Biology subjects

Agrawal, S.

Publications and source records attributed to Agrawal, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Single-cell Genomic Copy Number Evolution Reveals Frequent Loss of the Y chromosome in Esophageal Adenocarcinoma

The incidence of esophageal adenocarcinoma (EAC), an aggressive cancer, is rising in Western countries, and incidence is higher in men. To better understand the roles of DNA copy number evolution, tumor heterogeneity, and tumor microenvironment in EAC and its origins, we evaluated EAC, Barretts Esophagus (BE), and normal adjacent tissue using our previously developed ultra-sensitive, single-nucleus DNA copy number analysis (12,186 nuclei, 20 specimens) and single-nucleus RNA-sequencing (snRNA-seq; 87,027 nuclei, 32 specimens). Loss of Y (LOY) chromosome was rare in BE but markedly enriched in EAC. In genome unstable cells, complete LOY chromosome often coincided with X-chromosome duplication. We did not detect X-chromosome inactivation via XIST expression in cells with LOY, suggesting increased expression of X-chromosome genes and potential effects on tumor immune evasion. When considering patients clinical features, we identified distinct differences in tumor microenvironment by sex, age, and obesity status, particularly massively reduced immune cell composition in obese patients. Finally, our cell line models recapitulate LOY, and we identify the first candidate anti-LOY agent. Our findings highlight how LOY affects tumor subpopulations and shapes the tumor microenvironment during EAC progression.

cancer biology↗

bioRxiv2025

Structural Hijacking of FcRn by Human Astrovirus Spikes Reveals Conserved Epitopes for Broad-Spectrum Antivirals

Human astroviruses (HAstVs) are a leading cause of pediatric gastroenteritis and emerging systemic infections, yet no targeted therapies exist. A critical barrier to intervention has been the lack of molecular insights into viral entry, particularly the interaction between the HAstV capsid spike and its receptor, the neonatal Fc receptor (FcRn). Here, we report high-resolution crystal structures of the HAstV spike from classical serotypes 2 and 6 in complex with human FcRn, defining the conserved receptor-binding interface at atomic resolution. These structures reveal serotype-specific variations dictating receptor affinity and that reported neutralizing antibodies inhibit infection primarily by sterically blocking the receptor-binding site. Mapping conserved epitopes across classical HAstV serotypes provides a blueprint for design of broad-spectrum antivirals that disrupt viral entry. Notably, our structural data rationalize the potential repurposing of clinical FcRn inhibitors, such as Nipocalimab, to block HAstV infection, bridging critical gaps in astrovirus biology and antiviral development.

biophysics↗

bioRxiv2025

Structural characterization of MAM01 and other cost-effective engineered monoclonal antibodies for malaria prevention

Long-lasting and effective vaccines or monoclonal antibodies (mAbs) for malaria prevention are highly beneficial for people of all ages living in malaria-endemic regions. Recently, two highly protective human mAbs AB000224 (IGHV3-49/IGLV1-40) and AB007088 (IGHV3-33/IGKV1-5), which are encoded by different germline genes, were re-engineered and renamed as MAM01 and MS-1805, respectively, to improve their half-life, developability (including manufacturing), and cost-effectiveness according to WHO guidelines. MAM01 has completed phase 1 and 1b clinical trials (safety / PK / challenge) in US healthy naive adults and is in phase 1 trials (age-de-escalation studies) in Uganda. Here, we determined crystal structures of the antigen-binding fragments (Fabs) of engineered MAM01, MS-1805, and 7088 in complex with different regions of the Plasmodium falciparum (Pf) circumsporozoite protein (CSP), including junctional, minor, and major repeat regions. Notably, Fab 7088 features an extended CDRL3 comprising 10 amino acids (CDRL3:10) instead of the typical 8 amino acid CDRL3 (CDRL3:8) for VH3-33/VK1-5-encoded mAbs, revealing unique folding within its germline context. Structural comparisons showed that engineered antibodies retain the key molecular and hydrogen bond interactions with no significant conformational changes or loss in binding affinity. These findings demonstrate that the efficacy and affordability of human mAbs can be enhanced by selectively mutating residues in antibody framework regions without compromising binding affinity or epitope interaction.

biophysics↗

bioRxiv2025

Standardizing a Protocol for Streamlined Synthesis and Characterization of Lipid Nanoparticles to Enable Preclinical Research and Education

Lipid nanoparticles (LNPs) have revolutionized nucleic acid delivery, enabled the first FDA-approved RNAi therapy (Onpattro), and accelerated the development of mRNA vaccines during the COVID-19 pandemic. The success of LNP-based vaccines demonstrated the potential of these nanoparticles for broader therapeutic applications. As interest in LNP-based therapies expands, there is an urgent need for low-cost, reproducible synthesis protocols that can be readily implemented across a wide range of research settings. Traditional methods for LNP synthesis, such as pipette and vortex mixing, can yield inconsistent results. In contrast, microfluidic mixing offers better control over LNP properties but requires expensive equipment. This financial barrier prevents many laboratories from accessing cutting-edge LNP technology, slowing the pace of preclinical research and limiting the exploration of its full therapeutic potential. To address this, we developed a standardized protocol for microfluidic mixing using a syringe pump and a commercially available microfluidic chip. This approach offers a cost-effective and reproducible method for LNP synthesis. The protocol details the synthesis of LNPs, physical characterization via dynamic light scattering, encapsulation efficiency using the RiboGreen assay, and evaluation of in vitro transfection efficiency using the OneGlo assay, confocal microscopy, and a flow cytometer. We explored consistency through various experimental parameters, aiming to optimize the protocol and, importantly, tested user-to-user reproducibility with undergraduates without LNP experience with minimal supervision. We found consistency among assembly conditions, including mRNA/lipid concentration, flow rate, dialysis time, ionizable lipid type, and chip reusability. We tested the broader applicability of the method with four ionizable lipids (DLin-MC3-DMA, LP01, C12-200, and SM-102). High encapsulation efficiency (96-100%) was maintained across tested concentrations, with slightly larger particle sizes at lower doses. Based on the flow cytometry analysis, a 7.5 {micro}g dose was chosen for future use to conserve mRNA without sacrificing efficacy. Dialysis duration had minimal impact on encapsulation, but longer times increased particle size and reduced luminescence. The protocol consistently produced narrowly dispersed particles (PDI < 0.2), and equipment was reusable for up to six runs, potentially reducing the per-run cost. Even novice users achieved reproducible results, highlighting the protocols accessibility and potential to expand LNP research and applications.

bioengineering↗

bioRxiv2025

Beyond the Cut: Long-read sequencing reveals complex genomic and transcriptomic changes in AAV-CRISPR therapy for Duchenne Muscular Dystrophy

Adeno associated virus (AAV)-mediated delivery of CRISPR associated nucleases (AAV-CRISPR) is a promising solution to treat genetic diseases such as Duchenne Muscular Dystrophy (DMD) and is now in early clinical trials. However, genotoxicity and immunogenicity concerns have hindered clinical translation. Due to the complex etiology associated with DMD, the post-transduction consequences of double-stranded breaks induced by AAV-CRISPR in disease models are unclear. This barrier is partially conferred by conventional sequencing methods where common outcomes of AAV-CRISPR editing often escape detection. However, recent reports of novel long-read sequencing approaches permit comprehensive variant detection using a broader sequence context. Here, we comprehensively investigated genomic and transcriptomic post-AAV-CRISPR transduction consequences in myoblast cells and a DMD mouse model following intramuscular and intravenous AAV-CRISPR therapy using both long- and short-read sequencing techniques. Structural variant characterization indicates that unintended on-target large insertions and inversions are common editing outcomes. We demonstrate that combining adaptive sampling with nanopore Cas9-targeted sequencing (AS-nCATS) for long-read quantification of AAV integration is synergistic for detecting difficult-to-amplify editing events. This unbiased data suggests that full-length AAV integration is equally as probable as the on-target deletion. Further, we develop a Nanopore Rapid Amplification of cDNA Ends (nRACE-seq) pipeline for long-read detection of unknown 5 or 3 ends of edited transcripts. The nRACE-seq approach effectively detects the presence of AAV-Dmd chimeric transcripts, erroneous splicing events, and off-target AAV integration sites. In summary, our findings offer insights into the adaptation of AAV-CRISPR DSB-mediated therapeutics for monogenic diseases and promote the standardization of CRISPR evaluation. We highlight the importance of coupling polymerase-based and polymerase-free methods in long-read sequencing to assess editing outcomes as the field progresses toward clinical applications.

bioengineering↗

bioRxiv2025

Systematic optimization of Caenorhabditis elegans cryopreservation

Caenorhabditis elegans (C. elegans) is a non-parasitic roundworm widely utilized as a versatile model organism for studying fundamental biological processes. Despite the availability of multiple cryopreservation methods, variations in the selection of developmental stage, cryoprotectant composition, and storage conditions may sometimes cause inconsistencies and uncertainty among researchers. In this study, we followed a step-by-step approach to optimize the C. elegans cryopreservation process. Firstly, different larval stages (L1-gravid adult) of C. elegans were cryopreserved using a fixed concentration of trehalose-DMSO and glycerol, followed by the post-thaw survival rate assessment. We found that the starved L1 larvae has the highest survival rate among other developmental stages. Subsequently, the starved L1 larvae was cryopreserved using different concentrations (5%, 10%, and 15%) of DMSO and glycerol and found that 5% DMSO has the most effective cryoprotection. Finally, the impact of storage conditions on worm viability was evaluated by placing cryogenic vials in different storage boxes, including standard cardboard cryogenic boxes, Styrofoam boxes, and isopropanol-based freezing containers, and found that standard cardboard cryogenic boxes provided higher worm viability. The finalized protocol was then validated across multiple mutant and transgenic strains to confirm its reproducibility and broad suitability. Taken together, our results demonstrate that starved L1 larvae cryopreserved with 5% DMSO using a standard cardboard cryogenic box provided the highest survival rate (>90%) across wild-type, mutant, and transgenic strains. Here we propose a simple, reproducible, and strain-independent protocol for cryopreservation of C. elegans. The proposed protocol can be easily adopted in C. elegans research laboratories and stock centers globally.

genetics↗

bioRxiv2025

Integrative Transcriptomic and miRNA Analysis Reveals Immune Suppression and Metabolic Reprogramming in FGFR3-TACC3 Fusion-Positive versus Fusion-Negative Bladder Cancer

FGFR3-TACC3 gene fusion drives a molecularly distinct subset of bladder cancer characterized by post-transcriptional immune evasion and metabolic rewiring. To elucidate the transcriptomic and regulatory consequences of this fusion, we performed an integrative analysis of mRNA and miRNA expression profiles in fusion-positive versus fusion-negative bladder tumors using TCGA-derived RNA-Seq and miRNA-Seq data. Fusion-positive tumors exhibited upregulation of mitochondrial oxidative phosphorylation and ribosomal genes (e.g., COX5B, ATP5F1D, RPS15) and marked downregulation of immune regulatory genes (e.g., CD4, STAT3, IL6, CD86, PTPRC). Pathway enrichment revealed suppression of JAK/STAT, PD-L1 signaling, and cytokine networks, consistent with a fusion-driven immunosuppressive phenotype. We identified fusion-specific miRNA-mRNA regulatory interactions, including upregulated miRNAs (miR-1226-3p, miR-149-5p, miR-301b-3p) that target immune transcripts and downregulated miRNAs (miR-199b-5p, miR-214-3p) that normally suppress mitochondrial and ribosomal genes. In contrast, fusion-negative tumors displayed an immunoreactive signature marked by elevated IFNG, CXCL10, and IRF1 expression and downregulation of immune-suppressive miRNAs (miR-125a-5p, miR-23b-3p). Minimum free energy analysis and AlphaFold3-based AGO2-RISC docking confirmed structural feasibility of 3D duplex formation for 13 miRNA-target pairs, supporting active silencing complexes. Immune deconvolution revealed reduced infiltration of cytotoxic T cells, NK cells, and dendritic cells in fusion-positive tumors, while fusion-negative tumors showed robust immune cell presence. Key gene-immune cell correlations further confirmed differential immune architecture: SRC and CD4 were strongly linked to suppressed infiltration in fusion-positive tumors, while IFNG and CXCL10 positively correlated with T and NK cell abundance in fusion-negative cases. ROC analysis highlighted COMT, RPS15, and miR-187-3p as robust classifiers of fusion-positive tumors (AUC > 0.9), and CXCL10, CD74, and miR-142 for fusion-negative tumors. Connectivity Map analysis highlighted Lofexidine and Lenalidomide as candidate compounds predicted to reverse fusion-specific transcriptional programs. Collectively, our findings define immune suppression and metabolic reprogramming as core features of FGFR3-TACC3 fusion-positive bladder cancer and nominate fusion-specific miRNA-mRNA interactions as candidate biomarkers and therapeutic targets.

cancer biology↗

bioRxiv2025

Active learnings impact on student course performance in STEM varies by type and intensity

We updated a recent meta-analysis of active learnings impact on student achievement in undergraduate STEM courses by following the same protocol to evaluate studies published from 2010-2017. We screened 1659 papers, coded 1294, and found 210 that met five pre-established inclusion criteria and six pre-established criteria for methodological quality. After further dropping 76 studies with no exam scores data, 134 of these studies contained data on student performance on identical or equivalent exams. We found that on average, active learnings effect size on exam scores was 0.519 {+/-} 0.049, meaning that when students are in active learning classes, they perform roughly half a standard deviation higher on an identical exam. Funnel plots and sensitivity analyses indicated that these results were not due to sampling bias. Active learning had a positive impact on student outcomes regardless of class size, course level, or STEM discipline, though there was heterogeneity in the effects. All of these results are very similar when compared to earlier meta-analyses, however increased resolution in the studies analyzed here revealed two novel results. First, student performance was significantly better in courses that employed high-intensity active learning, defined as students being on task at least two-thirds of class time, versus lower-intensities. Additionally, there was significant heterogeneity in efficacy across different types of active learning employed. These results suggest that most, if not all types of active learning are effective, and that when innovating in their classes, instructors should continually work to increase active learning intensity. We urge caution in interpreting the results on active learning types, however, and propose a preliminary framework for making more-sophisticated and reliable analyses of variation in course design. Finally, the evidence presented here for active learnings impact on student outcomes creates a strong foundation for faculty professional development and administration.

scientific communication and education↗

bioRxiv2025

Distinct region-specific neutralization profiles of contemporary HIV-1 clade C against best-in-class broadly neutralizing antibodies

Broadly neutralizing antibodies (bnAb) have been clinically proven to be an excellent choice for HIV-1 prevention. However, the relative effectiveness of best-in-class bnAbs against regionally relevant circulating HIV-1 forms is not clear. In the present study, we compared the degree of neutralization sensitivity of contemporary HIV-1 Indian clade C with that of South African origin. Phylogenetic analysis revealed that these clade C viruses continue to evolve distinctly from one another. Env-pseudotyped viruses prepared using contemporary HIV-1 clade C env genes (N=115) obtained from nine geographically distinct sites in India (between 2020-2023) were found to be most sensitive to V3-directed bnAbs 10-1074 and BG18, and second generation CD4 binding site (CD4bs) directed bnAbs (VRC07, N6 and 1-18), however they were found to be significantly resistant to V1/V2 apex directed bnAbs. Moreover, we observed that the degree of sensitivity varied between contemporary Indian and South African clade C viruses. Differences in degree of neutralization susceptibility were associated with differences observed in key residues that form bnAb contact sites, gp120 loop lengths and the number of N-linked glycans in the V4 hypervariable region. Interestingly, the second generation CD4bs bnAbs (VRC07, N6, 1-18) showed neutralization of VRC01 and 3BNC117 resistant viruses but with 2-7-fold reduced potency compared to the VRC01 sensitive counterparts, likely due to the enrichment of resistance associated residues observed in loop D. Predictive analysis indicated that combination of BG18, N6 and PGDM1400 can provide over 95% neutralization coverage at 1g/mL of contemporary India clade C, an observation found to be distinct to that reported for the Africa clade C viruses. Taken together, we found distinct neutralization patterns and env signatures associated with resistance to key bnAbs. Our study highlights that towards achieving clinical effectiveness, both the complementarity of bnAb classes and the regionally relevant HIV forms need to be considered. Author summaryWhile the development of vaccines to prevent HIV infection remains a global priority, their potential effectiveness is limited by the extraordinarily diversified circulating forms of HIV-1. The prospect of best-in-class bnAbs as potential prevention option has been demonstrated in several studies including the Phase II Antibody Mediated Prevention (AMP) trial; however, to be broadly applicable, bnAbs will need to overcome the substantial variability of HIV env. The present study highlights that the contemporary HIV-1 clade C viruses are evolving to be less sensitive to the best-in-class bnAbs and HIV-1 clade C that predominates in India and South Africa vary in their degree of susceptibility to best-in-class clinically relevant bnAbs. This indicates differences in the antigenic properties between globally circulating HIV-1 clade C at a population level. Overall, the outcome of this study highlights the need for periodic assessment of sequence and neutralization profiles of the circulating regionally relevant HIV-1 forms towards prioritizing the bnAb combination suitable for effective intervention.

immunology↗

bioRxiv2024

Interplay of YEATS2 and GCDH mediates histone crotonylation and drives EMT in head and neck cancer

The regulation of gene expression is an integral cellular process orchestrated by epigenetic marks like histone modifications. Perturbations in the activity or abundance of epigenetic factors can lead to tumorigenesis. Remarkably, several metabolites influence the epigenetic landscape in cells. Here, we investigated the interplay between a highly expressed epigenetic factor, YEATS2, and a metabolic enzyme, GCDH, in regulating epithelial-to-mesenchymal transition in head and neck cancer. We report that the histone reader YEATS2 is responsible for increased invasive potential in head and neck cancer in an SP1-dependent manner. YEATS2 functions by maintaining histone crotonylation, and its abrogation leads to a global decrease in the H3K27cr mark. Mechanistically, we report that YEATS2 maintains high promoter H3K27cr levels by assisting in the recruitment of crotonyltransferase p300 at the promoter of the EMT-promoting gene SPARC. Further, we found that the addition of the H3K27cr mark is also dependent on the crotonyl-CoA-producing enzyme GCDH. Overall, we describe a novel mechanism of interplay between epigenetics and metabolism in head and neck tumorigenesis, which results in the enhanced expression of EMT-related genes in a histone crotonylation-dependent manner.

cancer biology↗

bioRxiv2024

Alternative mRNA Splicing and Polyadenylation Events are Synchronous with Podocyte Injury and Slit Diaphragm Dysregulation in Glomerular Disease Models

BackgroundGlomerular disease, characterized by podocyte injury and proteinuria, can lead to chronic kidney disease and end stage kidney disease. We hypothesized that the glomerular pathophysiology is associated with mRNA alternative splicing and polyadenylation of glomerular genes and of critical podocyte and slit diaphragm components that regulate the filtration barrier. MethodsGlomerular damage, accompanied by proteinuria, was induced by puromycin-aminonucleoside or adriamycin to mimic human minimal change disease or focal segmental glomerulosclerosis (FSGS), respectively, and RNA-seq analyses was performed. Alternatively spliced and alternatively polyadenylated events through differential exon and poly(A) site usage were queried in JunctionSeq and APATrap pipelines. These events were further mapped on podocyte and glomerular landscape, analyzed and modulated for slit diaphragm components, and cis- and trans-regulatory elements were identified. ResultsAltered glomerular mRNA processing by alternative splicing/polyadenylation was identified in 136/71 and 1875/746 genes in minimal change disease and FSGS models, respectively. Transcript annotation and prioritization of significant alternative splicing and polyadenylation identified key events in several podocyte and slit diaphragm genes with novel and established roles. Alternative splicing of critical slit diaphragm components, the junction protein TJP1/ZO1 and microtubule associating protein ITM2B was further characterized. Alternative polyadenylation of core members of the slit diaphragm, NPHS1, NPHS2 and NEPH1 was analyzed with potential alteration of microRNA binding sites between the proximal vs distal poly (A) site usage in their mRNAs. Concomitantly, dysregulation of trans-regulatory elements (polyadenylation and splicing factors), was discovered in these models of nephropathies. Additionally, beneficial proteinuria-reducing treatments, pioglitazone and GQ16 reversed many alternatively spliced and polyadenylated events. Moreover, GWAS SNPs as potential cis-regulatory elements were identified in several genes from the human nephrotic syndrome database. Finally, we demonstrated proof-of-concept principle of chemically modified splice switching oligonucleotides in modulating TJP1 alternative splicing in podocytes. ConclusionFindings from our studies identified that glomerular pathophysiology and disruption of the filtration barrier is associated with alternative splicing and alternative polyadenylation of glomerular genes, many of which are crucial determinants of podocyte structure and function and the slit diaphragm complex. Key PointsO_LIAlternative mRNA processing adds an important layer of transcriptome complexity in podocyte and slit diaphragm components during glomerular injury. C_LIO_LITrans- and cis-elements may offer potential mechanisms to regulate alternative splicing and polyadenylation during podocyte injury. C_LIO_LIAntisense oligonucleotides can regulate mRNA processing events, thus offering a potential RNA-based therapeutic strategy to treat glomerular disease. C_LI

molecular biology↗

bioRxiv2024

Human Replication Protein A complex is a Telomerase Processivity Factor Essential for Telomere Maintenance

Telomerase is crucial for maintaining telomere length and safeguarding genome stability. In this study, we identified Replication Protein A (RPA) as a novel telomerase processivity factor, functioning alongside the telomerase recruitment factor TPP1-POT1. AlphaFold2 predictions revealed that RPA and TPP1 interact with telomerase at distinct binding sites. Using separation- of-function mutants, we discovered that RPA-mediated telomerase stimulation is indispensable for telomere elongation, while TPP1-POT1 primarily functions in recruiting telomerase to telomeres. Furthermore, we demonstrated that short telomere disease-associated telomerase mutations compromise RPAs ability to stimulate telomerase, establishing a link between impaired RPA-dependent processivity and telomeropathies. Our findings redefine human telomerase regulation by establishing RPA as a critical regulator and provide new insights into the molecular basis of telomere-related diseases.

molecular biology↗

bioRxiv2024

A Dual-Fluorescence Assay for Gene Delivery Vehicle Screening in Macrophages with an Inflammation-Inducible Reporter Construct

BackgroundMacrophages are a promising target for therapeutics in various applications such as regenerative medicine and immunotherapy for cancer. Due to their plastic nature, macrophages can switch from a non-activated state to activated with the smallest environmental change. For macrophages to be effective in their respective applications, screening for phenotypic changes is necessary to elucidate the cell response to different delivery vehicles, vaccines, small molecules, and other stimuli. MethodsWe created a sensitive and dynamic high-throughput screening method for macrophages based on the activation of NF-{kappa}B. For this reporter, we placed an mRFP1 fluorescence gene under the control of an inflammatory promoter, which recruits NF-{kappa}B response elements to promote expression during the inflammatory response in macrophages. We characterized the inflammatory reporter based on key markers of an inflammatory response in macrophages including TNF- cytokine release and immunostaining for inflammatory and non-inflammatory cell surface markers. We compared gene delivery and inflammation of several clinically relevant viral vehicles and commercially available non-viral vehicles. Statistical analysis between groups was performed with a one-way ANOVA with post-hoc Tukeys test. ResultsThe reporter macrophages demonstrated a dynamic range after LPS stimulation with an EC50 of 0.61 ng/mL that was highly predictive of TNF- release. Flow cytometry revealed heterogeneity between groups but confirmed population level shifts in pro-inflammatory markers. Finally, we demonstrated utility of the reporter by showing divergent effects with various leading gene delivery vehicles. DiscussionThis screening technique developed here provides a dynamic, high-throughput screening technique for determining inflammatory response by mouse macrophages to specific stimuli. The method presented here provides insight into the inflammatory response in mouse macrophages to different viral and non-viral gene delivery methods and provides a tool for high-throughput screening of novel vehicles.

bioengineering↗

bioRxiv2024

Divergent neural circuits for proprioceptive and exteroceptive sensing of the Drosophila leg

Somatosensory neurons provide the nervous system with information about mechanical forces originating inside and outside the body. Here, we use connectomics from electron microscopy to reconstruct and analyze neural circuits downstream of the largest somatosensory organ in the Drosophila leg, the femoral chordotonal organ (FeCO). The FeCO has been proposed to support both proprioceptive sensing of the flys femur-tibia joint and exteroceptive sensing of substrate vibrations, but it was unknown which sensory neurons and central circuits contribute to each of these functions. We found that different subtypes of FeCO sensory neurons feed into distinct proprioceptive and exteroceptive pathways. Position- and movement-encoding FeCO neurons connect to local leg motor control circuits in the ventral nerve cord (VNC), indicating a proprioceptive function. In contrast, signals from the vibration-encoding FeCO neurons are integrated across legs and transmitted to mechanosensory regions in the brain, indicating an exteroceptive function. Overall, our analyses reveal the structure of specialized circuits for processing proprioceptive and exteroceptive signals from the fly leg. These findings are consistent with a growing body of work in invertebrate and vertebrate species demonstrating the existence of specialized limb mechanosensory pathways for sensing external vibrations.

neuroscience↗

bioRxiv2024

A conserved genetic basis for commensal-host specificity through live imaging of colonization dynamics

Animals throughout the metazoa selectively acquire specific symbiotic gut bacteria from their environment that aid host fitness. Current models of colonization suggest these bacteria use weakly specific receptors to stick to host tissues and that colonization results when they stick in a region of the host gut that overlaps with their nutritional niche. An alternative model is that unique receptor-ligand binding interactions provide specificity for target niches. Here we use live imaging of individual symbiotic bacterial cells colonizing the gut of living Drosophila melanogaster to show that Lactiplantibacillus plantarum specifically recognizes a distinct physical niche in the host gut. We find that recognition is controlled by a colonization island that is widely conserved in commensals and pathogens from the Lactobacillales to the Clostridia. Our findings indicate a genetic mechanism of host specificity that is broadly conserved. One-Sentence SummaryHost-symbiont specificity is encoded by a conserved colonization island that provides molecular precision to host niche access.

microbiology↗

bioRxiv2024

Precision and efficacy of RNA-guided DNA integration in high-expressing muscle loci

Gene replacement therapies primarily rely on adeno-associated viral (AAV) vectors for transgene expression. However, episomal expression can decline over time due to vector loss or epigenetic silencing. CRISPR-based integration methods offer promise for long-term transgene insertion. While the development of transgene integration methods has made substantial progress, identifying optimal insertion loci remains challenging. Skeletal muscle is a promising tissue for gene replacement owing to low invasiveness of intramuscular injections, relative proportion of body mass, the multinucleated nature of muscle, and the potential for reduced adverse effects. Leveraging endogenous promoters in skeletal muscle, we evaluated two high-expressing loci using homology-independent targeted integration (HITI) to integrate reporter or therapeutic genes in mouse myoblasts. We hijacked the muscle creatine kinase (Ckm) and myoglobin (Mb) promoters by co-delivering CRISPR-Cas9 and a donor plasmid with promoterless constructs encoding green fluorescent protein (GFP) or human Factor IX (hFIX). Additionally, we deeply profiled our genome and transcriptome outcomes from targeted integration and evaluated the safety of the proposed sites. This study introduces a proof-of-concept technology for achieving high-level therapeutic gene expression in skeletal muscle, with potential applications in targeted integration-based medicine and synthetic biology.

bioengineering↗

bioRxiv2023

Hummingbirds rapidly respond to the removal of vision and control a sequence of rate-commanded maneuvers in milliseconds

Hummingbird flight is the epitome of extreme aerial agility and controlled stability, as hummingbirds routinely exercise a variety of stunning aerobatic feats. Yet, the control of these amazing maneuvers is not well understood. Here we examined how hummingbirds control a sequence of maneuvers within milliseconds and tested whether and when their vision is active during this rapid process. We elicited escape flight in calliope hummingbirds and removed visible light at various instants during the maneuvers and quantified their flight kinematics and responses. We show that the escape maneuvers were composed of rapidly-controlled sequential modules, including evasion, reorientation, nose-down dive, forward flight, and nose-up to hover. The hummingbirds did not respond to the light removal during evasion and reorientation until a critical light-removal time; afterward they showed two categories of luminance-based responses that rapidly altered maneuvering modules to terminate the escape. We also show that hummingbird maneuvers are rate-commanded and require no active braking, i.e., their body angular velocities were proportional to the change of wing motion patterns, a trait that likely alleviates the computational demand on flight control. Together, this work uncovers the key traits of hummingbird agility, which can also inform and inspire designs for next-generation agile aerial systems.

animal behavior and cognition↗

bioRxiv2023

Dissecting Multiparametric Cerebral Hemodynamics using Integrated Ultrafast Ultrasound and Multispectral Photoacoustic Imaging

Understanding brain-wide hemodynamic responses to different stimuli at high spatiotemporal resolutions can help study neuro-disorders and brain functions. However, the existing brain imaging technologies have limited resolution, sensitivity, imaging depth and provide information about only one or two hemodynamic parameters. To address this, we propose a multimodal functional ultrasound and photoacoustic (fUSPA) imaging platform, which integrates ultrafast ultrasound and multispectral photoacoustic imaging methods in a compact head-mountable device, to quantitatively map cerebral blood volume (CBV), cerebral blood flow (CBF), oxygen saturation (SO2) dynamics as well as contrast agent enhanced brain imaging with high spatiotemporal resolutions. After systematic characterization, the fUSPA system was applied to quantitatively study the changes in brain hemodynamics and vascular reactivity at single vessel resolution in response to hypercapnia stimulation. Our results show an overall increase in brain-wide CBV, CBF, and SO2, but regional differences in singular cortical veins and arteries and a reproducible anti-correlation pattern between venous and cortical hemodynamics, demonstrating the capabilities of the fUSPA system for providing multiparametric cerebrovascular information at high-resolution and sensitivity, that can bring insights into the complex mechanisms of neurodiseases.

bioengineering↗