Biology subjects

Mohammed, A.

Publications and source records attributed to Mohammed, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Soda-baited RNAi Yeast Insecticides as Effective Attractive Targeted Sugar Baits (ATSBs) for Mosquito Control

BackgroundAttractive Targeted Sugar Baits (ATSBs) are a promising, environmentally friendly approach for mosquito control, but the direct field application, scalability and long-term effectiveness of ATSBs across diverse mosquito species remain significant challenges. Methodology/Principal FindingsWe assessed the efficacy of a genetically engineered RNA interference (RNAi) yeast strain (Sh.463_56.10R) formulated in three sugar baits, soda (Coca-ColaTM), 10% sucrose, and a commercial mosquito bait (BaitStabTM), on Aedes, Anopheles and Culex mosquitoes. All RNAi yeast bait formulations induced significantly higher mean mortality (87-100%) compared to the control groups (0-9%; P<0.0001), but mosquitoes exhibited a higher feeding preference for RNAi yeast-soda baits, which induced mortality rates of 94-100% (P < 0.0001) recorded across all mosquito species. Additionally, to assess the competitiveness of the RNAi yeast-soda bait to other tropical sugar sources, semi-field choice assays were conducted in Trinidad, West Indies using competing flowering plants and fruits typically found in residential environments. The RNAi yeast-soda ATSB continued to perform well in the presence of competing floral and fruit sugar sources during both Aedes albopictus and Culex quiquefasciatus trials, though the presence of several fruits and flowers did reduce A. aegypti mortality, suggesting that further field testing will be necessary. The residual activity of the Sh.463_56.10R + soda formulation was retained for at least 14 months, with sustained 100% mortality in C. quinquefasciatus and 93-100% mortality in Aedes aegypti, Anopheles gambiae and Anopheles stephensi. The RNAi yeast-soda ATSB also performed well in semi-field studies performed with a prototype soda bottle feeder. Conclusions/SignificanceThis study demonstrates the potential of soda-baited RNAi yeast as a potent, long-lasting, and scalable platform for ATSB-based mosquito control as a component of integrated vector management programs. Author SummaryMosquito-borne diseases continue to affect millions of people worldwide, and current mosquito control methods face challenges such as low public uptake, insecticide resistance and environmental concerns. Here we evaluated a new and environmentally friendly approach to mosquito control using ATSBs. We tested genetically engineered species-specific yeast producing RNAi molecules capable of killing mosquitoes that feed on it. We mixed the yeast with three different sugar baits, including soda (Coca-ColaTM), 10% sucrose, and the commercial mosquito bait BaitStabTM formulation, and evaluated how well they worked against different mosquitoes. The results showed that the RNAi yeast mixed with soda was the most effective, killing up to 100% of mosquitoes in laboratory and outdoor tests. The bait remained effective in the presence of many competing natural tropical fruit and floral sugar sources. Remarkably, the bait, which can be delivered in a soda bottle feeder, stayed active for at least 14 months under simulated field conditions. These findings suggest that soda-based RNAi yeast baits could provide a practical, long-lasting and scalable tool for mosquito control and may help strengthen future strategies to reduce mosquito-borne diseases.

genetics↗

bioRxiv2026

BRD4 recruitment desilences transcription without erasure or depletion of repressive chromatin

How genes are desilenced within mesoscale repressive chromatin is a crucial yet poorly understood phenomenon. Prevailing models posit that methylation of lysine-9 of histone H3 (H3K9me3) engages heterochromatin protein 1 (HP1) to drive chromatin compaction and transcriptional silencing. The erasure of this repressive mark and its replacement with acetyl/acyl groups recruits positive factors such as BRD4/BET to elicit gene transcription. We report that in Friedreichs ataxia, a synthetic gene regulator (SynGR1) drives transcription across repressive chromatin without removal or replacement of H3K9me3 or HP1. By selectively recruiting BRD4/BET into repressive GAA-repeats in frataxin (FXN), SynGR1 creates a paradoxical state where gene transcription and repressive chromatin co-exist. Contrary to convention, we find that BRD4 readily partitions into phase separated HP1 condensates in vitro and into HP1 puncta in patient-derived cells, thus presenting a mechanistic explanation for desilencing transcription without the dispersal of mesoscale repressive chromatin. Epigenetic drugs that gate sequential steps in transcription, synergistically stimulate FXN expression while concomitantly increasing, rather than eliminating, repressive H3K9me3 and HP1 levels. More broadly, this study highlights the dynamic nature of repressive chromatin and the context-dependence of epigenetic marks in regulating gene expression.

cell biology↗

bioRxiv2026

Complement modulation synergizes with therapeutic hypothermia in a rat model of neonatal HIE

BackgroundNeurodevelopmental impairment remains common in neonatal hypoxic-ischemic encephalopathy (HIE) despite treatment with the standard of care, therapeutic hypothermia (TH). The complement response activates at reperfusion and is known to exacerbate neuroinflammation and injury, though its full role and interaction with hypothermia are incompletely defined. We hypothesized that modulating the complement response could improve structural and functional outcomes in HIE, and tested a novel complement therapy (CT), consisting of C3a peptides and the C5a-receptor antagonist PMX205, as both a stand-alone treatment and as an adjuvant to TH. MethodsWistar rat pups were randomized to the following treatment groups: Sham (uninjured control), NT (uninjured, normothermia/not treated control), or injured and treated with either TH, CT, or CT+TH. At term-equivalence, mild-moderate hypoxic-ischemic injury was induced by Vannuccis method. To capture the short and long-term effects of the treatments, cohorts were harvested 3 or 66-72 days post-injury, respectively. Cerebral injury was measured by quantifying levels of inflammatory markers and cerebral tissue loss, and functional outcomes were assessed in a series of behavioral tests. The data were stratified to detect sexual dimorphisms. ResultsCT and TH treatments demonstrated test and sex-dependent differences in improvement compared to untreated, injured rats. In male rats, TH treatment worsened long-term hippocampal and thalamic brain injury and functional measures of ataxia and attention. CT-treatment worsened long-term thalamic loss in females. Combining the two treatments (CT+TH) demonstrated additive improvement in both sexes, including short and long-term cortical loss and ataxia. ConclusionsComplement modulation enhances the neuroprotective effects of TH after neonatal hypoxic-ischemic injury, with sex-specific effects on inflammation and behavior. Combining complement modulation with the standard of care often demonstrated synergistic improvement in both sexes, supporting complement-targeted therapy as a promising adjunct to hypothermia in neonatal HIE. Graphical abstract. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/717097v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1025d1forg.highwire.dtl.DTLVardef@2fa4e5org.highwire.dtl.DTLVardef@1f2c1c4org.highwire.dtl.DTLVardef@8f3410_HPS_FORMAT_FIGEXP M_FIG C_FIG Created with BioRender. Saadat, A. (2026) https://BioRender.com/siwm825.

neuroscience↗

bioRxiv2025

Comparing Multislice Simulations of MD Simulations with CryoEM Exposes Membrane Prediction Errors

Cryo-electron microscopy (cryoEM) is a powerful tool for atomic- and molecular-resolution structure determination, while molecular dynamics (MD) simulations are similarly powerful tools for predicting molecular trajectories. Given the challenges in estimating biomolecule dynamics with cryoEM alone, MD simulations are employed to forecast molecular motions and to interpret cryoEM reconstructions. Few methods, however, can evaluate MD predictions directly. Here, we use multislice wave propagation to project sampled snapshots of MD trajectories, either coarse-grained (CG) or all-atom (AA), into simulated cryoEM 3D reconstructions. We compared simulated and experimental images of low- and high-curvature membranes to show that MD simulations qualitatively reflect the fluidity and thus the contrast of biological membranes observed by cryoEM. MD simulations also correctly predicted bilayer dimensions for single component flat bilayers observed in cryoEM images. However, Martini3 CG-MD simulations failed to predict changes in membrane thickness induced by high curvature and with heterogeneous lipid compositions. We pinpointed the misbehavior of polyunsaturated lipid tails and cholesterol in Martini3 simulations as the main error sources contributing to inaccurate bilayer thicknesses. Our comparisons also explain membrane structure discrepancies between cryoEM and small angle X-ray scattering (SAXS). Further testing of MD predictions by direct comparisons between simulated and experimental cryoEM images should lead to the development of more accurate MD force fields. Statement of SignificanceMolecular dynamics (MD) simulations are frequently employed to predict the dynamics of biological macromolecules and assemblies, but these predictions remain difficult to validate experimentally. Cryo-electron microscopy (cryoEM) can be used to directly image the conformational ensemble of macromolecules, but images of Coulombic potential cannot be easily compared to snapshots of atoms from MD simulations. Here, we show that a physics-based multislice image projection algorithm accurately converts MD trajectories to simulated cryoEM 2D images and 3D reconstructions. Using this approach, we identify consistencies and discrepancies between MD simulations and cryoEM experiments. Notably, coarse-grained MD performs poorly compared to all-atom MD when simulating membranes composed of mixtures of lipids that include cholesterol and polyunsaturated lipids, providing observables for MD force field improvement.

biophysics↗

bioRxiv2025

Patterned 3D-printed hydrogel as a novel soilless substrate for plant cultivation

Plant roots need water, micronutrients, and oxygen to maintain cellular metabolism and tissue growth, yet traditional hydroponic systems often lack sufficient oxygen delivery. While 3D printing artificial substrates has been explored to mimic the physical structure of soil, it remains unclear which design parameters are critical for supporting full plant development. Here, we present a synthetic, soilless substrate based on 3D-printed hydrogels incorporating triply periodic minimal surface (TPMS) patterns to create internal air-filled channels. These channels are open to the atmosphere, enabling passive gas exchange throughout the substrate. We tested five TPMS geometries (Lidinoid, Split-P, Schwarz-D, Schwarz-P, and Schoen), each with a near-identical hydrogel volume but with different surface-to-volume ratios. Arabidopsis thaliana seeds germinated directly on the substrates and were monitored for vegetative and reproductive growth over five weeks. Among the designs, the Lidinoid substrate led to the highest number and surface area of leaves, exceeding the aerated hydroponic control in leaf area over most of the time course; it also produced the earliest and most complete flowering, which neither control achieved in the same amount of time. Our results indicate that the surface-to-volume ratio is a key parameter influencing substrate performance; we hypothesise that this reflects its effect on oxygen availability at the root interface, which we did not measure directly. Plants grown on substrates with higher surface areas transitioned to flowering more reliably and rapidly, with flowering efficiency showing a strong positive correlation with surface area. These findings suggest that interconnected air-channel architectures can support full plant development without active aeration, potentially by overcoming the oxygen limitations of traditional hydroponic systems. This work supports the use of additive manufacturing as a powerful tool for engineering soil analogues tailored for indoor agriculture. By combining passive aeration with hydration and nutrient delivery, patterned hydrogels offer a promising, scalable solution for sustainable soilless plant cultivation.

plant biology↗

bioRxiv2025

Human ribomes reveal DNA-embedded ribonucleotides as a new type of epigenetic mark

Ribonucleoside monophosphates (rNMPs) are abundant in DNA, but their distribution and function in human nuclear genomes remain unknown. Here, we mapped nearly one million rNMPs per genome across diverse human cell types, defining a nuclear "ribome" with non-random distribution patterns. rNMPs are enriched in C/G-rich sequences, epigenetically marked regions, and telomeres. Conserved ribonucleotide-enriched zones (REZs) overlap with CpG islands and R-loops. rNMP concentration near transcription start sites (TSSs) correlates positively with gene expression. Wild-type cells display a broader gene-expression range than ribonuclease H2A (RNH2A) knockouts, in which loss of rNMP cleavage causes pronounced retention of embedded rG and strand-biased rC near TSSs, both increasing with gene expression. These findings establish DNA-embedded rNMPs as a novel epigenetic mark that modulates human gene expression.

molecular biology↗

bioRxiv2025

Identity-by-descent captures Shared Environmental Factors at Biobank Scale

Genealogical ties leave traces beyond shared genetic material: relatives tend to share environmental exposures and cultural practices that persist across generations. Using genomic and electronic health record data from 13,143 individuals in the Biorepository for Integrative Genomics, we applied a hierarchical community detection algorithm to group individuals into four major communities and 17 subcommunities based on the proportion of their genome shared identical by descent (IBD). This approach captured a fine-scale demographic structure beyond traditional ancestry classifications. By integrating neighborhood-level geographic data with census-derived environmental metrics, we revealed unequal exposure to environmental stressors between subcommunities, which correlated with differential rates of health conditions, including elevated respiratory condition prevalence in subcommunities under greater pollution burden. Through an interventional mediation analysis we found that excess risk at the subcommunity-level substantially exceeded what measured environmental factors alone could explain. thus showing the contribution of unmeasured shared exposures that IBD-based grouping captures. Subcommunity-level disparities persisted in sensitivity analyses adjusting for self-reported race, indicating that IBD-based clustering captures health-relevant information beyond demographic categories commonly used as proxies for social determinants of health. We implemented an open-source dashboard for interactive exploration of IBD-defined communities, disease prevalence, and environmental exposures. Together, we demonstrate that IBD-based clustering jointly captures genetic and environmental determinants of health, offering a scalable framework for precision health and population genetics that translates biobank data into actionable insights.

genomics↗

bioRxiv2025

Conjugated GLP-1 and Estradiol Treatment as a Novel Treatment for Age-related Cognitive Decline in Males and Females

Middle age represents a critical window for metabolic and cognitive health, particularly in the context of rising obesity and diabetes rates. Glucagon-like peptide-1 (GLP-1)-based therapies, which regulate blood glucose and body weight, show sex-specific effects, with estradiol potentiating their metabolic benefits. However, research on GLP-1s cognitive and neuroprotective roles has largely been conducted in males. Here, we investigated the effects of GLP-1 conjugated to estradiol (GE2) on metabolism, cognition, cytokine levels and neurogenesis in the dentate gyrus of middle-aged male and female rats fed a standard (SD) or Western (WD) diet. In both sexes, WD increased body weight and plasma leptin levels, regardless of sex. GE2 treatment led to weight loss, enhanced cued and contextual fear memory, reduced cytokine levels in the hippocampus in SD rats, and increased neurogenesis in the dorsal dentate gyrus (DG), regardless of sex. Sex-specific differences were observed in fat distribution, glucose regulation, central cytokine levels, and neuroplasticity after WD and GE2 treatment. In females only, GE2 reduced visceral (gonadal) fat, reduced cytokines in the dorsal hippocampus, and improved basal blood glucose in response to a WD. In males only, GE2 restored neurogenesis in the DG after WD exposure, and reduced cytokine levels in the amygdala. These findings suggest that although WD increased body weight and GE2 improved associative learning in both sexes, both WD and GE2 had differential affects on metabolic hormones, insulin regulation, cytokine levels and neuroplasticity. Our findings underscore the importance of sex-specific approaches in metabolic and neuroprotective therapeutics in middle age.

neuroscience↗

bioRxiv2025

Proteomic characterisation of the early rheumatoid arthritis-cardiovascular disease multimorbid axis.

BackgroundInflammation contributes to the increased risk of cardiovascular disease (CVD) observed in people with rheumatoid arthritis (RA), with increased prevalence observed from time of diagnosis. Subclinical vascular and myocardial abnormalities can be detected with cardiovascular magnetic resonance (CMR) imaging in otherwise low risk individuals. Identifying associated circulating markers could enable diagnostics and implicate biological pathways of RA-CVD. The studys objective was to identify blood-based proteins associated with CMR measures of vascular and myocardial abnormality and associated inflammatory and/or cardio-metabolic pathways in a new-onset RA cohort. MethodsSerum samples (baseline/pre-treatment, N = 75 and year 1, N = 71) from CADERA (Coronary Artery Disease Evaluation in Rheumatoid Arthritis) participants, a subgroup of a randomised controlled trial, who underwent CMR, were used to measure 334 proteins across 4 pre-defined Olink panels (Inflammation, Cardiovascular-II, Cardiovascular-III, Cardiometabolic). Bayesian mixed effects regression analyses, unsupervised hierarchical clustering and protein network analyses were applied. Normalised protein expression from 334 Olink proteins were used as exposures in the regression analyses. CMR measures of vascular stiffness (aortic distensibility and stiffness index) and myocardial tissue characteristics (native T1, myocardial extracellular volume and late gadolinium enhancement) were each used as individual outcomes in regression analyses. Associations were considered significant at 95% threshold for credible intervals. An expanded physical protein-protein interaction (PPI) network created using the significant (seed) proteins was subjected to topological and enrichment analysis to identify enriched biological pathways. Results54/334 proteins were associated significantly with CMR measures at baseline (7 - vascular; 48 - myocardial tissue characteristics; Coagulation factor 11 with both). Two proteins, TNFSF13B (B-cell activating factor) and CRTAC1, were associated with CMR measures at baseline, were sensitive to change over time and co-varied with changes in CMR measure. Topological analysis of expanded PPI network revealed GRB2 as most connected (degree score=64; closeness score=118.83) and four key signalling pathways, including JAK-STAT and EGFR tyrosine kinase inhibitor resistance, emerged as significant. ConclusionsThis first proteomic study of treatment-naive early RA and subclinical cardiovascular pathology identifies proteins that could aid diagnostic test development and implicates signalling pathways in the RA-cardiovascular axis to inform on our understanding of the basis of RA-CVD.

systems biology↗

bioRxiv2025

Physiologically-relevant light exposure and light behaviour in Switzerland and Malaysia

Light synchronises the internal clock with the external light-dark cycle. Keeping this alignment benefits health and prevents diseases. Quantifying light exposure is, therefore, vital for effective prevention. Since light exposure depends on photoperiod, culture, and behaviour, we investigated objective light exposure and individual light-related behaviour in Switzerland and Malaysia. In this observational field study, participants (N=39) wore a calibrated melanopic light logger at chest level for 30 consecutive days. At baseline and study end, the Pittsburgh Sleep Quality Index was assessed, and every 3 to 4 days, the Light Exposure Behaviour Assessment (LEBA) was filled. Our pre-registered analyses reveal that participants in Switzerland experienced brighter days (+3.16 times the average mel EDI) and spent more time (x1.9 times the duration) in daylight levels per hour of daylight, had [~]1.5h later bright light exposure in the afternoon, and stayed over 1h longer in dim light conditions before bedtime. LEBA scores did not differ between Malaysia and Switzerland, and LEBA items were stable over time. LEBA items also correlated with objective light exposure variables in Switzerland but not Malaysia, with a medium effect size (range of absolute r=0.32-0.48). These results highlight cultural and geographical differences in light exposure. We showed that LEBA can be related to actual light exposure and is ecologically informative, but this varies by culture.

neuroscience↗

bioRxiv2024

Persistent changes in nociceptor translatomes govern hyperalgesic priming in mouse models

Hyperalgesic priming is a model system that has been widely used to understand plasticity in painful stimulus-detecting sensory neurons, called nociceptors. A key feature of this model system is that following priming, stimuli that do not normally cause hyperalgesia now readily provoke this state. We hypothesized that hyperalgesic priming occurs due to reorganization of translation of mRNA in nociceptors. To test this hypothesis, we used paclitaxel treatment as the priming stimulus and translating ribosome affinity purification (TRAP) to measure persistent changes in mRNA translation in Nav1.8+ nociceptors. TRAP sequencing revealed 161 genes with persistently altered mRNA translation in the primed state. We identified Gpr88 as upregulated and Metrn as downregulated. We confirmed a functional role for these genes, wherein a GPR88 agonist causes pain only in primed mice and established hyperalgesic priming is reversed by Meteorin. Our work demonstrates that altered nociceptor translatomes are causative in producing hyperalgesic priming.

neuroscience↗

bioRxiv2024

Invasion and spread of the neotropical leafhopper Curtara insularis (Hemiptera: Cicadellidae) in Africa and North America and the role of high-altitude windborne migration in invasive insects

Invasive insects threaten ecosystem stability, public health, and food security. Documenting newly invasive species and understanding how they reach into new territories, establish populations, and interact with other species remain vitally important. Here, we report on the invasion of the South American leafhopper, Curtara insularis into Africa, where it has established populations in Ghana, encroaching inland at least 350 km off the coast. Importantly, 80% of the specimens collected were intercepted between 160 and 190 m above ground. Further, the fraction of this species among all insects collected was also higher at altitude, demonstrating its propensity to engage in high-altitude windborne dispersal. Its aerial densities at altitude translate into millions of migrants/km over a year, representing massive propagule pressure. Given the predominant south-westerly winds, these sightings suggest an introduction of C. insularis into at least one of the Gulf of Guinea ports. To assess the contribution of windborne dispersal to its spread in a new territory, we examine records of C. insularis range-expansion in the USA. Reported first in 2004 from central Florida, it reached north Florida (Panhandle) by 2008-2011 and subsequently spread across the southeastern and south-central US. Its expansion fits a "diffusion-like" process with 200--300 km long "annual displacement steps"--a pattern consistent with autonomous dispersal rather than vehicular transport. Most "steps" are consistent with common wind trajectories from the nearest documented population, assuming 2--8 hours of wind-assisted flight at altitude. Curtara insularis has been intercepted at US ports and on trucks. Thus, it uses multiple dispersal modalities, yet its rapid overland spread is better explained by its massive propagule pressure linked with its high-altitude windborne dispersal. We propose that high-altitude windborne dispersal is common yet under-appreciated in invasive insect species.

ecology↗

bioRxiv2024

A Single-Short Partial Reprogramming of the Endothelial Cells decreases Blood Pressure via attenuation of EndMT in Hypertensive Mice

BackgroundSmall artery remodeling and endothelial dysfunction are hallmarks of hypertension. Growing evidence supports a likely causal association between cardiovascular diseases and the presence of endothelial-to-mesenchymal transition (EndMT), a cellular transdifferentiation process in which endothelial cells (ECs) partially lose their identity and acquire additional mesenchymal phenotypes. EC reprogramming represents an innovative strategy in regenerative medicine to prevent deleterious effects induced by cardiovascular diseases. MethodsUsing a partial reprogramming of ECs, via overexpression of Oct-3/4, Sox-2, and Klf-4 (OSK) transcription factors, we aimed to bring ECs back to a youthful phenotype in hypertensive mice. Primary ECs were infected with lentiviral vectors (LV) containing the specific EC marker cadherin 5 (Cdh5) and the fluorescent reporter enhanced green fluorescence protein (EGFP) with empty vector (LVCO) or with OSK (LV-OSK). Confocal microscopy and western blotting analysis were used to confirm the OSK overexpression. Cellular migration, senescence, and apoptosis were evaluated. Human aortic ECs (HAoECs) from male and female normotensive and hypertensive patients were analyzed after OSK or control treatments for their endothelial nitric oxide synthase (eNOS) levels, nitric oxide (NO), and genetic profile. Male and female normotensive (BPN/3J) and hypertensive (BPH/2J) mice were treated with an intravenous (i.v.) injection of LVCO or LV-OSK and evaluated 10 days post-infection. The blood pressure, cardiac function, vascular reactivity of small arteries, in vivo EGFP signal and EndMT inhibition were analyzed. ResultsOSK overexpression induced partial EC reprogramming in vitro, and these cells showed endothelial progenitor cell (EPC)-like features with lower migratory capability. OSK treatment of hypertensive BPH/2J mice normalized blood pressure and resistance arteries hypercontractility, via the attenuation of EndMT and elastin breaks. EGFP signal was detected in vivo in the prefrontal cortex of both BPN/3J and BPH/2J-treated mice, but OSK induced angiogenesis only in male BPN/3J mice. OSK-treated human ECs from hypertensive patients showed high eNOS activation and NO production, with low ROS formation. Single-cell RNA analysis showed that OSK alleviated EC senescence and EndMT, restoring their phenotypes in human ECs from hypertensive patients. ConclusionOverall, these data indicate that OSK treatment and EC reprogramming can decrease blood pressure and reverse hypertension-induced vascular damage.

physiology↗

bioRxiv2024

HIGH THROUGHPUT QUANTITATION OF HUMAN NEUTROPHIL RECRUITMENT AND FUNCTIONAL RESPONSES IN AN AIR-BLOOD BARRIER ARRAY

Dysregulated neutrophil recruitment drives many pulmonary diseases, but most preclinical screening methods are unsuited to evaluate pulmonary neutrophilia, limiting progress towards therapeutics. Namely, high throughput therapeutic screening systems typically exclude critical neutrophilic pathophysiology, including blood-to-lung recruitment, dysfunctional activation, and resulting impacts on the air-blood barrier. To meet the conflicting demands of physiological complexity and high throughput, we developed an assay of 96-well Leukocyte recruitment in an Air-Blood Barrier Array (L-ABBA-96) that enables in vivo-like neutrophil recruitment compatible with downstream phenotyping by automated flow cytometry. We modeled acute respiratory distress syndrome (ARDS) with neutrophil recruitment to 20 ng/mL epithelial-side interleukin 8 (IL-8) and found a dose dependent reduction in recruitment with physiologic doses of baricitinib, a JAK1/2 inhibitor recently FDA-approved for severe COVID-19 ARDS. Additionally, neutrophil recruitment to patient-derived cystic fibrosis sputum supernatant induced disease-mimetic recruitment and activation of healthy donor neutrophils and upregulated endothelial e-selectin. Compared to 24-well assays, the L-ABBA-96 reduces required patient sample volumes by 25 times per well and quadruples throughput per plate. Compared to microfluidic assays, the L-ABBA-96 recruits two orders of magnitude more neutrophils per well, enabling downstream flow cytometry and other standard biochemical assays. This novel pairing of high-throughput in vitro modeling of organ-level lung function with parallel high-throughput leukocyte phenotyping substantially advances opportunities for pathophysiological studies, personalized medicine, and drug testing applications.

bioengineering↗

bioRxiv2022

Single cell transcriptomics of human prenatal anterior foregut-derived organs identifies distinct developmental signatures directing commitment and specialization of the thymic epithelial stroma

The thymus instructs T cell immunity and central tolerance, yet its therapeutic potential remains untapped. The quest to regenerate thymic function for clinical application is lagging while the signals that drive thymic epithelial cell differentiation remain incompletely understood. Here, we elucidate pathways instructing commitment and specialization of the human thymic epithelial stroma through complementary single cell transcriptomic approaches. First, we identify gene regulatory networks that define fetal thymic epithelium in the thus far unexplored context of other anterior foregut-derived organs; then, we characterize lineage trajectories within the thymic epithelial compartment across embryonic, fetal, and early postnatal stages. Activation of interferon response gene regulatory networks distinguished epithelial cells of the thymus from those of all other anterior foregut-derived organs. Interferon signals were processed differentially within thymic cortical and medullary lineages, reflected in distinct NF{kappa}B and IRF signatures, respectively. Our study reveals novel, translatable insights into the developmental programs underlying thymic epithelial cell differentiation that may advance the field of regenerative cell therapies. SUMMARYSingle cell transcriptomics of anterior foregut-derived organs identifies pathways governing thymic epithelial commitment and specialization.

developmental biology↗

bioRxiv2022

Algorithmic design of 3D wireframe RNA polyhedra

We address the problem of de novo design and synthesis of nucleic acid nanostructures, a challenge that has been considered in the area of DNA nanotechnology since the 1980s and more recently in the area of RNA nanotechnology. Towards this goal, we introduce a general algorithmic design process and software pipeline for rendering 3D wireframe polyhedral nanostructures in single-stranded RNA. To initiate the pipeline, the user creates a model of the desired polyhedron using standard 3D graphic design software. As its output, the pipeline produces an RNA nucleotide sequence whose corresponding RNA primary structure can be transcribed from a DNA template and folded in the laboratory. As case examples, we design and characterize experimentally three 3D RNA nanostructures: a tetrahedron, a bipyramid and a prism. The design software is openly available, and also provides an export of the targeted 3D structure into the oxRNA molecular dynamics simulator for easy simulation and visualization.

bioengineering↗

bioRxiv2022

Linking Dynamic DNA Secondary Structures to Genome Instability

Genomic double-stranded DNA (dsDNA) becomes single-stranded (ssDNA) during replication, transcription, and DNA repair. ssDNA is therefore believed to be transient, occurring in only a fraction of the genome at a given time, and variable amongst a population of cells. These transiently formed ssDNA segments can also adopt alternative, dynamic DNA conformations, such as cruciform DNA, triplexes, quadruplexes and others. To determine whether there are stable and conserved regions of ssDNA, we utilized our previously developed method S1-END-seq 1 to convert ssDNA to DNA double strand breaks (DSBs), which are then processed for high-throughput sequencing. This approach revealed two predominant dynamic DNA structures: cruciform DNA formed by expanded (TA)n repeats that accumulated uniquely in microsatellite unstable human cancer cell lines, and DNA triplexes (H-DNA) formed by homopurine/homopyrimidine (hPu/hPy) mirror repeats common across a variety of human cell lines. Triplex-forming repeats accumulated during replication, blocked DNA synthesis and were hotspots of somatic mutation. In contrast, pathologically expanded (hPu/hPy) repeats in Friedreichs ataxia patient cells formed a replication-independent and transcription-inducible DNA secondary structure. Our results identify dynamic DNA secondary structures in vivo that contribute to elevated genome instability.

molecular biology↗

bioRxiv2021

Pancreatic cancer ductal cell of origin drives CD73-dependent generation of immunosuppressive adenosine

The microenvironment that surrounds pancreatic ductal adenocarcinoma (PDAC) is profoundly desmoplastic and immunosuppressive. Understanding initial triggers of immunosuppression during the process of pancreatic tumorigenesis would aid in establishing novel targets for effective prevention and therapy. Here, we interrogate the differential molecular mechanisms dependent on cell of origin and pathology subtype that determine immunosuppression during PDAC initiation and in established tumors. Transcriptomic analysis of cell of origin dependent-epithelial gene signatures revealed that Nt5e/CD73, a cell surface enzyme that is the pacemaker for extracellular adenosine generation, is one of the top 10% of genes over-expressed in murine tumors arising from ductal pancreatic epithelium as opposed to those rising from acinar cells. These findings were confirmed by Imaging Mass Cytometry and High-Performance Liquid Chromatography. Our data indicate that ductal activation of oncogenic mutant Kras results in loss of PTEN and elevated AKT signaling which ultimately releases CD73 suppression. Delivery of CD73 small molecule inhibitors through various delivery routes reduced tumor development and growth in genetically engineered and syngeneic mouse models. Analysis in human PDAC subtypes indicates that high Nt5e in murine ductal PDAC models overlaps with high NT5E in human PDAC Squamous and Basal Subtypes, considered to have the highest immunosuppression and worst prognosis. These findings highlight a molecular trigger of the immunosuppressive PDAC microenvironment which is dependent on ductal cell of origin, linking biology with pathological subtype classification, critical components to personalized approaches for PDAC prevention and immunotherapeutic intervention. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/470415v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@14625org.highwire.dtl.DTLVardef@1a43372org.highwire.dtl.DTLVardef@12dc0c8org.highwire.dtl.DTLVardef@cbb835_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗