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bioRxiv2025

F-53B damages cell viability leading to impaired root development in Arabidopsis

The emerging contaminant chlorinated polyfluoroalkyl ether sulfonate (Cl-PFAES/F-53B) has been detected in many plant species, however, the impact of F-53B on the development in plants and the underpinning mechanisms remain largely unelucidated. In this study, we demonstrate by molecular biology, cytogenetics, and fluorescence microscopy mythologies that F-53B impairs cell viability leading to disrupted root development in Arabidopsis (Arabidopsis thaliana). We show that F-53B suppresses root length and the inhibitory effect is pronunanced at increasing concentrations. F-53B disrupts cell wall and microtubule-mediated cell plate formation and configuration in meristem cells, revealing its toxicity to cell division and cytoskeleton organization. F-53B induces cell death predominantly at the meristematic zone, however, gene exression and genetic studies indicate that F-53B does not trigger DNA double-strand breaks, implying a divergent effect on DNA stability across species. Moreover, we show that F-53B damages nuclei stability and viability, and induces Autophagy-Related 8 protein foci formation implying that it triggers autophagy-mediated cell responses. Taken together, this study provides cellular and molecular insights into the toxicity of F-53B to plant cells, which highlights its damage to agriculture and ecology security that deserve imperative environmental management actions. Environmental ImplicationF-53B has been detected in many plant species, which raises increasing concerns on its toxicity to plants and thus agriculture and ecology safety. However, the toxicity of F-53B to development in plants and the underpinning mechanisms remain largely unelucidated. Here, we demonstrate that F-53B damages nuclei stability and viability leading to cell death and impaired root development in the model plant species Arabidopsis thaliana. Our study provides cytogenetic and molecular insights into the F-53B toxicity to plant cells, which highlights imperative environmental management actions and can be referenced for developing F-53B control and management policies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/653422v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@159b211org.highwire.dtl.DTLVardef@1b99a95org.highwire.dtl.DTLVardef@53b964org.highwire.dtl.DTLVardef@b089be_HPS_FORMAT_FIGEXP M_FIG C_FIG One-sentence summaryF-53B triggers DNA double-strand break-free cell unviability leading to impaired meristematic cell division and root development in plants. HIGHLIGHTSO_LIF-53B impairs root development in Arabidopsis. C_LIO_LIF-53B prohibits mitotic cell division at root tips. C_LIO_LIF-53B damages cell viability predominantly at the meristematic zone in seedlings. C_LIO_LIF-53B does not induce DNA double-strand breaks in Arabidopsis seedlings. C_LIO_LIF-53B damages nuclei viability and stability in seedlings. C_LI

developmental biology↗

bioRxiv2026

Undergraduate Biophysical Chemistry Series: Teaching through a Combination of a Purpose-built Textbook, Research-derived Biomolecular Samples and Computer Labs

Biophysics is a rapidly advancing field with an incredible breadth of topics. Thus, undergraduate biophysics instructors have to strategize and decide what topics they will cover in their courses. Educational institutions utilize a variety of biophysics textbooks. A common deficiency of each of the existing texts is that it serves well a given set of topics (theory, illustrations, practice problems) and leaves out other areas. A typical example includes good theory and problems for thermodynamics and kinetics while presenting molecular dynamics and various spectroscopic methods in a lacking or outdated way. The authors of this manuscript teach a capstone Biophysical Chemistry three-quarter series (Western Washington University/WWU, Bellingham, WA) which ideally should resonate with the general and major-specific courses the students take within their major at WWU. To achieve this goal and to enrich the traditional lecture-based delivery, the instructors have developed and brought together key pedagogical elements: purpose-built online textbook with a uniform structure of the academic content and practice problems, a study sample (oligopeptide) of biophysical significance with a growing set of experimental and computational data and student-centric in-class activities including computer labs. Our Biophysical series emphasizes concepts and methods of computational structural biology (Molecular Dynamics) and spectroscopic approaches (IR, UV and NMR). Here we describe the details of our integrative approach, summarize key outcomes and chart ways to advance the biophysical chemistry series further. Our textbook can be found through LibreText.

scientific communication and education↗

bioRxiv2023

CUR(E)ating a New Approach to Study Fungal Effectors and Enhance Undergraduate Education through Authentic Research

Course-based Undergraduate Research Experiences (CUREs) integrate active, discovery-based learning into undergraduate curriculums, adding tremendous value to Biochemistry and Molecular Biology (BMB) education. There are multiple challenges in transforming a research project into a CURE, such as the readiness of students, the time commitment of the instructor, and the productivity of the research. In this article, we report a CURE course developed and offered in the University of Massachusetts Amherst BMB Department since 2018 that addresses these challenges. Our CURE focuses on fungal effectors which are proteins secreted by a destructive pathogenic fungus Fusarium oxysporum, one of the top five most devastating plant pathogens. By studying this group of proteins, students are connected to real-world problems and participate in the search for potential solutions. A three-week "standard Bootcamp" is implemented to help students familiarize themselves with all basic techniques and boost their confidence. Next, molecular cloning, a versatile technique with modularity and repeatability, is used as the bedrock of the course. Our past five years of experience have confirmed that we have developed a novel and feasible CURE protocol. Measurable progress documented by students who took this course includes stimulated active learning and increased career trajectory to pursue hypothesis-based research to address societal needs. In addition, data generated through the course advance ongoing lab research. Collectively, we encourage the implementation of CURE among research-intensive faculty to provide a more inclusive research experience to all students, an important element in predicting career success.

scientific communication and education↗

bioRxiv2021

Correlative microscopy reveals the nanoscale morphology of E. coli-derived supported lipid bilayers

Supported lipid bilayers (SLBs) made from reconstituted lipid vesicles are an important tool in molecular biology. A breakthrough in the field has come with the use of vesicles derived from cell membranes to form SLBs. These new supported bilayers, consisting both of natural and synthetic components, provide a physiologically relevant system on which to study protein-protein interactions as well as protein-ligand interactions and other lipid membrane properties. These complex bilayer systems hold promise but have not yet been fully characterised in terms of their composition, ratio of natural to synthetic component and membrane protein content. Here, we describe a method of correlative atomic force (AFM) with structured illumination microscopy (SIM) for the accurate mapping of complex lipid bilayers that consist of a synthetic fraction and a fraction of lipids derived from Escherichia coli outer membrane vesicles (OMVs). We exploit the enhanced resolution and molecular specificity that SIM can offer to identify areas of interest in these bilayers and the atomic scale resolution that the AFM provides to create detailed topography maps of the bilayers. We are thus able to understand the way in which the two different lipid fractions (natural and synthetic) mix within the bilayers, quantify the amount of bacterial membrane incorporated in the bilayer and directly visualise the interaction of these bilayers with bacteria-specific, membrane-binding proteins. Our work sets the foundation for accurately understanding the composition and properties of OMV-derived SLBs and establishes correlative AFM/ SIM as a method for characterising complex systems at the nanoscale.

microbiology↗

bioRxiv2023

Dynamic local mRNA distribution and translation influence the postnatal molecular maturation of perivascular astrocytic processes

Astrocytes (the main glial cells in the brain) are highly ramified and send out perivascular processes (PvAPs) that entirely sheathe the brains blood vessels. PvAPs are equipped with an enriched molecular repertoire that sustains astrocytic regulatory functions at the vascular interface. In the mouse, PvAP development starts after birth and is essentially complete by postnatal day (P) 15. Progressive molecular maturation also occurs over this period, with the acquisition of proteins enriched in PvAPs. The mechanisms controlling the development and molecular maturation of PvAPs have not been extensively characterized. We reported previously that mRNAs are distributed unequally in mature PvAPs and are locally translated. Since dynamic mRNA distribution and local translation influence the cells polarity, we hypothesized that they might sustain the postnatal maturation of PvAPs. Here, we used a combination of molecular biology and imaging approaches to demonstrate that the development of PvAPs is accompanied by the transport of mRNA and polysomal mRNA into PvAPs, the development of a rough endoplasmic reticulum (RER) network and Golgi cisternae, and local translation. By focusing on genes and proteins that are selectively or specifically expressed in astrocytes, we characterized the developmental profile of mRNAs, polysomal mRNAs and proteins in PvAPs from P5 to P60. Furthermore, we found that distribution of mRNAs in PvAPs is perturbed in a mouse model of megalencephalic leukoencephalopathy with subcortical cysts. Lastly, we found that some polysomal mRNAs polarized progressively towards the PvAPs. Our results indicate that dynamic mRNA distribution and local translation influence the postnatal maturation of PvAPs. Summary statementLocal translation operates during the postnatal development of perivascular astrocyte processes and might contribute to their molecular maturation.

neuroscience↗

bioRxiv2024

THRESHOLD: A Comprehensive Transcriptomic Analysis Tool for Evaluating Gene Saturation and Impact in Disease Progression

Gene expression studies serve as a foundational tool in molecular biology, providing insights into developmental, physiological, and pathological processes. Variations in gene expression can indicate disease states, which are vital in understanding disease progression, subtype manifestations, and identifying therapeutic targets based on detailed expression patterns. To effectively investigate gene expression patterns, especially in large datasets, a robust and precise analysis tool is crucial. In response to this critical analytical need, we developed THRESHOLD, a novel tool that goes beyond traditional gene expression analysis by introducing the concept of gene saturation. Unlike conventional methods that focus on absolute expression levels or binary differential expression, THRESHOLD quantifies the consistency of gene expression across patients, revealing co-regulation patterns that may otherwise be overlooked. This novel metric offers a unique perspective on gene expression patterns by highlighting consistent trends across patient samples, which are critical for understanding disease mechanisms and stratifying patients based on molecular signatures. The tool offers several features, including user-defined parameters, statistical comparisons, and interactive data visualization. THRESHOLD has uncovered compelling insights into disease progression using TCGA Cancer Datasets. For instance, bladder urothelial carcinoma demonstrated increasing upregulated gene saturation in progressive cancer stages (p < 0.00001). Moreover, THRESHOLD identified heightened gene saturation in patients with earlier onset of prostate adenocarcinoma (p < 0.0001) and revealed a critical fusion transcript, SLC45A2-AMACR, implicated in prostate adenocarcinoma progression, recurrence, and metastasis. Additionally, novel biomarkers and potential candidates for drug therapies were identified through protein-protein interaction networks and functional analyses of saturation data in colon adenocarcinoma and breast invasive carcinoma. Collectively, THRESHOLD advances our understanding of patient stratification and molecular signatures by offering a more detailed view of gene expression dynamics. The THRESHOLD tool is publicly available at: https://github.com/alperuzun/THRESHOLD.

bioinformatics↗

bioRxiv2025

Catalytic Mechanism and Differential Alarmone Regulation of a Conserved Stringent Nucleosidase

Understanding how bacteria rapidly adapt their metabolism in response to external stimuli is key to addressing the present crisis of antibiotics-resistant infections. In the Gram-negative bacterium Escherichia coli, the universal stringent response is elicited in response to some antibiotics and involves production of the global alarmones, (p)ppGpp, which bind directly to many cellular targets. The nucleosidase PpnN that cleaves nucleotides into 5-phosphate ribose and nucleobase, was shown to be a target of (p)ppGpp and control the delicate balance of bacterial fitness and persistence to fluoroquinolone antibiotics, thus conferring optimal survival strategies to bacteria during antibiotic selective pressure. Although both pppGpp and ppGpp stimulate the enzymatic activity of PpnN, they exert distinct effects on the enzymes cooperativity. The molecular mechanism underlying this subtle difference as well as the precise catalytic mechanism of PpnN, remain obscure. In this study, we provide mechanistic insights into the interaction of PpnN with substrate analogue, reaction products and alarmone molecules, which allows us to understand the catalytic mechanism of this family of nucleosidases and the differential modes of regulation by ppGpp and pppGpp, respectively. Comparison with the homologous LOG proteins involved in cytokinin production in plants reveals an ancient, universal mechanism for cleaving purine monophosphates that bacteria have incorporated regulatory controls through alarmones upon stringent responses. IMPORTANCEThis study explores the distinct manners that the stringent alarmones pppGpp and ppGpp interact with the nucleosidase PpnN, which diverge from their typical, consistent effects on other target proteins. Furthermore, PpnN plays a key role in balancing bacterial fitness and tolerance to antibiotics, yet its catalytic mechanism has remained unclear. Through a combination of structural biology, molecular simulations, biochemistry, mutagenesis, and physiological analyses, this research uncovers the mechanistic differences in how both alarmones differentially impact enzyme cooperativity of PpnN and activation under stress conditions. Additionally, by analysing structures of PpnN complexed with substrate analogues, reaction products, and alarmones, as well as conducting bioinformatic comparisons, we propose a conserved catalytic mechanism shared with its homologue, LOG, a protein involved in cytokinin signalling - a critical growth hormone in plants. These insights underscore the nuanced regulatory roles of alarmones and PpnN/LOG group of nucleosidases, highlighting their evolutionary diversification to meet the varied environmental survival needs across organisms.

biochemistry↗

bioRxiv2025

Reproducible Boolean model analyses and simulations with the CoLoMoTo software suite: a tutorial.

This tutorial provides stepwise instructions to install the 20 tools integrated in the CoLoMoTo software suite, to develop reproducible dynamical analyses of logical models of complex biological molecular networks. The tutorial specifically focuses on the analysis of a previously published model of the regulatory network controlling mammalian cell proliferation. It includes chunks of python code to reproduce several of the results and figures published in the original article and further extend these results with the help of a selection of tools included in the CoLoMoTo suite. The notebook covers the visualisation of the network with the tool GINsim, an attractor analysis with bioLQM, the computation of synchronous attractors with BNS, the extraction of modules from the full model, MaBoSS simulations of the wild-type model, as well as of selected mutants, and finally the delineation of compressed probabilistic state transition graphs. The integration of all these analyses in an executable Jupyter notebook greatly eases their reproducibility, as well as the inclusion of further extensions. This notebook can further be used as a template and enriched with other ColoMoTo tools to enable comprehensive dynamical analyses of biological network models.

bioinformatics↗

bioRxiv2026

Student-Driven Microbiome Exploration: A Low-Cost 16S rRNA Sequencing Curriculum for Undergraduate Biology Education

Microbiome science is increasingly important in modern biology education because microbial communities influence human health, ecosystems, and environmental processes. However, undergraduate microbiome instruction is often limited by the high cost and technical complexity of sequencing-based workflows, restricting opportunities for authentic student-driven research. To address this challenge, we developed a low-cost, inquiry-based curriculum that enables undergraduate students to conduct complete microbiome studies using 16S rRNA gene sequencing. The module integrates project design, environmental sample collection, microbial cell processing, PCR amplification, sequencing, and bioinformatic analysis using open-source tools such as QIIME 2. Cost-reduction strategies included centrifugation-based cell collection and a surfactant-assisted direct PCR workflow that eliminated the need for commercial DNA extraction kits. Students designed independent research projects investigating microbial communities in local environments, including campus water sources and gym equipment surfaces. Assessment data from post-course surveys, knowledge checks, and student research products demonstrated strong learning gains in microbiome concepts, molecular biology techniques, scientific communication, and computational analysis. Students reported high confidence in PCR, experimental design, and microbiome interpretation, while also identifying bioinformatics as the most challenging yet rewarding component of the curriculum. All participants expressed increased interest in future research in microbiology or bioinformatics. Overall, this curriculum provides an accessible, scalable framework for integrating next-generation sequencing into undergraduate education while promoting inquiry-driven learning, student ownership, and engagement in authentic scientific research.

microbiology↗

bioRxiv2025

It is Not Always Crystal Clear: In-Pocket Analysis for Understanding Binding Site Microenvironment Properties

Structural biology enabled key advances in biochemistry, drug discovery, and AI-driven three-dimensional structure modelling. Despite its importance, reliable and general computational tools for chemical validation of structural models remain limited, especially when small-molecule ligands are involved. This challenge is exacerbated when experimental resolution is insufficient to define interaction chemistry, leaving critical interpretations ambiguous. We introduce In-Pocket, a quantum mechanical optimisation method biased by experimental data that evaluates the chemical nature of protein-ligand binding interfaces. In-Pocket improves model quality, corrects chemically inconsistent structures, and identifies the energetically most favourable ligand-conformers within biomacromolecular complexes. The approach is generally applicable to a wide range of systems, including those containing transition metals. Benchmarking demonstrates that In-Pocket enhances the reliability of legacy and newly solved structures. With automated, user-friendly routine chemical validation, In-Pocket offers unique opportunities to improve the integrity of structural databases and impact diverse domains: from molecular biology to rational drug design and application of generative AI.

bioinformatics↗

bioRxiv2019

A novel role for the tumor suppressor gene ITF2 in lung tumorigenesis through the action of Wnt signaling pathway

Despite often leading to platinum resistance, platinum-based chemotherapy continues to be the standard treatment for many epithelial tumors. In this study we analyze the cytogenetic alterations that arise after cisplatin treatment providing novel insights into the molecular biology and the cellular mechanisms involved in the acquired resistance in these tumor types. MethodsIn this study, we used 1 million array-CGH and qRT-PCR methodologies to identify and validate cytogenetic alterations that arise after cisplatin treatment in four lung and ovarian paired cisplatin-sensitive/resistant cell lines. We used whole transcriptome sequencing (RNA-seq), functional transfection assays and gene-pathway activity analysis in our experimental cellular models and in fresh frozen primary NSCLC tumors to identify genes with a potential role in the development of this malignancy. Results were further explored in 55 lung and ovarian primary tumors and control samples and in two extensive in silico databases (TCGA and KMplotter) with 1,926 NSCLC and 1,425 additional epithelial tumors. ResultsLong-term cell exposure to platinum induces the frequent deletion of ITF2 gene. Restoration of ITF2 expression re-sensitizes tumor cells to platinum and recovers the levels of Wnt/{beta}-catenin transcriptional activity. ITF2 expression was also frequently downregulated in NSCLC, ovarian and other epithelial tumors, predicting a worse overall survival. We also identified an inverse correlation in expression between ITF2 and HOXD9, revealing that NSCLC patients with lower expression of HOXD9 have a better overall survival rate that was independent of the tumor histology. ConclusionWe have defined the implication of ITF2 as a molecular mechanism behind the development of cisplatin resistance probably through the activation of the Wnt-signaling pathway. Our translational data suggest that ITF2 could be used as a general epithelial tumor platinum-predictive marker and have identified HOXD9 as a potential prognostic biomarker in NSCLC, a gene which expression is induced by Wnt signaling. Furthermore, this data highlights the possible role of ITF2 and HOXD9 as a novel therapeutic target for platinum resistant tumors.

cancer biology↗

bioRxiv2023

High-quality genome assemblies of four members of the Podospora anserina species complex

The filamentous fungus Podospora anserina is a model organism used extensively in the study of molecular biology, senescence, prion biology, meiotic drive, mating-type chromosome evolution, and plant biomass degradation. It has recently been established that P. anserina is a member of a complex of seven, closely related species. In addition to P. anserina, high-quality genomic resources are available for two of these taxa. Here we provide chromosome-level annotated assemblies of the four remaining species of the complex, as well as a comprehensive dataset of annotated assemblies from a total of 28 Podospora genomes. We find that all seven species have genomes of around 35 Mbp arranged in seven chromosomes that are mostly collinear and less than 2% divergent from each other at genic regions. We further attempt to resolve their phylogenetic relationships, finding significant levels of phylogenetic conflict as expected from a rapid and recent diversification. SignificanceHere we provide a dataset of 28 annotated genomes from the P. anserina species complex, including chromosome-level assemblies of four species that lacked a reference genome. With this dataset in hand, biologists can take advantage of the molecular tools available for P. anserina to study evolutionary dynamics at the interphase between micro- and macroevolution, with particular emphasis on trait evolution, genome architecture, and speciation.

genomics↗

bioRxiv2022

Hepatobiliary organoids derived from leporids support the replication of hepatotropic lagoviruses

Lagoviruses, family Caliciviridae, are some of the most virulent vertebrate viruses known. They infect leporids, i.e., rabbits (Oryctolagus spp.), hares (Lepus spp.) and cottontails (Sylvilagus spp.) and rapidly kill over 95% of susceptible animals. Pathogenic lagoviruses are hepatotropic and induce a fulminant hepatitis that typically leads to disseminated intravascular coagulation within 24-72 hours of infection. However, the pathophysiological mechanisms governing this extreme phenotype and other aspects of the fundamental biology of these viruses are poorly understood due to a lack of cell culture systems. Here, we report on a robust and reliable ex vivo model for the cultivation of hepatotropic lagoviruses. We show that three rabbit haemorrhagic disease virus (RHDV) variants, RHDV1, RHDV2 and RHDVa-K5, replicate in monolayer cultures derived from rabbit hepatobiliary organoids. Viral replication was demonstrated by a (i) increase in viral RNA levels of greater than one log10 over a 23-hour period, (ii) detection of viral structural and non-structural proteins, and (iii) detection of double-stranded RNA viral replication intermediates. Furthermore, we generated hepatobiliary organoids from a feral cat (Felis domesticus), a wild mouse (Mus musculus) and a European brown hare (Lepus europaeus) and showed that monolayer cultures derived from the cat and mouse organoids were not permissive for lagovirus infection, while those derived from the hare organoids only supported replication of RHDV2, recapitulating the species tropism that has been observed with these viruses. Our organoid culture system will facilitate future studies into the molecular biology of lagoviruses, which will have considerable import for the conservation of endangered leporid species in Europe and North America and the biocontrol of overabundant rabbit populations in Australia and New Zealand.

microbiology↗

bioRxiv2023

Highly resolved genome assembly and comparative transcriptome profiling reveal genes related to developmental stages of the tapeworm Ligula intestinalis

Ligula intestinalis (Cestoda: Diphyllobothriidae) is an emerging model organism for studies on parasite population biology and host-parasite interactions. However, a well resolved genome and catalogue of its gene content has not been previously developed. Here, we present the first genome assembly of L. intestinalis, based on Oxford Nanopore Technologies, Illumina and Omni-C sequencing methodologies. We use transcriptome profiling to compare plerocercoid larvae and adult worms and identify differentially expressed genes associated with these life stages. The genome assembly is 775.5 Mbp in size, with scaffold N50 value of 118 Mbp and encodes 27,256 predicted protein-coding sequences. Over 60% of the genome consists of repetitive sequences. Synteny analyses showed that the 10 largest scaffolds representing 75% of the genome display high correspondence to full chromosomes of cyclophyllidean tapeworms. Mapping RNA-seq data to the new reference genome we identified 3,922 differentially expressed genes in adults compared to plerocercoids. Gene Ontology analyses revealed overrepresented genes involved in reproductive development of the adult stage (e.g. sperm production) and significantly enriched DEGs associated with immune evasion of plerocercoids in their fish host. This study provides the first insights into the molecular biology of L. intestinalis and provides the most highly contiguous assembly to date of a diphyllobothriid tapeworm useful for population and comparative genomic investigations of parasitic flatworms.

genomics↗

bioRxiv2023

Exploring functional conservation in silico: a new machine learning approach to RNA-editing

Around 50 years from now, molecular biology opened the path to understand changes in forms, adaptations, complexity, or the basis of human diseases, through myriads of reports on gene birth, gene duplication, gene expression regulation, and splicing regulation, among other relevant mechanisms behind gene function. Here, with the advent of big data and artificial intelligence (AI), we focus on an elusive and intriguing mechanism of gene function regulation, RNA editing, in which a single nucleotide from an RNA molecule is changed with a remarkable impact in the increase of the complexity of transcriptome and proteome. We present a new generation approach to assess the functional conservation of the RNA-editing targeting mechanism using two AI learning algorithms, random forest (RF) and bidirectional long short-term memory (biLSTM) neural networks with attention layer. These algorithms combined with RNA-editing data coming from databases and variant calling from same-individual RNA and DNA-seq experiments from different species, allowed us to predict RNA-editing events using both primary sequence and secondary structure. Then, we devised a method for assessing conservation or divergence in the molecular mechanisms of editing completely in silico: the cross-training analysis. This novel method not only helps to understand the conservation of the editing mechanism through evolution but could set the basis for understanding how it is involved in several human diseases.

genetics↗

bioRxiv2025

SAND-MEDIATED DNA EXTRACTION FROM ORCHIDS FOR GENOMIC APPLICATIONS

DNA extraction is an essential routine procedure for downstream applications in genetics and molecular biology. Since DNA was extracted for the first time, countless protocols have been developed yielding excellent results; however, the diversity of plant families and the distribution in remote areas represent a challenge for these protocols. Orchidaceae, one of the most species-rich plant families in the world, is a group with abundant polyphenols and polysaccharides in its tissues. In addition, sample collection in remote areas represents a challenge, consequently, conventional protocols can be inappropriate for obtaining DNA. In this way, CTAB-based gel preservation has emerged as an excellent alternative to easily collect, store and transport samples, moreover, alternatives such as sterile sand and DNA stabilization solutions during grinding allow a suitable DNA extraction. Here, we combined saturated NaCI-CTAB gels, sterile sand, sucrose-based DNA stabilization buffer, and conventional CTAB solution for obtaining DNA from fresh and stored samples for up to 32 days at room temperature, and described, from sample collection to spectrophotometer-obtained quantification, a non-organic method for obtaining DNA from orchids species for genomic applications. This protocol allows not only the extraction of suitable quantities of high molecular weight DNA of adequate purity and integrity from different orchid species without the use of expensive equipment and cumbersome reagents such as liquid nitrogen and toxic phenols, and/or time-consuming procedures, but also, the use of this DNA in downstream applications such as PCR-based genotyping, restriction enzyme analysis, and most genomic applications.

genetics↗

bioRxiv2023

Direct, quantitative, and comprehensive analysis of tRNA acylation using intact tRNA liquid-chromatography mass-spectrometry

Aminoacyl-tRNA synthetases (aaRSs) provide the functional and essential link between the sequence of an mRNA and the protein it encodes. aaRS enzymes catalyze a two-step chemical reaction that acylates specific tRNAs with a cognate -amino acid. In addition to their role in translation, acylated tRNAs contribute to non-ribosomal natural product biosynthesis and are implicated in multiple human diseases. From the standpoint of synthetic biology, the acylation of tRNAs with a non-canonical -amino acid (ncAA) or more recently, a non--amino acid monomer (nAA) is a critical first step in the incorporation of these monomers into proteins, where they can be used for fundamental and applied science. These endeavors all demand an understanding of aaRS activity and specificity. Although a number of methods to monitor aaRS function in vitro or in vivo have been developed, many evaluate only the first step of the two-step reaction, require the use of radioactivity, or are slow, difficult to generalize, or both. Here we describe an LC-MS assay that rapidly, quantitatively, and directly monitors aaRS activity by detecting the intact acyl-tRNA product. After a simple tRNA acylation reaction workup, acyl- and non-acyl-tRNA molecules are resolved using ion-pairing reverse phase chromatography and their exact masses are determined using high-resolution time-of-flight mass spectrometry. The intact tRNA assay we describe is fast, simple, and quantifies reaction yields as low as 0.23%. The assay can also be employed on tRNAs acylated with flexizyme to detect products that are undetectable using standard techniques. The protocol requires basic expertise in molecular biology, mass spectrometry, and RNAse-free techniques.

biochemistry↗

bioRxiv2015

Wikidata as a semantic framework for the Gene Wiki initiative

Open biological data is distributed over many resources making it challenging to integrate, to update and to disseminate quickly. Wikidata is a growing, open community database which can serve this purpose and also provides tight integration with Wikipedia.\n\nIn order to improve the state of biological data, facilitate data management and dissemination, we imported all human and mouse genes, and all human and mouse proteins into Wikidata. In total, 59,530 human genes and 73,130 mouse genes have been imported from NCBI and 27,662 human proteins and 16,728 mouse proteins have been imported from the Swissprot subset of UniProt. As Wikidata is open and can be edited by anybody, our corpus of imported data serves as the starting point for integration of further data by scientists, the Wikidata community and citizen scientists alike. The first use case for this data is to populate Wikipedia Gene Wiki infoboxes directly from Wikidata with the data integrated above. This enables immediate updates of the Gene Wiki infoboxes as soon as the data in Wikidata is modified. Although Gene Wiki pages are currently only on the English language version of Wikipedia, the multilingual nature of Wikidata allows for a usage of the data we imported in all 280 different language Wikipedias. Apart from the Gene Wiki infobox use case, a powerful SPARQL endpoint and up to date exporting functionality (e.g. JSON, XML) enable very convenient further use of the data by scientists.\n\nIn summary, we created a fully open and extensible data resource for human and mouse molecular biology and biochemistry data. This resource enriches all the Wikipedias with structured information and serves as a new linking hub for the biological semantic web.

Bioinformatics↗