Biology subjects

Mai, D.

Publications and source records attributed to Mai, D..

14 recordsLinked to original sources

bioRxiv2026

Evolutionary contingency facilitates scent divergence in an emergent orchid population

Reproductive isolation can evolve rapidly even among populations with extensive genetic similarity, but the mechanisms underlying this divergence remain an open question in evolutionary biology. Here, we investigate the genetic basis of floral scent divergence, a key trait mediating differential pollinator attraction, in a group of nascent Gongora orchid lineages that occur in sympatry. We combined whole-genome resequencing and floral transcriptomics to characterize the transition from the monoterpene-emitting 'M chemotype' to the pollinator-isolated phenylpropanoid-emitting 'A chemotype' nested within it. While overall genetic differentiation was low, a few highly differentiated regions contained candidate genes in biosynthetic pathways. Of particular interest was a putative o-methyltransferase exhibiting evidence of a recent selective sweep in the A chemotype, with much higher expression in scent-emitting floral tissues of A than in M. Transcriptomic comparison with a more distantly related monoterpene-emitting lineage ('X chemotype') further revealed the coordinated and elevated expression of phenylpropanoid biosynthetic genes in M despite its absence of aromatic floral volatiles, including a putative key enzyme potentially involved in directing metabolic flux towards monolignol biosynthesis. Together, our results support a model in which pre-existing activity in a metabolic pathway may facilitate the evolution of a novel floral scent phenotype, potentially making transitions across the adaptive landscape of scent more accessible than others. We propose that such evolutionary contingency may help explain how divergent scent profiles, and consequently, reproductive isolation, can evolve rapidly via few genetic changes.

evolutionary biology↗

bioRxiv2026

A pooled screening approach reveals bacterial chemoreceptors for short-chain carboxylic acids

Bacterial chemotaxis is a key process in host colonization and virulence, mediated by large repertoires of chemoreceptors. Despite their physiological and ecological importance, mapping these chemoreceptors to their cognate metabolic ligands remains a major challenge due to the vast number of potential interactions. To address this, we developed a pooled screening assay that enriches functional chemoreceptors from a gene library. Using this approach, we identified a previously uncharacterized group of chemoreceptors in Pseudomonas species with Cache_3-Cache_2 domains that sense short-chain C3 carboxylic acids. Sequence and computational structural analyses revealed that these chemoreceptors exhibit domain features similar to a recently reported C1 formate chemoreceptor despite substantial sequence divergence. Functional assays of representative chemoreceptors confirmed robust chemotactic responses to C3 carboxylic acids, with limited responses to formate. Integrating structural and molecular dynamics analyses suggests that increased binding pocket size and altered flexibility, relative to the formate chemoreceptor, facilitate recognition of larger C3 carboxylic acid ligands. Together, our approach provides a simple and scalable framework for mapping ligand-chemoreceptor interactions and enables systematic characterization across diverse metabolites.

microbiology↗

bioRxiv2025

Bioinspired Oxidized mRNA Lipid Nanoparticles for Ex Vivo Engineering of Chimeric Antigen Receptor Macrophages Targeting Solid Tumors

Solid tumors remain difficult to treat via conventional and novel therapeutic strategies. Immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy have been remarkably effective in treating hematological cancers, but their efficacy is limited in solid tumors. Recently, CAR macrophages (CAR-Ms) have emerged as a promising solid tumor immunotherapy, primarily for their intrinsic tumor infiltration and effector functions. However, CAR-Ms are engineered using viral transduction, which is associated with aberrant immunogenicity and toxicity. To overcome these challenges, we developed a bioinspired oxidized lipid nanoparticle (LNP) platform for mRNA-based engineering of human CAR-Ms. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened for delivery to human macrophages. The composition of the top LNP was subsequently optimized using an orthogonal design of experiments (DoE) and physicochemical properties, such as size and mRNA encapsulation, were tuned via optimization of microfluidic mixing parameters, yielding a particle that significantly outperformed a gold standard C12-200 LNP. Utilizing small molecule and antibody inhibitors, we demonstrate that uptake of optimized LNPs into macrophages is driven by apolipoprotein E (ApoE) independent macropinocytosis, which is further supported by potent extrahepatic spleen tropism upon intravenous administration to mice. Lastly, we demonstrate the translatability of this LNP platform and utilize it to engineer functional primary human HER2-CAR-Ms ex vivo with potent antigen-specific tumor killing, validated in an ex vivo co-culture with ovarian cancer cells. This bioinspired oxidized LNP platform can potentially be utilized to engineer a range of human CAR-M immunotherapies to treat various types of solid tumors.

bioengineering↗

bioRxiv2025

Capsule-Based Single-Cell Genome Sequencing

Single-cell genomics methods have unveiled the heterogeneity present in seemingly homogenous populations of cells, however, these techniques require meticulous optimization. How exactly does one handle and manipulate the biological contents from a single cell? Here, we introduce and characterize a novel semi-permeable capsule (SPC), capable of isolating single cells and their contents while facilitating biomolecular exchange based on size-selectivity. These capsules maintain stability under diverse physical and chemical conditions and allow selective diffusion of biomolecules, effectively retaining larger biomolecules including genomic DNA, and cellular complexes, while permitting the exchange of smaller molecules, including primers and enzymes. We demonstrate the utility of SPCs for single cell assays by performing the simultaneous culture of over 500,000 cellular colonies, demonstrating efficient and unbiased nucleic acid amplification, and performing combinatorial indexing-based single-cell whole genome sequencing (sc-WGS). Notably, SPC-based sc-WGS facilitates uniform genome coverage and minimal cross-contamination allowing for the detection of genomic variants with high sensitivity and specificity. Leveraging these properties, we conducted a proof-of-concept lineage tracing experiment using cells harboring the hypermutator polymerase {varepsilon} allele (POLE P286R). Sequencing of 1000 single cell genomes at low depth facilitated the capture of lineage marks deposited throughout the genome during each cell division and the subsequent reconstruction of cellular genealogies. Capsule-based sc-WGS expands the single-cell genomics toolkit and will facilitate the investigation of somatic variants, resolved to single cells at scale.

genomics↗

bioRxiv2025

c-Myc inhibits macrophage antimycobacterial response in Mycobacterium tuberculosis infection

Mycobacterium tuberculosis (MTB) is a major global cause of mortality worldwide, responsible for over a million deaths annually. Despite this burden, natural immunity prevents disease in more than 90% of exposed individuals. Previous studies have identified interferon-gamma (IFN-{gamma}) as a key regulator of innate immune defense against MTB. Here, we investigate the impact of IFN-{gamma} timing on macrophage-mediated control of MTB infection. We demonstrate that IFN-{gamma} exposure before infection enhances macrophage antibacterial activity, whereas post-infection exposure does not. Further investigation into this phenotype revealed a strong association between c-Myc signaling and macrophage function in MTB control, as identified using unbiased in vitro systems approaches. Given the challenge of perturbing c-Myc in primary cells, we developed a lentiviral system for c-Myc inhibition and overexpression. Using a tetracycline-inducible Omomyc system - a small peptide inhibitor of c-Myc - we show that c-Myc inhibition promotes a pro-inflammatory macrophage phenotype with enhanced antimycobacterial activity. Mechanistically, c-Myc inhibition induces metabolic reprogramming via increased mTORC1 activity, leading to upregulated inducible nitric oxide synthase and improved bacterial control. In vivo analyses, including murine models and human clinical histopathology, reveal a strong correlation between c-Myc expression and MTB persistence, as well as active tuberculosis (TB), suggesting a role for c-Myc in immune evasion. These findings reveal c-Myc as a potential mediator of immune privilege in MTB infection and highlight its role as a promising target for novel TB therapies aimed at enhancing macrophage function.

immunology↗

bioRxiv2024

CRAGE-RB-PI-seq enables transcriptional profiling of rhizobacteria during plant-root colonization

Plant roots release a wide array of metabolites into the rhizosphere, shaping microbial communities and their functions. While metagenomics has expanded our understanding of these communities, little is known about the physiology of their members in host environments. Transcriptome analysis via RNA sequencing is a common approach to learning more, but its use has been challenging because plant RNA masks bacterial transcripts. To overcome this, we developed randomly-barcoded promoter-library insertion sequencing (RB-PI-seq) and combined it with chassis-independent recombinase assisted genome engineering (CRAGE), using Pseudomonas simiae WCS417 as a model rhizobacterium. This method enables targeted amplification of barcoded transcripts, bypassing plant RNA interference and allowing measurement of thousands of promoter activities during root colonization. Our analysis revealed time-resolved dynamics of promoter activities, highlighting early transcriptional reprogramming as a key determinant of successful colonization. Additionally, we discovered that transcriptional activation of xanthine dehydrogenase and a lysozyme inhibitor are crucial for evading plant immune system defenses. This framework is scalable to other bacterial species and provides new opportunities for understanding rhizobacterial gene regulation in native environments.

microbiology↗

bioRxiv2024

Absence of c-Maf and IL-10 enables Type I IFN enhancement of innate responses to low-dose LPS in alveolar macrophages

Alveolar macrophages (AMs) are lower-airway resident myeloid cells and are among the first to respond to inhaled pathogens. Here, we interrogate AM innate sensing to Pathogen Associated Molecular Patterns (PAMPs) and determine AMs have decreased responses to low- dose LPS compared to other macrophages, as measured by TNF, IL-6, Ifnb, and Ifit3. We find the reduced response to low-dose LPS correlates with minimal TLR4 and CD14 surface expression, despite sufficient internal expression of TLR4. Additionally, we find that AMs do not produce IL-10 in response to a variety of PAMPs due to low expression of transcription factor c- Maf and that lack of IL-10 production contributes to an enhancement of pro-inflammatory responses by Type I IFN. Our findings demonstrate that AMs have cell-intrinsic dampened responses to LPS, which is enhanced by type I IFN exposure. These data implicate conditions where AMs may have reduced or enhanced sentinel responses to bacterial infections. HIGHLIGHTSO_LIAlveolar macrophages (AMs) do not produce TNF or IL-6 in response to low-dose LPS due to minimal surface expression of TLR4 and CD14 C_LIO_LILack of AM IL-10 production is dependent on low c-Maf expression C_LIO_LIExogenous c-Maf expression increases AM IL-10 production C_LIO_LIIFN{beta} enhances AM TNF and IL-6 responses to low-dose LPS and this is dependent on a lack of IL-10 C_LI

immunology↗

bioRxiv2022

Mycobacterial exposure remodels alveolar macrophages and the early innate response to Mycobacterium tuberculosis infection

Alveolar macrophages (AMs) play a critical role during Mycobacterium tuberculosis (Mtb) infection as the first cells in the lung to encounter bacteria. We previously showed that AMs initially respond to Mtb in vivo by mounting a cell-protective, rather than pro-inflammatory response. However, the plasticity of the initial AM response was unknown. Here, we characterize how previous exposure to mycobacterium, either through subcutaneous vaccination with Mycobacterium bovis (scBCG) or through a contained Mtb infection (coMtb) that mimics aspects of concomitant immunity, impacts the initial response by AMs. We find that both scBCG and coMtb accelerate early innate cell activation and recruitment and generate a stronger pro-inflammatory response to Mtb in vivo by AMs. Within the lung environment, AMs from scBCG vaccinated mice mount a robust interferon-associated response, while AMs from coMtb mice produce a broader inflammatory response that is not dominated by Interferon Stimulated Genes. Using scRNAseq, we identify changes to the frequency and phenotype of airway-resident macrophages following mycobacterium exposure, with enrichment for both interferon-associated and pro-inflammatory populations of AMs. In contrast, minimal changes were found for airway-resident T cells and dendritic cells after exposures. Ex vivo stimulation of AMs with Pam3Cys, LPS and Mtb reveal that scBCG and coMtb exposures generate stronger interferon-associated responses to LPS and Mtb that are cell-intrinsic changes. However, AM profiles that were unique to each exposure modality following Mtb infection in vivo are dependent on the lung environment and do not emerge following ex vivo stimulation. Overall, our studies reveal significant and durable remodeling of AMs following exposure to mycobacterium, with evidence for both AM-intrinsic changes and contributions from the altered lung microenvironments. Comparisons between the scBCG and coMtb models highlight the plasticity of AMs in the airway and opportunities to target their function through vaccination or host-directed therapies. Author SummaryTuberculosis, a disease caused by the bacteria Mycobacterium tuberculosis (Mtb), claims around 1.6 million lives each year, making it one of the leading causes of death worldwide by an infectious agent. Based on principles of conventional immunological memory, prior exposure to either Mtb or M. bovis BCG leads to antigen-specific long-lasting changes to the adaptive immune response that can be effective at protecting against subsequent challenge. However, how these exposures may also impact the innate immune response is less understood. Alveolar macrophages are tissue-resident myeloid cells that play an important role during Mtb infection as innate immune sentinels in the lung and the first host cells to respond to infection. Here, we examined how prior mycobacterium exposure, either through BCG vaccination or a model of contained Mtb infection impacts the early innate response by alveolar macrophages. We find that prior exposure remodels the alveolar macrophage response to Mtb through both cell-intrinsic changes and signals that depend on the altered lung environment. These findings suggest that the early innate immune response could be targeted through vaccination or host-directed therapy and could complement existing strategies to enhance the host response to Mtb.

immunology↗

bioRxiv2021

Dynamics and impacts of transposable element proliferation during the Drosophila nasuta species group radiation

Transposable element (TE) mobilization is a constant threat to genome integrity. Eukaryotic organisms have evolved robust defensive mechanisms to suppress their activity, yet TEs can escape suppression and proliferate, creating strong selective pressure for host defense to adapt. This genomic conflict fuels a never-ending arms race that drives the rapid evolution of TEs and recurrent positive selection of genes involved in host defense; the latter has been shown to contribute to postzygotic hybrid incompatibility. However, how TE proliferation impacts genome and regulatory divergence remains poorly understood. Here, we report the highly complete and contiguous (N50=33.8Mb - 38.0Mb) genome assemblies of seven closely-related Drosophila species that belong to the nasuta species group - a poorly studied group of flies that radiated in the last 2 million years. We constructed a high quality de novo TE library and gathered germline RNA-seq data, which allowed us to comprehensively annotate and compare insertion patterns between the species, and infer the evolutionary forces controlling their spread. We find a strong negative association between TE insertion frequency and expression of genes nearby; this likely reflects survivor-bias from reduced fitness impact of TE inserting near lowly expressed, non-essential genes, with limited TE-induced epigenetic silencing. Phylogenetic analyses of insertions of 147 TE families reveal that 53% of them show recent amplification in at least one species. The most highly amplified TE is an non-autonomous DNA element DINE which has gone through multiple bouts of expansions with thousands of full length copies littered throughout each genome. Across all TEs, we find that TEs expansions are significantly associated with high expression in the expanded species consistent with suppression escape. Altogether, our results shed light on the heterogenous and context-dependent nature in which TEs affect gene regulation and the dynamics of rampant TE proliferation amidst a recently radiated species group.

evolutionary biology↗

bioRxiv2021

Neo-sex chromosome evolution shapes sex-dependent asymmetrical introgression barrier

It is increasingly recognized that sex chromosomes are not only the "battlegrounds" between sexes, but also the "Great Walls" fencing-off introgression between diverging lineages. Here we describe conflicting roles of nascent sex chromosomes on patterns of introgression in an experimental hybrid swarm. Drosophila nasuta and D. albomicans are recently diverged, fully fertile sister species that have different sex chromosome systems. The fusion between an autosome (Muller CD) with the ancestral X and Y gave rise to neo-sex chromosomes in D. albomicans, while Muller CDs remains unfused in D. nasuta. We found that a large block containing overlapping inversions on the neo-sex chromosome stood out as the strongest barrier to introgression. Intriguingly, the neo-sex chromosome introgression barrier is asymmetrical in a sex-dependent manner. Female hybrids showed significant D. albomicans biased introgression on Muller CD (neo-X excess), while males showed heterosis with excessive (neo-X, D. nasuta Muller CD) genotypes. While the neo-Y is a more compatible pairing partner of the neo-X, it also shows moderate levels of degeneration and may thus be selectively disfavored, and sex ratio assay revealed heterospecific meiotic drive. We used a population genetic model to dissect the interplay of sex chromosome drive, heterospecific pairing incompatibility between the neo-sex chromosomes and unfused Muller CD, neo-Y disadvantage, and neo-X advantage in generating the observed neo-X excess in females and heterozygous (neo-X, D. nasuta Muller CD) genotypes in males. We show that moderate neo-Y disadvantage and D. albomicans specific meiotic drive are required to counteract the effect of heterospecific meiotic drive observed in our cross, in concert with pairing incompatibility and neo-X advantage to explain observed genotype frequencies. Together, this hybrid swarm between a young species pair shed light onto the dual roles of neo-sex chromosome evolution in creating a sex-dependent asymmetrical introgression barrier at species boundary.

evolutionary biology↗

bioRxiv2021

Molecular characterization of inversion breakpoints in the Drosophila nasuta species group

Chromosomal inversions are fundamental drivers of genome evolution. In the Drosophila genus, inversions have been widely characterized cytologically, and play an important role in local adaptation. Here, we characterize chromosomal inversions in the Drosophila nasuta species group using chromosome-level, reference-quality assemblies of seven species and subspecies in this clade. Reconstruction of ancestral karyotypes allowed us to infer the order in which the 22 identified inversions occurred along the phylogeny. We found a higher rate of inversions on the X chromosome, and heterogeneity in the rate of accumulation across the phylogeny. We molecularly characterize the breakpoints of six autosomal inversions, and found that repeated sequences are associated with inversion breakpoints in four of these inversions, suggesting that ectopic recombination is an important mechanism in generating inversion. Characterization of inversions in this species group provides a foundation for future population genetic and functional studies in this recently diverged species group.

evolutionary biology↗

bioRxiv2019

Latent Mycobacterium tuberculosis infection provides protection for the host by changing the activation state of the innate immune system

An efficacious vaccine against adult tuberculosis (TB) remains elusive. Progress is hampered by an incomplete understanding of the immune mechanisms that protect against infection with Mycobacterium tuberculosis (Mtb), the causative agent of TB1. Over 90% of people who become infected with Mtb mount an immune response that contains the bacteria indefinitely, leading to a state known as "latent TB infection" (LTBI)2. A significant body of epidemiologic evidence indicates that LTBI protects against active TB after re-exposure, offering an intriguing avenue to identifying protective mechanisms3, 4. We show that in a mouse model, LTBI is highly protective against infection with Mtb for up to 100 days following aerosol challenge. LTBI mice are also protected against heterologous bacterial challenge (Listeria monocytogenes) and disseminated melanoma suggesting that protection is in part mediated by alterations in the activation state of the innate immune system. Protection is associated with elevated activation of alveolar macrophages (AM), the first cells that respond to inhaled Mtb, and accelerated recruitment of Mtb-specific T cells to the lung parenchyma upon aerosol challenge. Systems approaches, including transcriptome analysis of both naive and infected AMs, as well as ex vivo functional assays, demonstrate that LTBI reconfigures the response of tissue resident AMs.. Furthermore, we demonstrate that both LTBI mice and latently infected humans show similar alterations in the relative proportions of circulating innate immune cells, suggesting that the same cellular changes observed in the LTBI mouse model are also occurring in humans. Therefore, we argue that under certain circumstances, LTBI could be beneficial to the host by providing protection against subsequent Mtb exposure.

immunology↗

bioRxiv2019

Alveolar macrophages up-regulate a non-classical innate response to Mycobacterium tuberculosis infection in vivo

Alveolar macrophages (AMs) are the first cells to be infected during Mycobacterium tuberculosis (Mtb) infection. Thus the AM response to infection is the first of many steps leading to initiation of the adaptive immune response, which is required for efficient control of infection. A hallmark of Mtb infection is the delay of the adaptive response, yet the mechanisms responsible for this delay are largely unknown. We developed a system to identify, sort and analyze Mtb-infected AMs from the lung within the first 10 days of infection. In contrast to what has been previously described using in vitro systems, we find that Mtb-infected AMs up-regulate a cell-protective antioxidant transcriptional signature that is dependent on the lung environment and not dependent on bacterial virulence. Computational approaches including pathway analysis and transcription factor binding motif enrichment analysis identify Nrf2 as a master regulator of the response of AMs to Mtb infection. Using knock-out mouse models, we demonstrate that Nrf2 drives the expression of the cell protective transcriptional program and impairs the ability of the host to control bacterial growth over the first 10 days of infection. Mtb-infected AMs exhibit a highly delayed pro-inflammatory response, and comparisons with uninfected AMs from the same infected animals demonstrate that inflammatory signals in the lung environment are blocked in the Mtb-infected cells. Thus, we have identified a novel lung-specific transcriptional response to Mtb infection that impedes AMs from responding rapidly to intracellular infection and thereby hinders the overall immune response.\n\nOne Sentence SummaryIn response to Mtb infection in vivo, alveolar macrophages fail to up-regulate the canonical pro-inflammatory innate response and instead induce an Nrf2-dependent cell protective transcriptional program, which in turn impairs the hosts control of bacterial growth.

immunology↗

bioRxiv2018

An essential mycolate remodeling program for mycobacterial adaptation in host cells

The success of Mycobacterium tuberculosis (MTB) stems from its ability to remain hidden from the immune system within macrophages. Here, we report a new technology (Path-seq) to sequence miniscule amounts of MTB transcripts within up to million-fold excess host RNA. Using Path-seq we have discovered a novel transcriptional program for in vivo mycobacterial cell wall remodeling when the pathogen infects alveolar macrophages in mice. We have discovered that MadR transcriptionally modulates two mycolic acid desaturases desA1/A2 to initially promote cell wall remodeling upon in vitro macrophage infection and, subsequently, reduces mycolate biosynthesis upon entering dormancy. We demonstrate that disrupting MadR program is lethal to diverse mycobacteria making this evolutionarily conserved regulator a prime antitubercular target for both early and late stages of infection.\n\nOne Sentence SummaryNovel technology (Path-seq) discovers cell wall remodeling program during Mycobacterium tuberculosis infection of macrophages

microbiology↗