced proteins were shared across all abiotic constraints. Stress-specific and non-specific stress-responsive proteins identified were categorized in terms of core biological processes, molecular functions, and cellular localization. Data AccessAll data have also been submitted to the PRIDE data repository, and will be available with project identifier PXD037280.

plant biology↗

bioRxiv2022

METTL3 maintains epithelial homeostasis through m6A-dependent regulation of chromatin modifiers

The balance between epithelial stemness and differentiation requires the precise regulation of gene expression programs. Epitranscriptomic RNA modifications have been implicated in both epithelial development as well as cancers. However, the underlying mechanisms are poorly understood. Here, we show that deletion of the m6A methyltransferase, METTL3, impairs the m6A-mediated degradation of numerous mRNA transcripts encoding critical chromatin modifying enzymes, resulting in widespread gene expression abnormalities as well as both aberrant cutaneous and oral epithelial phenotypes in vivo. Collectively, these results offer new insights into a new layer of gene regulation within epithelial surface tissues and will inform future epitranscriptomic studies within epithelial cancer and developmental biology.

molecular biology↗

bioRxiv2023

Super-resolution fluorescence imaging of cryosamples does not limit achievable resolution in cryoEM

Correlated super-resolution cryo-fluorescence and cryo-electron microscopy (cryoEM) has been gaining popularity as a method to investigate biological samples with high resolution and specificity. A concern in this combined method (called SR-cryoCLEM), however, is whether and how fluorescence imaging prior to cryoEM acquisition is detrimental to sample integrity. In this report, we investigated the effect of high-dose laser light irradiation on apoferritin samples prepared for cryoEM with excitation wavelengths commonly used in fluorescence microscopy, and comparing these samples to controls that were kept in the dark. We found that laser illumination, of equal duration and intensity as used in super-resolution cryomicroscopy and in the presence of high concentrations of fluorescent protein, did not affect the achievable resolution in cryoEM, with final reconstructions reaching resolutions of ~1.8 [A] regardless of the illumination conditions. The finding that super-resolution fluorescence imaging of cryosamples prior to cryoEM data acquisition does not limit the achievable resolution suggests that super-resolution cryo-fluorescence microscopy and in situ structural biology using cryoEM are entirely compatible. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗

bioRxiv2024

Widespread destabilization of C. elegans microRNAs by the E3 ubiquitin ligase EBAX-1

MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to form complexes that direct mRNA repression. miRNAs are also the subject of regulation. For example, some miRNAs are destabilized through a pathway in which pairing to specialized transcripts recruits the ZSWIM8 E3 ubiquitin ligase, which polyubiquitinates AGO, leading to its degradation and exposure of the miRNA to cellular nucleases. Here, we found that 22 miRNAs in C. elegans are sensitive to loss of EBAX-1, the ZSWIM8 ortholog in nematodes, implying that these 22 miRNAs might be subject to this pathway of target-directed miRNA degradation (TDMD). The impact of EBAX-1 depended on the developmental stage, with the greatest effect on the miRNA pool (14.5%) observed in L1 larvae and the greatest number of different miRNAs affected (17) observed in germline-depleted adults. The affected miRNAs included the miR-35-42 family, as well as other miRNAs among the least stable in the worm, suggesting that TDMD is a major miRNA-destabilization pathway in the worm. The excess miR-35-42 molecules that accumulated in ebax-1 mutants caused increased repression of their predicted target mRNAs and underwent 3' trimming over time. In general, however, miRNAs sensitive to EBAX-1 loss had no consistent pattern of either trimming or tailing. Replacement of the 3' region of miR-43 substantially reduced EBAX-1 sensitivity, a result that differed from that observed previously for miR-35. Together, these findings broaden the implied biological scope of TDMD-like regulation of miRNA stability in animals, and indicate that a role for miRNA 3' sequences is variable in the worm.

molecular biology↗

bioRxiv2025

Longitudinal and large-scale monitoring of transcriptome and RBP-RNA interactome in living cells by engineered protein nanocages

Nondestructive sequencing of RNA from live cells is essential for monitoring and understanding dynamic biological processes. However, most existing RNA sequencing methods rely on cell lysis or fixation, limiting their applicability for longitudinal studies. Here, we introduce POND-seq (Protein nanocage-empOwered Non-Destructive sequencing), a novel approach that employs secretory protein nanocages fused with RNA-binding proteins (RBPs) to capture the RBP-RNA interactome and transcriptome in live cells. POND-seq reliably identifies RNA targets of canonical RBPs across multiple cell types. By fusing poly(A)-binding protein (PABPC1) to the nanocage, we demonstrate that POND-seq can monitor transcriptomic changes in response to signaling stimuli and selectively capture cell-type-specific transcriptomes from mixed populations. Additionally, POND-seq facilitates the dissection of RNA-binding domains and key amino acid residues critical for RBP-RNA interactions. We further highlight its utility in large-scale screening, offering compelling evidence for the pathogenicity of FMR1 variants. POND-seq represents a transformative advancement in RNA biology, cell biology and precision medicine, enabling unprecedented insights into cellular dynamics and disease mechanisms.

molecular biology↗

bioRxiv2025

A unique mechanism explaining the outstanding performance of a newly discovered doxycycline riboswitch

Synthetic riboswitches offer a versatile and protein-independent solution for conditional gene regulation. They consist of a regulatory domain and an aptamer domain that binds a specific ligand, with their performance largely depending on the ability of the aptamer to control the regulatory domain. Expanding the range of synthetic riboswitches therefore requires the discovery and characterization of new regulatory aptamers. In the present study, we identified a doxycycline-binding aptamer with outstanding regulatory properties in both yeast and human cells which are based on a unique structural dynamic upon ligand binding. Single-molecule force spectroscopy revealed that doxycycline binding strongly stabilizes an intermediate aptamer conformation offering mechanistic insights into its function. The identification of the aptamer through a combination of parallel SELEX and subsequent in vivo screening in yeast, also provided valuable insights into selection dynamics and the first proof for the effectiveness of RNA Capture-SELEX in aptamer selection. Together, the presented data deepen our understanding of regulatory aptamer selection and functionality while adding a high-performing doxycycline-responsive aptamer to the synthetic biology toolbox.

molecular biology↗

bioRxiv2019

A consensus set of genetic vulnerabilities to ATR inhibition

The response to DNA replication stress in eukaryotes is under the control of the ataxia-telangiectasia and Rad3-related (ATR) kinase. ATR responds to single-stranded (ss) DNA to stabilize distressed DNA replication forks, modulate DNA replication firing and prevent cells with damaged DNA or incomplete DNA replication from entering into mitosis. Furthermore, inhibitors of ATR are currently in clinical development either as monotherapies or in combination with agents that perturb DNA replication. To gain a genetic view of the cellular pathways requiring ATR kinase function, we mapped genes whose mutation causes hypersensitivity to ATR inhibitors with genome-scale CRISPR/Cas9 screens. We delineate a consensus set of 117 genes enriched in DNA replication, DNA repair and cell cycle regulators that promote survival when ATR kinase activity is suppressed. We validate 14 genes from this set and report genes not previously described to modulate response to ATR inhibitors. In particular we found that the loss of the POLE3/POLE4 proteins, which are DNA polymerase e accessory subunits, results in marked hypersensitivity to ATR inhibition. We anticipate that this 117-gene set will be useful for the identification of genes involved in the regulation of genome integrity, the characterization of new biological processes involving ATR, and may reveal biomarkers of ATR inhibitor response in the clinic.

molecular biology↗

bioRxiv2019

The yeast proteases Ddi1 and Wss1 are both involved in the DNA replication stress response.

Genome integrity and cell survival are dependent on proper replication stress response. Multiple repair pathways addressing obstacles generated by replication stress arose during evolution, and a detailed understanding of these processes is crucial for treatment of numerous human diseases. Here, we investigated the strong negative genetic interaction between two proteases involved in the DNA replication stress response, yeast Wss1 and Ddi1. While Wss1 proteolytically acts on DNA-protein crosslinks, mammalian DDI1 and DDI2 proteins remove RTF2 from stalled forks via a proposed proteasome shuttle hypothesis. We show that the double-deleted {Delta}ddi1, {Delta}wss1 yeast strain is hypersensitive to the replication drug hydroxyurea and that this phenotype can be complemented only by catalytically competent Ddi1 protease. Furthermore, our data show the key involvement of the helical domain preceding the Ddi1 protease domain in response to replication stress caused by hydroxyurea, offering the first suggestion of this domains biological function. Overall, our study provides a basis for a novel dual protease-based mechanism enabling yeast cells to counteract DNA replication stress.

molecular biology↗

bioRxiv2019

Human PUM1 and PUM2 exhibit regulation of divergent mRNA targets in male germ cells

Mammalian Pumilio (PUM) proteins are sequence-specific, RNA-binding proteins with wide-ranging roles, including germ cell development that has functional implications in fertility. Although human PUM1 and PUM2 are closely related to each other and recognize the same RNA binding motif, there is some evidence for functional diversity, particularly related to their roles in fertility. Here, by RNA sequencing (RNA-Seq) approaches, we identified separate mRNA pools regulated by PUM1 and PUM2 proteins in human male germ cells. Using global mass spectrometry-based profiling, we identified distinct PUM1- and PUM2-bound putative protein cofactors, most of them involved in RNA processing. Combinatorial analysis of RNA-Seq and mass spectrometry findings revealed that PUM1 and PUM2 may form distinct RNA-regulatory networks, with different roles in human reproduction and testicular tumorigenesis. Our findings highlight the functional divergence and versatility of PUM paralogue-based post-transcriptional regulation, offering insight into the mechanisms underlying their diverse biological roles and diseases resulting from their dysfunction.

molecular biology↗

bioRxiv2025

Functional Mapping and Engineering of the Sec Translocon Unlocked by a Cell-Free System

Almost all proteins are inserted or translocated across membranes by the universally conserved Sec translocon. Despite its central role, experimental access to Sec function has remained limited. Here, we present a cell-free protein synthesis platform that inserts SecYEG into synthetic vesicles, enabling direct testing of Sec in real-time and high-throughput, circumventing longstanding viability constraints. Screening 300 Sec variants in a single experiment, we consolidate three decades of Sec research, while vastly expanding mutant diversity for structure-function insights. Mapping over 30 functionally critical regions that modulate Sec activity across three orders of magnitude, we uncover dozens of super-active variants. We further leverage our system to increase membrane protein quality and nanobody export, highlighting the potential of our system for advancing applications in synthetic biology and biotechnology.

molecular biology↗