Biological Questions Research Articles

Parahydrogen Polarization in Reverse Micelles and Application to Sensing of Protein-Ligand Binding.

A medium containing reverse micelles supports non-hydrogenative parahydrogen induced polarization (nhPHIP) in the organic phase while solubilizing a protein in the aqueous phase. Strongly enhanced NMR signals from iridium hydride complexes report on a ligand, 4-amino-2-benzylaminopyrimidine, which crosses the phase boundary and interacts with the thiaminase protein TenA. The calculation of binding equilibria reveals a KD of 39.7 ± 8.9 μM for protein binding. The nanoscale separation of the two phases allows the separate optimization of the parahydrogen polarization and solubilization of a biological macromolecule. The reverse micelles may be used to study other biological questions using signal enhancement by parahydrogen polarization, such as enzyme reactions, protein-protein interactions, and protein binding epitopes.

Application of the Rasch Model to the Evaluation of Biological Problems

Evaluation of student abilities in Biology subjects often faces challenges in ensuring that the instruments used actually measure what they are supposed to measure. The data analysis used is the Rasch model, which is part of item response theory, offering a more accurate approach in analyzing evaluation data. This model allows the identification of items that are inappropriate or too difficult, as well as providing more in-depth information about individual student abilities. Through analysis of instruments and question items from 30 multiple choice questions that emphasize critical thinking skills, the aim of this research is to assess the quality of Biology questions for class X SMA. Testing was carried out using WinStep software and Rasch modeling analysis on 166 students from five high schools/vocational schools. The research method used is quantitative. The results of the instrument investigation show that the issue has very good dependability and the unidimensionality of the instrument is indicated by unexplained changes in the first differentiation. These results show that the Rasch test can help educators in selecting questions that meet the evaluation criteria and contribute to improving the quality of national education. The implications of this research are By applying the Rasch Model, educators can develop more accurate and fair evaluation tools, which are able to identify individual student difficulties and strengths.

Transcriptome and Evolutionary Analysis of Pseudotrichomonas keilini, a Free-Living Anaerobic Eukaryote.

The early evolution of eukaryotes and their adaptations to low-oxygen environments are fascinating open questions in biology. Genome-scale data from novel eukaryotes, and particularly from free-living lineages, are the key to answering these questions. The Parabasalia are a major group of anaerobic eukaryotes that form the most speciose lineage of Metamonada. The most well-studied are parasitic parabasalids, including Trichomonas vaginalis and Tritrichomonas foetus, but very little genome-scale data are available for free-living members of the group. Here, we sequenced the transcriptome of Pseudotrichomonas keilini, a free-living parabasalian. Comparative genomic analysis indicated that P. keilini possesses a metabolism and gene complement that are in many respects similar to its parasitic relative T. vaginalis and that in the time since their most recent common ancestor, it is the T. vaginalis lineage that has experienced more genomic change, likely due to the transition to a parasitic lifestyle. Features shared between P. keilini and T. vaginalis include a hydrogenosome (anaerobic mitochondrial homolog) that we predict to function much as in T. vaginalis and a complete glycolytic pathway that is likely to represent one of the primary means by which P. keilini obtains ATP. Phylogenomic analysis indicates that P. keilini branches within a clade of endobiotic parabasalids, consistent with the hypothesis that different parabasalid lineages evolved toward parasitic or free-living lifestyles from an endobiotic, anaerobic, or microaerophilic common ancestor.

A timed epigenetic switch balances T and ILC lineage proportions in the thymus.

How multipotent progenitors give rise to multiple cell types in defined numbers is a central question in developmental biology. Epigenetic switches, acting at single gene loci, can generate extended delays in the activation of lineage-specifying genes and impact lineage decisions and cell type output. Here, we analyzed a timed epigenetic switch controlling expression of mouse Bcl11b, a transcription factor that drives T-cell commitment, but only after a multi-day delay. To investigate roles for this delay in controlling lineage decision making, we analyzed progenitors with a deletion in a distal Bcl11b enhancer, which extends this delay by ∼3 days. Strikingly, delaying Bcl11b activation reduces T-cell output but enhances innate lymphoid cell (ILC) generation in the thymus by redirecting uncommitted progenitors to the ILC lineages. Mechanistically, delaying Bcl11b activation promoted ILC redirection by enabling upregulation of the ILC-specifying transcription factor PLZF. Despite the upregulation of PLZF, committed ILC progenitors could subsequently express Bcl11b, which is also needed for type 2 ILC differentiation. These results show that epigenetic switches can control the activation timing and order of lineage-specifying genes to modulate cell type numbers and proportions.

Stochastic to Deterministic: A Straightforward Approach to Create Serially Perfusable Multiscale Capillary Beds.

Generation of in vitro tissue models with serially perfused hierarchical vasculature would allow greater control of fluid perfusion throughout the network and enable direct mechanistic investigation of vasculogenesis, angiogenesis, and vascular remodeling. In this work, we have developed a method to produce a closed, serially perfused, multiscale vessel network fully embedded within an acellular hydrogel, where flow through the capillary bed is required prior to fluid exit. We confirmed that the acellular and cellular gel-gel interface was functionally annealed without preventing or biasing cell migration and endothelial self-assembly. Multiscale connectivity of the vessel network was validated via high-resolution microscopy techniques to confirm anastomosis between self-assembled and patterned vessels. Lastly, using a simple acrylic cassette and fluorescently labeled microspheres, the multiscale network was demonstrated to be perfusable. Directed flow from inlet to outlet mandated flow through the capillary bed. This method for producing closed, multiscale vascular networks was developed with the intention of straightforward fabrication and engineering techniques so as to be a low barrier to entry for researchers who wish to investigate mechanistic questions in vascular biology. This ease of use offers a facile extension of these methods for incorporation into organoid culture, organ-on-a-chip (OOC) models, and bioprinted tissues.

Dissecting neurofilament tail sequence-phosphorylation-structure relationships with multicomponent reconstituted protein brushes

Neurofilaments (NFs) are multisubunit, bottlebrush-shaped intermediate filaments abundant in the axonal cytoskeleton. Each NF subunit contains a long intrinsically disordered tail domain, which protrudes from the NF core to form a "brush" surrounding each NF. Precisely how the tails' variable charge patterns and repetitive phosphorylation sites mediate their conformation within the brush remains an open question in axonal biology. We address this problem by grafting recombinant NF tail protein constructs NF-Light, -Medium, and -Heavy (NFL, NFM, and NFH) to surfaces, yielding protein brushes of defined stoichiometry that can be phosphorylated in vitro. Atomic force microscopy measurements reveal that brush height depends on composition monotonically but not always linearly for binary NFL:NFM or NFL:NFH systems, and that NFM-based brushes are highly extended, while brushes incorporating the much larger NFH are surprisingly compact even after multisite phosphorylation. Complementary self-consistent field theory (SCFT) predicts multilayer brush morphologies for NFM and phosphorylated NFH brushes. Further experiments and SCFT analysis with designed mutants reveal that N-terminal negative charges in the NFH tail repel phosphorylated residues to generate the multilayer morphology, while the C-terminal charge-neutral region contributes to multilayer brush morphology but not total brush height. Charge-shuffled NFM variants show that charge segregation promotes brush collapse near physiological ionic strengths. Collectively, this study supports a role for NFM in establishing a dynamic range for NF brush conformation, lending insight into previous in vitro and in vivo findings. More broadly, this work establishes a platform for dissecting contributions of disordered protein sequence to conformation at interfaces.

A global database of butterfly species native distributions.

Butterflies represent a diverse group of insects, playing key ecosystem roles such as pollination and their larval form engage in herbivory. Despite their importance, comprehensive global distribution data for butterfly species are lacking. This lack of comprehensive global data has hindered many large-scale questions in ecology, evolutionary biology, and conservation at the regional and global scales. Here, I use an integrative workflow that combines occurrence records, alpha hull polygons, species' dispersal capacity, and natural habitat and environmental variables within a framework of species distribution models to generate species-level native distributions for butterflies at a global scale in the contemporary period. The database releases native range maps for 10,372 extant species of butterflies at a spatial grain resolution of 5 arcmin (~10 km). This database has the potential to allow unprecedented large-scale analyses in ecology, biogeography, and conservation of butterflies. The maps are available in the WGS84 coordinate reference system (EPSG:4326 code) and stored as vector polygons in the GEOPACKAGE format for maximum compression, allowing easy data manipulation using a standard computer. I additionally provide each species' spatial raster. All maps and R scripts are open access and available for download in Dryad and Zenodo, respectively, and are guided by FAIR (Findable, Accessible, Interoperable, and Reusable) data principles. By making these data available to the scientific community, I aim to advance the sharing of biological data to stimulate more comprehensive research in ecology, biogeography, and conservation of butterflies.

PIPETS: a statistically informed, gene-annotation agnostic analysis method to study bacterial termination using 3′-end sequencing

BackgroundOver the last decade the drop in short-read sequencing costs has allowed experimental techniques utilizing sequencing to address specific biological questions to proliferate, oftentimes outpacing standardized or effective analysis approaches for the data generated. There are growing amounts of bacterial 3′-end sequencing data, yet there is currently no commonly accepted analysis methodology for this datatype. Most data analysis approaches are somewhat ad hoc and, despite the presence of substantial signal within annotated genes, focus on genomic regions outside the annotated genes (e.g. 3′ or 5′ UTRs). Furthermore, the lack of consistent systematic analysis approaches, as well as the absence of genome-wide ground truth data, make it impossible to compare conclusions generated by different labs, using different organisms.ResultsWe present PIPETS, (Poisson Identification of PEaks from Term-Seq data), an R package available on Bioconductor that provides a novel analysis method for 3'-end sequencing data. PIPETS is a statistically informed, gene-annotation agnostic methodology. Across two different datasets from two different organisms, PIPETS identified significant 3'-end termination signal across a wider range of annotated genomic contexts than existing analysis approaches, suggesting that existing approaches may miss biologically relevant signal. Furthermore, assessment of the previously called 3′-end positions not captured by PIPETS showed that they were uniformly very low coverage.ConclusionsPIPETS provides a broadly applicable platform to explore and analyze 3′-end sequencing data sets from across different organisms. It requires only the 3′-end sequencing data, and is broadly accessible to non-expert users.

TOMOMAN: a software package for large-scale cryo-electron tomography data preprocessing, community data sharing and collaborative computing.

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) are becoming the preferred methodologies for investigating subcellular and macromolecular structures in native or near-native environments. Although cryo-ET is amenable to a wide range of biological problems, these problems often have data-processing requirements that need to be individually optimized, precluding the notion of a one-size-fits-all processing pipeline. Cryo-ET data processing is also becoming progressively more complex due to the increasing number of packages for each processing step. Though each package has its strengths and weaknesses, independent development and different data formats make them difficult to interface with one another. TOMOMAN (TOMOgram MANager) is an extensible package for streamlining the interoperability of packages, enabling users to develop project-specific processing workflows. TOMOMAN does this by maintaining an internal metadata format and wrapping external packages to manage and perform preprocessing, from raw tilt-series data to reconstructed tomograms. TOMOMAN can also export these metadata between various STA packages. TOMOMAN includes tools for archiving projects to data repositories, allowing subsequent users to download TOMOMAN projects and directly resume processing. By tracking essential metadata, TOMOMAN streamlines data sharing, which improves the reproducibility of published results, reduces computational costs by minimizing reprocessing, and enables the distribution of cryo-ET projects between multiple groups and institutions. TOMOMAN provides a way for users to test different software packages in order to develop processing workflows that meet the specific needs of their biological questions and to distribute their results to the broader scientific community.

Short-sighted evolution of virulence for invasive gut microbes: From hypothesis to tests

Why microbes harm their hosts is a fundamental question in evolutionary biology with broad relevance to our understanding of infectious diseases. Several hypotheses have been proposed to explain this "evolution of virulence." In this perspective, we reexamine one of these hypotheses in the specific context of the human gut microbiome, namely short-sighted evolution. According to the short-sighted evolution hypothesis, virulence is a product of niche expansion within a colonized host, whereby variants of commensal microbes establish populations in tissues and sites where the infection causes morbidity or mortality. This evolution is short-sighted in that the evolved variants that infect those tissues and sites are not transmitted to other hosts. The specific hypothesis that we propose is that some bacteria responsible for invasive infections and disease are the products of the short-sighted evolution of commensal bacteria residing in the gut microbiota. We present observations in support of this hypothesis and discuss the challenges inherent in assessing its general application to infections and diseases associated with specific members of the gut microbiota. We then describe how this hypothesis can be tested using genomic data and animal model experiments and outline how such studies will serve to provide fundamental information about both the evolution and genetic basis of virulence, and the bacteria of intensively studied yet poorly understood habitats including the gut microbiomes of humans and other mammals.

Multifaceted Landscape ofOmics Data

Multifaceted Landscape ofOmics Data

Past, Present, and Future of RNA Modifications in Infectious Disease Research.

In early 2024, the National Academies of Sciences, Engineering, and Medicine (NASEM) released a roadmap for the future of research into mapping ribonucleic acid (RNA) modifications, which underscored the importance of better defining these diverse chemical changes to the RNA macromolecule. As nearly all mature RNA molecules harbor some form of modification, we must understand RNA modifications to fully appreciate the functionality of RNA. The NASEM report calls for massive mobilization of resources and investment akin to the transformative Human Genome Project of the early 1990s. Like the Human Genome Project, a concerted effort in improving our ability to assess every single modification on every single RNA molecule in an organism will change the way we approach biological questions, accelerate technological advance, and improve our understanding of the molecular world. Consequently, we are also at the start of a revolution in defining the impact of RNA modifications in the context of host-microbe and even microbe-microbe interactions. In this perspective, we briefly introduce RNA modifications to the infection biologist, highlight key aspects of the NASEM report and exciting examples of RNA modifications contributing to host and pathogen biology, and finally postulate where infectious disease research may benefit from this exciting new endeavor in globally mapping RNA modifications.

Cutting the sap: First molecular phylogeny of twig‐girdler longhorn beetles (Coleoptera: Cerambycidae: Lamiinae: Onciderini) suggests shifts in host plant attack behaviours contributed to morphological evolution

AbstractUnderstanding how novel adaptive traits arise, evolve and impact other aspects of an organism's phenotype is a foundational question in evolutionary biology. We explore this by focusing on Onciderini (Coleoptera: Cerambycidae: Lamiinae), a tribe of longhorn beetles commonly referred to as twig girdlers because the females of some species girdle live trees to deposit their eggs. We reconstructed the first time‐calibrated phylogeny of the Onciderini, based on three genetic markers (cytochrome c oxidase subunit 1 ‘cox1’, Wingless ‘Wg’ and carbamoyl‐phosphate synthase domain of the CAD locus ‘CPS’), and used morphometric data, employing multiple models of trait evolution and phylogenetic regressions, to assess whether girdling behaviour predicts head size or head size sexual dimorphism. Our results indicate that onciderines originated c. 49 million years ago ‘Ma’ (95% highest posterior densities ‘HPD’: 44.1–54.57 Ma) and consist of two major lineages, which we formalize as subtribes Hypsiomina and Onciderina. Additionally, our analyses revealed several taxonomic inconsistencies within the tribe, which we rectify by proposing new synonymies, including Jamesia Jekel as a synonym of Hypselomus Perty, Cipriscola Dillon & Dillon of Hypsioma Audinet‐Serville and Psyllotoxus Thomson and Taricanus Thomson as a synonym of Oncideres Lacordaire, and a new combination for Periergates kenjii Nearns & Swift as Oncideres kenjii (Nearns & Swift) comb. nov. Using this new phylogenetic framework, we identified five independent unidirectional origins of girdling behaviour within the tribe. Overall, our results suggest that girdling influences head size evolution in Onciderini, as it is associated with an increase in both male and female head size of girdler species. Despite this, girdling and non‐girdling lineages do not consistently differ in head size sexual dimorphism. This study refines the classification of Onciderini and marks a significant step in understanding the evolutionary dynamics shaping the diversity of twig‐girdler beetles, a group with notable ecological and economic importance.

Can Transcriptomics Elucidate the Role of Regulation in Invasion Success?

When a species invades a novel environment, it must bridge the environment-phenotype mismatch in its new range to persist. Contemporary invasion biology research has focused on the role that trait variation and adaptation, and their underlying genomic factors, play in a species' adaptive potential, and thus facilitating invasion. Empirical studies have provided valuable insights into phenotypes that persist and arise in novel environments, coupled with 'omics tools that further the understanding of the contributions of genomic architecture in species establishment. Particularly, the use of transcriptomics to explore the role of plasticity in the initial stages of an invasion is growing. Here, we assess the role of various mechanisms relating to regulation and functional adaptation (often measured via the transcriptome) that support trait-specific plasticity in invasive species, allowing phenotypic variability without directly altering genomic diversity. First, we present a comprehensive review of the studies utilising transcriptomics in invasion biology. Second, we collate the evidence for and against the role of a range of regulatory processes in contributing to invasive species plasticity. Finally, we pose open questions in invasion biology where the use of transcriptome data may be valuable, as well as discuss the methodological limitations.

Genomic Architecture Underlying the Striking Colour Variation in the Presence of Gene Flow for the Guinan Toad-Headed Lizard.

How divergence occurs between closely related organisms in the absence of geographic barriers to gene flow stands as one of the long-standing questions in evolutionary biology. Previous studies suggested that the interplay between selection, gene flow and recombination strongly affected the process of divergence with gene flow. However, the extent to which these forces interact to drive divergence remains largely ambiguous. Guinan toad-headed lizards (Phrynocephalus guinanensis) in the Mugetan Desert exhibit striking colour differences from lizards outside the desert and provide an excellent model to address this question. Through extensive sampling and whole genome sequencing, we obtained genotypes for 191 samples from 14 populations inside and outside the desert. Despite the colour differences, continuous and asymmetric gene flow was detected across the desert border. More importantly, 273 highly diverged regions (HDRs) were identified between them, accounting only for 0.47% of the genome but widely distributed across 20 (out of the total 24) chromosomes. Strong signatures of selection were identified in HDRs, and local recombination rates were repressed. Furthermore, five HDRs exhibited significantly higher divergence, which contained key genes associated with crucial functions in animal coloration, including pteridine and melanocyte pigmentation. Genes related to retinal cells and steroid hormones were identified in other HDRs, which might have also contributed to the formation of colour variation in the presence of gene flow. This study provided novel insights into the understanding of the evolutionary mechanisms of genetic divergence in the presence of gene flow.

Optogenetic Control of Phosphate-Responsive Genes Using Single-Component Fusion Proteins in Saccharomyces cerevisiae.

Blue light illumination can be detected by light-oxygen-voltage (LOV) photosensing proteins and translated into a range of biochemical responses, facilitating the generation of novel optogenetic tools to control cellular function. Here, we develop new variants of our previously described VP-EL222 light-dependent transcription factor and apply them to study the phosphate-responsive signaling (PHO) pathway in the budding yeast Saccharomyces cerevisiae, exemplifying the utilities of these new tools. Focusing first on the VP-EL222 protein itself, we quantified the tunability of gene expression as a function of light intensity and duration and demonstrated that this system can tolerate the addition of substantially larger effector domains without impacting function. We further demonstrated the utility of several EL222-driven transcriptional controllers in both plasmid and genomic settings, using the PHO5 and PHO84 promoters in their native chromosomal contexts as examples. These studies highlight the utility of light-controlled gene activation using EL222 tethered to either artificial transcription domains or yeast activator proteins (Pho4). Similarly, we demonstrate the ability to optogenetically repress gene expression with EL222 fused to the yeast Ume6 protein. We finally investigated the effects of moving EL222 recruitment sites to different locations within the PHO5 and PHO84 promoters, as well as determining how this artificial light-controlled regulation could be integrated with the native controls dependent on inorganic phosphate (Pi) availability. Taken together, our work expands the applicability of these versatile optogenetic tools in the types of functionalities that they can deliver and the biological questions that can be probed.

Simultaneous detection of membrane contact dynamics and associated Ca2+ signals by reversible chemogenetic reporters

Membrane contact sites (MCSs) are hubs allowing various cell organelles to coordinate their activities. The dynamic nature of these sites and their small size hinder analysis by current imaging techniques. To overcome these limitations, we here design a series of reversible chemogenetic reporters incorporating improved, low-affinity variants of splitFAST, and study the dynamics of different MCSs at high spatiotemporal resolution, both in vitro and in vivo. We demonstrate that these versatile reporters suit different experimental setups well, allowing one to address challenging biological questions. Using these probes, we identify a pathway in which calcium (Ca2+) signalling dynamically regulates endoplasmic reticulum-mitochondria juxtaposition, characterizing the underlying mechanism. Finally, by integrating Ca2+-sensing capabilities into the splitFAST technology, we introduce PRINCESS (PRobe for INterorganelle Ca2+-Exchange Sites based on SplitFAST), a class of reporters to simultaneously detect MCSs and measure the associated Ca2+ dynamics using a single biosensor.

Awakening the Genome: CRISPR Activation to Unravel the Origin and Vulnerabilities of Cancer

CRISPR activation (CRISPRa) co-opts nuclease-dead Cas-molecule DNA-binding capabilities to direct transcriptional activators to specific loci, driving gene expression. CRISPRa technology has advanced rapidly in the past few years, and it is now applicable to a wide range of biological questions, including the study of cancer. In this review, we discuss the different forms of CRISPRa technologies, their in vitro and in vivo applications, and recent studies that have used CRISPRa in their cancers of choice in highly diverse studies. We further discuss the different CRISPRa tools that are available, including mouse models and single-guide RNA libraries. Finally, we examine the maturation of CRISPRa, as its potential therapeutic applications are beginning to be explored.

TMIC-66. A NEW MOUSE MODEL THAT COMBINES DRIVER MUTATIONS TO ACCURATELY RECAPITULATE THE DIVERSE PHENOTYPES OF HUMAN GLIOBLASTOMA

Abstract To effectively beat glioblastoma, new pre-clinical models are needed: to dissect the biology of processes like migration and therapy resistance; to understand immune suppression and immunotherapy; and to use as a model for testing in therapeutic development. We have developed a new panel of glioblastoma mouse models, which engineer well-characterised glioblastoma driver mutations into an immortalised eGFP murine astrocyte-like cell. These “GEM-CLeM” are Genetically Engineered Mouse Cell Line Models. When transplanted intra-cranially into immunocompetent mice, these lines formed tumors with the key characteristics of glioblastoma – migratory and invasive, with rapid proliferation and infiltration of suppressive immune cells. The eGFP expression allowed detection of glioblastoma cells in situ and ex vivo, and the ability to readily distinguish tumor and stromal components of the tumors. Importantly, different combinations of driver mutations led to diverse tumor phenotypes. Loss of Pten with over-expression of mutant RAS generated tumors with 100% efficiency and the same time to tumor onset as the commonly used GL261 cell line. However, unlike GL261 tumors the Pten-RASV12 tumors had a highly invasive edge and extensive migration, and were full of pro-tumorigenic macrophages and monocytes, with very few T-cells or dendritic cells. Together, this recapitulated the phenotype of human wildtype IDH1 glioblastoma. In contrast, the very common EGFRvIII mutant did not drive tumor formation in combination with Pten loss. We also modelled mutant IDH glioblastoma by combining loss of p53 with overexpression of IDH1R132H. These were slow growing, densely packed tumors with extensive microglial involvement in addition to pro-tumorigenic macrophages. This GEM-CLeM system combines the benefits of the traditional genetically engineered mouse models with accessible, rapid, reproducible tumor formation. The GEM-CLeM lines are amenable to further genetic manipulation, so cells can be customised with other mutations to ask important biological questions in the presence of an intact tumor immune microenvironment.

Rapid speciation in the holopelagic ctenophore Mnemiopsis following glacial recession.

Understanding how populations diverge is one of the oldest and most compelling questions in evolutionary biology. An in depth understanding of how this process operates in planktonic marine animals, where barriers for gene flow are seemingly absent, is critical to understanding the past, present, and future of ocean life. Mnemiopsis plays an important ecological role in its native habitat along the Atlantic coast of the Americas and is highly destructive in its non-native habitats in European waters. Although historical literature described three species of Mnemiopsis , the lack of stable morphological characters has led to the collapse of this group into a single species, Mnemiopsis leidyi . We generate high-quality reference genomes and use a whole-genome sequencing approach to reveal that there are two species of Mnemiopsis along its native range and show that historical divergence between the two species coincides with historical glacial melting. We define a hybridization zone between species and highlight that environmental sensing genes likely contribute to the invasive success of Mnemiopsis . Overall, this study provides insights into the fundamental question of how holopelagic species arise without clear barriers to gene flow and sheds light on the genomic mechanisms important for invasion success in a highly invasive species.