Scholarly Works, Fralin Life Sciences Institute

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Scaffold-free 3D-Cell Culture Model System for the Study of Metastatic Cell Behavior in the Brain TME
Sarkar, Pratistha; Ahuja, Shreya; Lazar, Iuliana M. (Cold Spring Harbor Laboratory, 2025-11-03)
Cancer is a complex disease involving dynamic interactions between cancer, stromal, and infiltrating immune cells, as well as between these cells and the extracellular matrix components of the tumor microenvironment. Brain metastases arise primarily from solid tumors and often result in fatal outcomes. An in-depth understanding of the complex intercellular interactions that evolve in the brain microenvironment is essential to enabling early cancer diagnostics and improving patient outcomes. The protected tumor microenvironment of the brain hinders, however, direct access, impeding the execution of mechanistic studies and limiting the ability to derive meaningful insights. Several in vitro 2D and 3D model systems have been developed to circumvent this problem, none, however, without limitations. The 2D models fail to recapitulate the 3D architecture of the in-vivo environment lacking therefore physiological relevance, while the 3D models present challenges related to the lack of control over cell positioning, lack of vascularization, contamination from non-human scaffolds, batch-to-batch reproducibility, and high production costs. To overcome some of these limitations, we developed an in vitro scaffold-free 3D tumor model system to simulate the in vivo brain metastatic niche. The model was constructed from human brain endothelial cells (HBEC-5i) and two different cancer cell lines derived from breast (MDA-MB-231/triple negative and SK-BR-3/HER2+) and aggressive ovarian (SK-OV-3) cancers. The development of the model relies on a newly identified affinity between the endothelial and cancer cells that enables them to self-assemble in 3D networked constructs, a feature facilitated by the high collagen production by endothelial cells and the secretion of key chemokines by both endothelial and cancer cells. The model mimics the attachment of metastasized cancer cells to the brain microvasculature, enabling the study of temporal changes in endothelial morphology and molecular signaling processes that sustain cancer cell migration, survival, proliferation, and angiogenic processes. Moreover, the model exhibits long-term stability, reproducibility, and effectiveness in evaluating anti-cancer agents. Altogether, the scaffold-free, simple 3D in vitro model systems provides a low cost, physiologically relevant tool for studying the dynamic molecular crosstalk between cancer and brain endothelial cells, and for investigating the fundamental biological processes that unfold in the tumor microenvironment.
The Species-Specific Inversion Polymorphism of the X Chromosome in Anopheles messeae and Anopheles daciae Is Based on the Common Ancestral Variant X1
Soboleva, Evgeniya S.; Sharakhova, Maria V.; Sharakhov, Igor V.; Artemov, Gleb N. (MDPI, 2025-12-19)
Background/Objectives: Chromosomal inversions play an important role in the evolution of insects by forming genetic barriers between closely related species and facilitating local adaptation. Polymorphic inversions in malaria mosquitoes of the Maculipennis subgroup have been studied for over 50 years, yet the evolutionary ancestry of the gene orders remains unknown. In this study, we mapped the genes flanking the breakpoints of two polymorphic X-chromosome inversions in the cryptic species Anopheles messeae and Anopheles daciae of the Maculipennis subgroup. Methods: We used an iterative mapping approach to define the breakpoint regions, selecting flanking markers based on the genome assembly of the reference species, Anopheles atroparvus. To identify the ancestral X chromosomal arrangement in An. messeae and An. daciae, we developed and implemented the genomic inversion calculator (GIC), which uses greedy heuristics to determine the shortest evolutionary scenario of rearrangements. Results: Our knowledge of the relative genomic positions of the inversion breakpoints in An. daciae and An. messeae enabled us to use the An. atroparvus genome as an outgroup and the GIC tool to show that the X0 and X2 arrangements emerged independently along the evolutionary lineages of An. daciae and An. messeae, respectively, based on the X1 arrangement. Conclusions: These results refine the structure and boundaries of the X chromosome rearrangements and reconstruct the sequence of evolutionary events in the cryptic complex An. messeae–An. daciae, demonstrating that the X1 arrangement is ancestral. This study lays the groundwork for analyzing the molecular organization of breakpoints, the mechanisms of inversion formation, and their role in speciation.
Perinatal citalopram exposure alters the gut composition and microbial metabolic profiles of Sprague-Dawley rat dams and female offspring but not male offspring
Kropp, Dawson R.; Glover, Matthew E.; Samanta, Rupabali; Unroe, Keaton A.; Clinton, Sarah M.; Hodes, Georgia E. (2025-12-03)
Background: Selective serotonin reuptake inhibitors are widely prescribed during pregnancy. Their main route of administration is through the gut. However, their impact on the maternal and offspring gut microbiome and microbial metabolic pathways remains poorly understood. This study used metagenomic shotgun sequencing to examine the effects of perinatal citalopram exposure in rat dams and their offspring on gut composition and downstream metabolic pathways. Methods: We treated pregnant and nursing rat dams with either citalopram or vehicle (water). Their feces were collected, DNA from these samples was extracted and then sequenced using shotgun metagenomic sequencing. The BioBakery suite of microbiome analysis tools was utilized in tandem with RStudio to analyze the gut composition and microbial metabolic pathways of the rat dams and their offspring. Results: Pregnant and nursing dams treated with citalopram exhibited marked shifts in microbial community structure, including phylum-level alterations in Proteobacteria and Defferibacteria. Citalopram treated dams displayed significantly altered beta diversity. Species level alterations due to treatment were composed of five significantly altered microbes, two of which belong to the Proteobacteria phylum. These changes were highly diverse and were not congruent with microbe-level alterations observed in offspring. Alpha diversity of microbial metabolic pathways was compared using the Gini-Simpson index, which was significantly increased in dams suggesting greater metabolic functional diversity with age. Female offspring perinatally exposed to citalopram showed significant changes in gut beta diversity, with seven significant alterations at the microbe level. These microbial shifts were accompanied by twenty-one significantly altered microbial metabolic pathways. In contrast, male offspring showed no significant differences in microbial composition or beta diversity and only minor metabolic changes. Conclusions: These findings demonstrate that maternal citalopram exposure during pregnancy and lactation has lasting, sex-specific impacts on the offspring’s gut microbiome and microbial metabolic pathways. The pronounced alterations in female, but not male offspring, suggest that host sex may be a critical determinant in the developmental response to citalopram exposure. This work underscores the value of metagenomic approaches in uncovering complex host-microbiome interactions and highlights the need to consider offspring sex in evaluating the safety and long-term effects of antidepressant use during pregnancy.
Chromosomal inversions and their potential impact on the evolution of arboviral vector Aedes aegypti
Liang, Jiangtao; Rose, Noah; Brusentsov, Ilya; Lukyanchikova, Varvara; Karagodin, Dmitriy; Feng, Yifan; Yurchenko, Andrey; Sharakhov, Igor V.; McBride, Carolyn; Sharakhova, Maria V. (Oxford University Press, 2024-06-29)
Chromosomal inversions play a crucial role in evolution and have been found to regulate epidemiologically significant traits in malaria mosquitoes. However, they have not been characterized in Aedes aegypti, the primary vector of arboviruses, due to the poor structure of its polytene chromosomes. The Hi-C proximity ligation approach was used to identify chromosomal inversions in 25 strains of A. aegypti obtained from its worldwide distribution and in one strain of Aedes mascarensis. The study identified 21 multimegabase polymorphic inversions ranging in size from 5 to 55 Mbp. Inversions were more abundant in African than in non-African strains, 15 versus 3 inversions, with the highest number observed in West Africa. All inversions were grouped into two geographic clusters of African or non-African origin, suggesting their association with A. aegypti subspecies. Inversions were unevenly distributed along chromosomal arms, with the highest number found in the 1q and 3p arms homologous to the inversion-rich 2R chromosomal arm in the malaria vector Anopheles gambiae. Direct comparison of inversions between A. aegypti and An. gambiae revealed significant overlap in their genomic locations. This finding may explain the parallel evolution of the two species under similar environmental conditions. Some of the inversions colocalized with chemoreceptor genes and quantitative trait loci associated with pathogen infection, suggesting their potential role in host preference and disease transmission. Our study revealed the large pool of structural variations in the A. aegypti genome and provides the foundation for future studies of their impact on the biology of this important arboviral vector.
Chromosomal inversions and their impact on insect evolution
Sharakhov, Igor V.; Sharakhova, Maria V. (Elsevier, 2024-12)
Insects can adapt quickly and effectively to rapid environmental change and maintain long-term adaptations, but the genetic mechanisms underlying this response are not fully understood. In this review, we summarize studies on the potential impact of chromosomal inversion polymorphisms on insect evolution at different spatial and temporal scales, ranging from long-term evolutionary stability to rapid emergence in response to emerging biotic and abiotic factors. The study of inversions has recently been advanced by comparative, population, and 3D genomics methods. The impact of inversions on insect genome evolution can be profound, including increased gene order rearrangements on sex chromosomes, accumulation of transposable elements, and facilitation of genome divergence. Understanding these processes provides critical insights into the evolutionary mechanisms shaping insect diversity.
Chromatin landscape, transcriptomic and ChIP-seq profiling of Anopheles stephensi MSQ43 cell line
Lukyanchikova, Varvara; Nuriddinov, Miroslav; Khabarova, Anna; Gridina, Maria; Popov, Andrey; Belokopytova, Polina; Sharakhov, Igor V.; Fishman, Veniamin (Springer, 2025-12-01)
Anopheles mosquitoes are known as dominant vectors of malaria parasites and other viral and bacterial pathogens. Deciphering Anopheles genomes has opened a new era of research and allowed in-depth investigation of several molecular mechanisms involved in pathogen transmission. However, there is a lack of appropriate model systems to study specific vector-pathogen interactions at the molecular level. Even inbred mosquito colonies represent genetically heterogeneous population and require special care facilities. Insect cell cultures could serve as a useful alternative due to the ease of handling and maintenance, especially for genomic studies requiring millions of cells per experiment. Here, we provide chromatin contact maps and a dataset of epigenetic characteristics, including histone mark profiles and RNA-seq data, as well as transfection conditions and promoter specificity for the mosquito cell line MSQ43.
Protocol for Hi-C-based identification of chromosomal inversions in mosquitoes
Lukyanchikova, Varvara; Brusentsov, Ilya I.; Karagodin, Dmitry A.; Sharakhova, Maria V.; Sharakhov, Igor V. (Elsevier, 2025-12-19)
Chromosomal inversions play an important role in the genomic evolution and adaptation of mosquito species. Here, we present a protocol for detecting chromosomal inversions in mosquitoes using Hi-C technology and chromatin contact heatmaps. We describe the steps for Hi-C library preparation using the Hi-C Arima+ kit, along with the Arima Library Prep Module, and provide several adaptations for mosquito samples. We then outline procedures for Hi-C data analysis, Hi-C heatmap generation, and the identification of polymorphic and fixed inversions. For additional details on the use and execution of this protocol, please refer to Lukyanchikova et al.¹
Noninvasive Analysis of Peptidoglycan from Living Animals
Ocius, Karl L.; Kolli, Sree H.; Ahmad, Saadman S.; Dressler, Jules M.; Chordia, Mahendra D.; Jutras, Brandon L.; Rutkowski, Melanie R.; Pires, Marcos M. (American Chemical Society, 2024-04-09)
The role of the intestinal microbiota in host health is increasingly revealed in its contributions to disease states. The host-microbiome interaction is multifactorial and dynamic. One of the factors that has recently been strongly associated with host physiological responses is peptidoglycan from bacterial cell walls. Peptidoglycan from gut commensal bacteria activates peptidoglycan sensors in human cells, including the nucleotide-binding oligomerization domain-containing protein 2. When present in the gastrointestinal tract, both the polymeric form (sacculi) and depolymerized fragments can modulate host physiology, including checkpoint anticancer therapy efficacy, body temperature and appetite, and postnatal growth. To utilize this growing area of biology toward therapeutic prescriptions, it will be critical to directly analyze a key feature of the host-microbiome interaction from living hosts in a reproducible and noninvasive way. Here we show that metabolically labeled peptidoglycan/sacculi can be readily isolated from fecal samples collected from both mice and humans. Analysis of fecal samples provided a noninvasive route to probe the gut commensal community including the metabolic synchronicity with the host circadian clock. Together, these results pave the way for noninvasive diagnostic tools to interrogate the causal nature of peptidoglycan in host health and disease.
Genetically Encoded, Noise-Tolerant, Auxin Biosensors in Yeast
Chaisupa, Patarasuda; Rahman, Md Mahbubur; Hildreth, Sherry B.; Moseley, Saede; Gatling, Chauncey; Bryant, Matthew R.; Helm, Richard F.; Wright, R. Clay (American Chemical Society, 2024-08-28)
Auxins are crucial signaling molecules that regulate the growth, metabolism, and behavior of various organisms, most notably plants but also bacteria, fungi, and animals. Many microbes synthesize and perceive auxins, primarily indole-3-acetic acid (IAA, referred to as auxin herein), the most prevalent natural auxin, which influences their ability to colonize plants and animals. Understanding auxin biosynthesis and signaling in fungi may allow us to better control interkingdom relationships and microbiomes from agricultural soils to the human gut. Despite this importance, a biological tool for measuring auxin with high spatial and temporal resolution has not been engineered in fungi. In this study, we present a suite of genetically encoded, ratiometric, protein-based auxin biosensors designed for the model yeast Saccharomyces cerevisiae. Inspired by auxin signaling in plants, the ratiometric nature of these biosensors enhances the precision of auxin concentration measurements by minimizing clonal and growth phase variation. We used these biosensors to measure auxin production across diverse growth conditions and phases in yeast cultures and calibrated their responses to physiologically relevant levels of auxin. Future work will aim to improve the fold change and reversibility of these biosensors. These genetically encoded auxin biosensors are valuable tools for investigating auxin biosynthesis and signaling in S. cerevisiae and potentially other yeast and fungi and will also advance quantitative functional studies of the plant auxin perception machinery, from which they are built.
Membrane Composition Modulates Vp54 Binding: A Combined Experimental and Computational Study
Guo, Wenhan; Dong, Rui; Okedigba, Ayoyinka O.; Sanchez, Jason E.; Agarkova, Irina V.; Abisamra, Elea-Maria; Jelinsky, Andrew; Riekhof, Wayne; Noor, Laila; Dunigan, David D.; Van Etten, James L.; Capelluto, Daniel G. S.; Xiao, Chuan; Li, Lin (MDPI, 2025-10-03)
The recruitment of peripheral membrane proteins is tightly regulated by membrane lipid composition and local electrostatic microenvironments. Our experimental observations revealed that Vp54, a viral matrix protein, exhibited preferential binding to lipid bilayers enriched in anionic lipids such as phosphatidylglycerol (PG) and phosphatidylserine (PS), compared to neutral phosphatidylcholine/phosphatidylethanolamine liposomes, and this occurred in a curvature-dependent manner. To elucidate the molecular basis of this selective interaction, we performed a series of computational analyses including helical wheel projection, electrostatic potential calculations, electric field lines simulations, and electrostatic force analysis. Our results showed that the membrane-proximal region of Vp54 adopted an amphipathic α-helical structure with a positively charged interface. In membranes containing PG or PS, electrostatic potentials at the interface were significantly more negative, enhancing attraction with Vp54. Field line and force analyses further confirmed that both the presence and spatial clustering of anionic lipids intensify membrane–Vp54 electrostatic interactions. These computational findings align with experimental binding data, jointly demonstrating that membrane lipid composition and organization critically modulate Vp54 recruitment. Together, our findings highlight the importance of electrostatic complementarity and membrane heterogeneity in peripheral protein targeting and provide a framework applicable to broader classes of membrane-binding proteins.
Combining graph theory and spatially-explicit, individual-based models to improve invasive species control strategies at a regional scale
Drake, Joseph; O'Malley, Grace; Kraft, John; Mims, Meryl C. (Springer, 2024-10-25)
Context: Invasive species cause widespread species extinction and economic loss. There is an increasing need to identify ways to efficiently target control efforts from local to regional scales. Objectives: Our goal was to test whether prioritizing managed habitat using different treatments based on spatial measures of connectivity, including graph-theoretic measures, can improve management of invasive species and whether the level of control effort affects treatment performance. We also explored how uncertainty in biological variables, such as dispersal ability, affects measures performance. Methods: We used a spatially-explicit, individual-based model (sIBM) based on the American bullfrog (Lithobates catesbeianus), a globally pervasive invasive species. Simulations were informed by geographic data from part of the American bullfrog's non-native range in southeastern Arizona, USA where they are known to pose a threat to native species. Results: We found that total bullfrog populations and occupancy declined in response to all treatments regardless of effort level or patch prioritization methods. The most effective spatial prioritization was effort-dependent and varied depending on spatial context, but frequently a buffer strategy was most effective. Treatments were also sensitive to dispersal ability. Performance of treatments prioritizing habitat patches using betweenness centrality improved with increasing dispersal ability, while performance of eigenvalue centrality improved as dispersal ability decreased. Conclusions: With the careful application of connectivity measures to prioritize control efforts, similar reductions in invasive species population size and occupancy could be achieved with less than half the effort of sub-optimal connectivity measures at higher effort rates. More work is needed to determine if trait-based generalities may define appropriate connectivity measures for specific suites of dispersal abilities, demographic traits, and population dynamics.
Obesity's Unexpected Influence: Reduced Alphavirus Transmission and Altered Immune Activation in the Vector
Rai, Pallavi; Webb, Emily M.; Paulson, Sally L.; Kang, Lin; Weger-Lucarelli, James (Wiley, 2024-11-01)
Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are emerging/re-emerging alphaviruses transmitted by Aedes spp. mosquitoes and responsible for recent disease outbreaks in the Americas. The capacity of these viruses to cause epidemics is frequently associated with increased mosquito transmission, which in turn is governed by virus-host-vector interactions. Although many studies have explored virus-vector interactions, significant gaps remain in understanding how vertebrate host factors influence alphavirus transmission by mosquitoes. We previously showed that obesity, a ubiquitous vertebrate host biological factor, reduces alphavirus transmission potential in mosquitoes. We hypothesized that alphavirus-infected obese bloodmeals altered immune genes and/or pathways in mosquitoes, thereby inhibiting virus transmission. To test this, we conducted RNA sequencing (RNA-seq) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) on midgut RNA from mosquitoes fed on alphavirus-infected lean and obese mice. This approach aimed to identify potential antiviral or proviral genes and pathways altered in mosquitoes after consuming infected obese bloodmeals. We found upregulation of the Toll pathway and downregulation of several metabolic and other genes in mosquitoes fed on alphavirus-infected obese bloodmeals. Through gene knockdown studies, we demonstrated the antiviral role of Toll pathway and proviral roles of AAEL009965 and fatty acid synthase (FASN) in the transmission of alphaviruses by mosquitoes. Therefore, this study utilized obesity to identify factors influencing alphavirus transmission by mosquitoes and this research approach may pave the way for designing broadly effective antiviral measures to combat mosquito-borne viruses, such as releasing transgenic mosquitoes deficient in the identified genes.
Differential elimination of marked sex chromosomes enables production of nontransgenic male mosquitoes in a single strain
Compton, Austin; Sharma, Atashi; Hempel, Melanie; Aryan, Azadeh; Biedler, James K.; Potters, Mark B.; Chandrasegaran, Karthikeyan; Vinauger, Clément; Tu, Zhijian (National Academy of Sciences, 2025-05-08)
Diverse genetic strategies are being pursued to control mosquito-borne infectious diseases. These strategies often rely on the release of nonbiting males to either reduce the target mosquito population or render them resistant to pathogens. Male-only releases are important as any contaminating females can bite and potentially transmit pathogens. Despite significant efforts, it remains a major bottleneck to reliably and efficiently separate males from females, especially when nontransgenic males are preferred. In the yellow fever mosquito Aedes aegypti, sex is determined by a pair of homomorphic sex chromosomes, with the dominant male-determining locus (the M locus) and its counterpart (the m locus) embedded in an M-bearing and an m-bearing chromosome 1, respectively. We utilized both naturally occurring and engineered sex-linked recessive lethal alleles (RLAs) to create sex separation strains for Ae. aegypti on the basis of differential elimination of marked sex chromosomes (DeMark). DeMark strains are self-sustaining and produce nontransgenic males that are readily separated from individuals carrying RLA-and transgene-marked m chromosomes. For example, the marked m chromosome in the heterozygous mother in some strains was only inherited by her female progeny due to RLA-mediated incompatibility with the M-bearing chromosome in the father, producing nontransgenic males and transgenic females, generation after generation. We further explore strategies to conditionally eliminate females that contain marked sex chromosomes. We also discuss DeMark designs that are applicable for efficient sex separation in organisms with well-differentiated X and Y chromosomes, such as the Anopheles mosquitoes.
Mechanistic modeling of mitosis: Insights from three collaborative case studies
Chen, Jing; Cimini, Daniela (Elsevier, 2025-11-01)
Mechanistic mathematical modeling has become an essential tool in modern biological research due to its powerful ability to integrate diverse data, generate hypotheses, and guide experimental design. It is particularly valuable for studying complex cellular mechanisms involving numerous interacting components. While the full dynamics of such systems usually elude direct experimental observation, modeling provides a means to integrate fragmented data with reasonable and/or informed assumptions into coherent mechanistic frameworks, simulate system behavior, and identify promising directions for further experimentation. When closely integrated with experiments, modeling can greatly accelerate progress in cell biology. However, the value of modeling is not automatic—it must be earned through careful model construction, critical interpretation of results, and thoughtful design of follow-up experiments. To demystify this process, we review three of our collaborative projects in mitosis, drawing on our experiences as a modeler and an experimentalist. We describe how the projects were initiated, why specific modeling approaches were chosen, how models were developed and refined, how model predictions guided new experiments, and how integrated modeling and experimentation led to deeper mechanistic insights. Finally, we emphasize that at the heart of every successful collaboration lies human connection. Productive cross-disciplinary communication is fundamental to bridging experimental and modeling perspectives and fully realizing the potential of integrative approaches in modern cell biology.
Elevated EGR1 binding at enhancers in excitatory neurons correlates with neuronal subtype-specific epigenetic regulation
Yin, Liduo; Xu, Xiguang; Conacher, Benjamin; Lin, Yu; Carrillo, Gabriela L.; Cun, Yupeng; Fox, Michael A.; Lu, Xuemei; Xie, Hehuang (2025-08-11)
Background: Brain development and neuronal cell specification are accompanied by epigenetic changes that enable the regulation of diverse gene expression patterns. During these processes, transcription factors interact with cell-type-specific epigenetic marks, binding to unique sets of cis-regulatory elements in different cell types. However, the detailed mechanisms through which cell-type-specific gene regulation is established in neurons remain to be explored. Results: In this study, we conducted a comparative histone modification analysis between excitatory and inhibitory neurons. Our results revealed that neuronal cell-type-specific histone modifications are enriched in super enhancer regions that contain abundant EGR1 motifs. Further CUT&RUN assay confirmed that excitatory neurons exhibit more EGR1 binding sites, primarily located in enhancers. Integrative analysis demonstrated that EGR1 binding is strongly correlated with various epigenetic markers of open chromatin regions and is linked to distinct gene pathways specific to neuronal subtypes. In inhibitory neurons, most genomic regions containing EGR1 binding sites become accessible during early embryonic stages, whereas super enhancers in excitatory neurons, which also host EGR1 binding sites, gain accessibility during postnatal stages. Conclusions: This study highlights the crucial role of transcription factor binding, such as EGR1, to enhancer regions, which may be key to establishing cell-type-specific gene regulation in neurons.
Soybean Lectin Cross-Links Membranes by Binding Sulfatide in a Curvature-Dependent Manner
Okedigba, Ayoyinka O.; Ng, Emery L.; Deegbey, Mawuli; Rosso, M. Luciana; Ngo, William; Xiao, Ruoshi; Huang, Haibo; Zhang, Bo; Welborn, Valerie Vaissier; Capelluto, Daniel G. S. (American Chemical Society, 2025-05-24)
Soybean (Glycine max) is a key source of plant-based protein, yet its nutritional value is impacted by antinutritional factors, including lectins. Whereas soybean lectin is known to bind N-acetyl-d-galactosamine (GalNAc), its lipid interactions remain unexplored. Using a novel purification method, we isolated lectin from soybean meals and characterized its interactions with GalNAc and the glycosphingolipid sulfatide. Isothermal titration calorimetry revealed micromolar affinity for GalNAc, whereas most GalNAc derivatives displayed weak or no binding. Lectin exhibited high-affinity binding to sulfatide in a membrane curvature-dependent manner. Binding of lectin to sulfatide promoted cross-linking of sulfatide-containing vesicles. Whereas sulfatide interaction was independent of GalNAc binding, suggesting distinct binding sites, vesicle cross-linking was inhibited by the sugar. Molecular dynamics simulations identified a consensus sulfatide-binding site in lectin. These findings highlight the dual ligand-binding properties of soybean lectin and may provide strategies to mitigate its antinutritional effects and improve soybean meal processing.
Larval environment reshapes mosquito disease risk via phenotypic and molecular plasticity
Chandrasegaran, Karthikeyan; Walker, Melody; Marano, Jeffrey M.; Rami, Spruha; Bisese, Adaline; Weger-Lucarelli, James; Lahondère, Chloé; Robert, Michael A.; Childs, Lauren M.; Vinauger, Clément (2025-06-21)
Early-life environmental conditions can exert profound, lasting effects on adult phenotypes, with major consequences for fitness and disease transmission, especially in holometabolous insects like mosquitoes, which are a key vector species. Yet, the molecular mechanisms through which juvenile environments shape adult physiology and behavior via transstadial effects remain largely unresolved. Here, we demonstrate that larval competition, a key ecological stressor, profoundly alters adult body size, survival, reproductive output, host-seeking behavior, olfactory neurophysiology, and vector competence in the mosquito Aedes aegypti. Crucially, using transcriptomic profiling and integrative network analyses, we identify seven regulatory hub genes whose expression is strongly associated with size-dependent variation in olfactory behavior, reproductive investment, and Zika virus transmission potential. These hub genes belong to gene modules enriched for functions in chemosensory processing, metabolic regulation, and signal transduction, revealing a molecular framework mediating environmentally induced plasticity across metamorphosis. Integrating these empirical findings into a transmission model, we show that incomplete larval control can inadvertently increase outbreak risk by producing larger, longer-lived, and more competent vectors. Our results uncover molecular mechanisms underpinning phenotypic plasticity in disease vectors and highlight the critical need to account for transstadial effects in models of vector-borne disease transmission.
Spatial Transcriptomics Reveals Regional and Temporal Dynamics of Gene Expression in the Mouse Brain Across Development and Aging
Conacher, Benjamin; Moore, Amanda; Yin, Liduo; Lin, Yu; Xu, Xiguang; Mao, Qinwen; Xie, Hehuang (MDPI, 2025-06-18)
Investigating transcriptomic changes during healthy development and aging provides insights into the molecular mechanisms that regulate the maturation of brain functions and drive age-related decline. Although it has been speculated that aging may represent a reversal of late-stage brain development, direct molecular comparisons between these two processes have remained limited. This study employs spatial transcriptomics to analyze the mouse brain at three key timepoints: postnatal day 21 (P21), 3 months (adult), and 28 months (aged), to identify region-specific differential gene expression dynamics. We identify widespread transcriptional changes across both brain development and aging, with all brain regions exhibiting distinct, region-specific gene expression dynamics that reflect divergent regulatory trajectories across the lifespan. During development, gene expression patterns were strongly enriched for neurogenesis, synaptic plasticity, and myelination, reflecting active circuit formation and white matter maturation. In contrast, aging was characterized by a decline in myelination-related gene expression and a pronounced increase in inflammatory and glial activation pathways, particularly within the hippocampus. While both development and aging involved changes in myelination-associated genes, the underlying mechanisms appear distinct: developmental upregulation supports circuit establishment and refinement, whereas aging-related downregulation may reflect secondary consequences of neuroinflammation and reactive gliosis. These findings underscore that, despite some overlap in affected pathways, neural maturation and age-related decline are driven by fundamentally different regulatory programs. These findings establish a novel spatial transcriptomic reference for brain development and aging, offering a valuable data resource for investigating neurodevelopmental and neurodegenerative mechanisms.
Systematic evaluation of parameters in RNA bisulfite sequencing data generation and analysis
Johnson, Zachary; Xu, Xiguang; Pacholec, Christina; Xie, Hehuang (Oxford University Press, 2022-03-31)
The presence of 5-methylcytosine (m5C) in RNA molecules has been known for decades and its importance in regulating RNA metabolism has gradually become appreciated. Despite recent advances made in the functional and mechanistic understanding of RNA m5C modifications, the detection and quantification of methylated RNA remains a challenge. In this study, we compared four library construction procedures for RNA bisulfite sequencing and implemented an analytical pipeline to assess the key parameters in the process of m5C calling. We found that RNA fragmentation after bisulfite conversion increased the yield significantly, and an additional high temperature treatment improved bisulfite conversion efficiency especially for sequence reads mapped to the mitochondrial transcriptome. Using Unique Molecular Identifiers (UMIs), we observed that PCR favors the amplification of unmethylated templates. The low sequencing quality of bisulfite-converted bases is a major contributor to the methylation artifacts. In addition, we found that mitochondrial transcripts are frequently resistant to bisulfite conversion and no p-m5C sites with high confidence could be identified on mitochondrial mRNAs. Taken together, this study reveals the various sources of artifacts in RNA bisulfite sequencing data and provides an improved experimental procedure together with analytical methodology.
Egr2 Deletion in Autoimmune-Prone C57BL6/lpr Mice Suppresses the Expression of Methylation-Sensitive Dlk1-Dio3 Cluster MicroRNAs
Wang, Zuhang; Heid, Bettina; He, Jianlin; Xie, Hehuang; Reilly, Christopher M.; Dai, Rujuan; Ahmed, S. Ansar (Oxford University Press, 2023-12)
We previously demonstrated that the upregulation of microRNAs (miRNAs) at the genomic imprinted Dlk1-Dio3 locus in murine lupus is correlated with global DNA hypomethylation. We now report that the Dlk1-Dio3 genomic region in CD4+ T cells of MRL/lpr mice is hypomethylated, linking it to increased Dlk1-Dio3 miRNA expression. We evaluated the gene expression of methylating enzymes, DNA methyltransferases (DNMTs), and demethylating ten-eleven translocation proteins (TETs) to elucidate the molecular basis of DNA hypomethylation in lupus CD4+ T cells. There was a significantly elevated expression of Dnmt1 and Dnmt3b, as well as Tet1 and Tet2, in CD4+ T cells of three different lupus-prone mouse strains compared to controls. These findings suggest that the hypomethylation of murine lupus CD4+ T cells is likely attributed to a TET-mediated active demethylation pathway. Moreover, we found that deletion of early growth response 2 (Egr2), a transcription factor gene in B6/lpr mice markedly reduced maternally expressed miRNA genes but not paternally expressed protein-coding genes at the Dlk1-Dio3 locus in CD4+ T cells. EGR2 has been shown to induce DNA demethylation by recruiting TETs. Surprisingly, we found that deleting Egr2 in B6/lpr mice induced more hypomethylated differentially methylated regions at either the whole-genome level or the Dlk1-Dio3 locus in CD4+ T cells. Although the role of methylation in EGR2-mediated regulation of Dlk1-Dio3 miRNAs is not readily apparent, these are the first data to show that in lupus, Egr2 regulates Dlk1-Dio3 miRNAs, which target major signaling pathways in autoimmunity. These data provide a new perspective on the role of upregulated EGR2 in lupus pathogenesis.

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