Biology subjects

Perez, M.

Publications and source records attributed to Perez, M..

At least 19 recordsLinked to original sources

bioRxiv2026

EXO1 Facilitates MiDAS and Prevents Genome Instability and Cell Death in Ewing Sarcoma

Ewing sarcoma (EwS) is an aggressive malignancy driven by EWSR1::ETS fusions, predominantly EWSR1::FLI1. Previous efforts using both direct and indirect approaches to target these chimeric oncoproteins have yielded limited clinical benefit. Although EWSR1::FLI1 is a well-known source of replication stress and genome instability, targeting DNA damage response (DDR) factors that mitigate these effects remain poorly understood. Here, we identified a marked dependency of EwS cells on exonuclease 1 (EXO1). We demonstrate that EXO1 is essential for EwS cell survival and tumor growth, highlighting its potential as a novel therapeutic target. Intriguingly, we unveil that EXO1 loss impairs mitotic DNA synthesis (MiDAS), promoting EWSR1::FLI1-associated genome instability and cell death. Collectively, our results support the idea that targeting DDR factors, which counteract replication stress and/or DNA damage induced by fusion oncoproteins, represents a promising therapeutic option for EwS.

cancer biology↗

bioRxiv2026

Muscle-specific motor unit firing characteristics in elbow flexors and extensors after cervical spinal cord injury

Individuals with cervical spinal cord injury (SCI) often exhibit asymmetric recovery of upper-limb function, with greater weakness in elbow extensors than flexors. To determine whether muscle-specific changes in motor unit (MU) behavior contribute to this disparity, we identified MU firing instants from high-density surface electromyography to characterize MU firing characteristics in the biceps brachii (BIC) and triceps brachii (TRI) of individuals with cervical SCI (n = 20) and non-injured controls (n = 18). We quantified rate-coding behavior and metrics related to persistent inward currents (PICs), including onset-offset hysteresis ({Delta}F), ascending firing rate nonlinearity, and self-sustained firing. At the group level, BIC MUs in SCI participants showed reduced rate coding and altered ascending firing rate nonlinearity relative to controls. In contrast, TRI MUs showed no clear group-level differences. However, subgroup analysis revealed that SCI participants with low-strength during extension (n = 9) exhibited lower {Delta}F and longer self-sustained firing durations in TRI MUs than those with high-strength (n = 6). In BIC, SCI participants with low-strength during flexion (n = 8) showed reduced rate-coding behavior relative to high-strength SCI participants (n = 9), with no differences in PIC-related metrics. Together, these results demonstrate muscle-specific alterations in MU firing after cervical SCI that may relate to strength recovery or preservation and underscore the need for nuanced analyses in heterogeneous SCI populations. Key pointsO_LIRate coding and nonlinear firing behaviors are significantly altered in the biceps brachii, but not triceps brachii, of participants with cervical spinal cord injury. C_LIO_LIStrength based subgroup analyses revealed muscle-specific differences in motor unit behaviors that may be associated with strength preservation or recovery following spinal cord injury. C_LIO_LIFunctional heterogeneity following spinal cord injury may mask group differences in motor unit behaviors and warrants careful interpretation of results of future studies. C_LI

neuroscience↗

bioRxiv2025

Umbilical Cord Blood Cell Transcriptional and Methylation Signatures at Birth Are Associated with BPD Development.

Bronchopulmonary dysplasia (BPD) is the most common respiratory disease in preterm infants born at less than 28 weeks gestation. Most existing clinical prediction models for BPD show limited accuracy in predicting BPD development when validated using external data, stressing the need for novel biomarkers to identify at-risk infants for early and effective interventions. We leveraged existing frozen umbilical cord blood samples from the Northwestern University Cord Blood Biobank (NUCord) to perform parallel transcriptional and DNA methylation profiling. BPD-associated differentially expressed genes (DEGs) in our cohort included markers previously established in clinical and animal BPD studies, such as genes related to NF-{kappa}B signaling and immune responses. We also identified that BPD development is associated with disrupted methylation signatures in microRNA genes and genes associated with glucose metabolism. Our results suggest that BPD development is associated with distinct transcriptomic and epigenetic signatures when compared with healthy term and preterm infants. These signatures may represent biomarkers measurable at birth that predict BPD development during a time window when preventative or therapeutic interventions could be applied.

genomics↗

bioRxiv2025

Domain-Invariant Feature Learning for Patient-Level Phenotype Prediction from Single-Cell Data

Accurate prediction of patient-level disease status from single-cell RNA sequencing (scRNA-seq) data is critical to enabling precision diagnostics. However, study-specific artifacts induce spurious correlations that limit generalization and interpretability. We studied this problem in the context of Multiple Instance Learning (MIL), a framework where each patient is modeled as a set of single-cell profiles. To improve robustness to domain shifts, we propose an adversarial and metric-based approach that learns domain-invariant representations while preserving task-relevant biological variation. We benchmarked our method on a systemic lupus erythematosus (SLE) dataset with synthetically added spurious features and evaluated its performance on two real-world scRNA-seq atlases: a cross-tissue immune dataset and a COVID-19 severity atlas. Across all settings, we observed consistent improvements in out-of-domain accuracy and more biologically faithful model attributions. Our findings establish a new standard for robust, interpretable patient-level prediction under domain shifts using scRNA-seq.

bioinformatics↗

bioRxiv2025

Gene model for the ortholog of Ilp5 in Drosophila erecta

Gene model for the ortholog of Insulin-like peptide 5 (Ilp5) in the Dere_CAF1 Genome Assembly (GenBank Accession: GCA_000005135.1) of Drosophila erecta. This ortholog was characterized as part of a developing dataset to study the evolution of the insulin/insulin-like growth factor signaling pathway across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

genomics↗

bioRxiv2025

Control of fall armyworm with Spodoptera frugiperda multiple nucleopolyhedrovirus in sweet corn cultivated under agroecological conditions

The fall armyworm (FAW), Spodoptera frugiperda (JE Smith) is a polyphagous pest documented on more than 80 plant species. In vegetable production systems, sweet corn is their preferred host, but also infests other plants such as chilli, tomato, pumpkins, cucumber, beans and eggplant. Control strategies are commonly carried out by the use of chemical insecticides even though resistance of S. frugiperda populations to several insecticides has been reported. Last decades, have registered rapid adoption of organic and agroecological practices in horticulture, particularly those that rely on bioinputs such as biopesticides and biofertilizers. The baculovirus Spodoptera frugiperda Multiple Nucleopolyhedrovirus (SfMNPV) is a pathogenic agent for the fall armyworm and an alternative tool used for its control in sustainable pest management strategies. In this work, we evaluate two indigenous SfMNPV-based liquid formulations sprayed on sweet corn cultivated under agroecological conditions. Larvae fed with leaves treated with SfMNPV OBs showed mortality rates ranging from 55 to 68 %. Offspring from adult surviving to OBs treatments was analyzed by PCR and the results confirmed sublethal infections and vertical transmission in the fall armyworm. Corn ears harvested from each treatment were individually observed for insect presence, where the lowest rate of fall armyworm per corn ear was registered in plants treated with SfMNPV OBs. Analysis of secondary plagues in corn ears identified the sap beetle Carpophilus dimidiatus and the maize-infesting fly larvae Euxesta mazorca as the most abundant insect pests. These results show that indigenous isolates of SfMNPV produce a significant control on FAW under agroecological conditions, suggesting that the use of formulations including this virus might be a valuable tool for pest management in sustainable vegetable production systems. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/666974v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@12e8d59org.highwire.dtl.DTLVardef@1420401org.highwire.dtl.DTLVardef@11516b7org.highwire.dtl.DTLVardef@186750b_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

On the use of generative models for evolutionary inference of malaria vectors from genomic data

Malaria in sub-Saharan Africa is transmitted by mosquitoes, in particular the Anopheles gambiae complex. Efforts to control the spread of malaria have often focused on these vectors, but less is known about the demographic history of populations and species in the Anopheles complex. Here, we first quantify the genetic structure of mosquito populations in sub-Saharan Africa using unsupervised machine learning. We then adapt and apply an innovative generative deep learning algorithm to infer the joint evolutionary history of populations sampled in Guinea and Burkina Faso, West Africa. We further develop a novel model selection approach and discover that an evolutionary model with migration fits this pair of populations better than a model without post-split migration. For the migration model, we find that our method accurately captures population genetic differentiation. These findings demonstrate that machine learning and generative models are a valuable direction for future understanding of the evolution of malaria vectors, including the joint inference of demography and natural selection. Understanding changes in population size, migration patterns, and adaptation in hosts, vectors, and pathogens will assist malaria control interventions, with the ultimate goal of predicting nuanced outcomes from insecticide resistance to population collapse.

genomics↗

bioRxiv2025

Lipid nanoemulsion incorporating DOTAP reverse micelles as clinically translatable carriers of ALDH inhibitors for lung cancer therapy

Lung cancer remains the most prevalent malignancy worldwide and the leading cause of cancer-related deaths. In this study, we designed and optimized a lipid nanosystem incorporating the cationic lipid DOTAP by utilizing reverse micelle structures to enhance pulmonary tropism. Fluorescence quenching assays confirmed the incorporation of reverse micelles into the oily core, thus validating the structural integrity of the system. This nanosystem was tailored for the encapsulation of ABD0171, a potent inhibitor of ALDH1A3, an enzyme strongly associated with chemoresistance in lung cancer. Physicochemical characterization revealed robust colloidal properties with a particle diameter of 60 nm, a surface charge of +45 mV, and a drug encapsulation yield of 99%. In vitro, formulations with/without DOTAP demonstrated high efficacy against epithelial-like H358 cells derived from human bronchioalveolar carcinoma, with IC50 values < 5 {micro}M. Chicken ChorioAllantoic Membrane (CAM) evaluations demonstrated good tolerability of the DOTAP-containing formulation and significant H358 tumor reduction by 28% compared with the untreated control, while maintaining a favorable safety profile. By combining industrially feasible methods with FDA-approved components, this study demonstrated the potential of DOTAP-containing lipid nanosystems as scalable platforms for the delivery of ALDH inhibitors for lung cancer treatment. These findings provide a pathway for future applications in cancer therapy, bridging the gap between nanosystem innovation and clinical translation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/659868v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@dbc8org.highwire.dtl.DTLVardef@5d5187org.highwire.dtl.DTLVardef@9391f3org.highwire.dtl.DTLVardef@52fd54_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

bioengineering↗

bioRxiv2025

Hierarchical small molecule inhibition of MYST acetyltransferases

MYST lysine acetyltransferases (KATs) is a class of epigenetic enzymes critical for cellular function that constitute an emerging therapeutic target in cancer. Recently, several drug-like MYST inhibitors have been reported that show promise in a variety of preclinical models as well as in clinical trials of breast cancer. However, the comparative properties of these small molecules remains to be directly assessed. Here we apply an integrated profiling strategy to systematically define the potency and selectivity of drug-like MYST KAT inhibitors. First, we use optimized chemoproteomic profiling and histone acetylation biormarkers to study the industry-developed KAT inhibitor PF-9363. This reveals dose-dependent engagement of native KAT complexes, with hierarchical inhibition following the order KAT6A/B > KAT7 >> KAT8 > KAT5. Next, we demonstrate how PF-9363s ability to disrupt capture of MYST complex members in chemoproteomic experiments can be leveraged to identify new candidate members of these complexes, including the transcription factor FOXK2. Applying insights from these studies to WM-8014, WM-1119 and WM-3835, which have been extensively applied in the literature as MYST probes, highlights unexpected cross-inhibition and suggests a new framework for how these small molecules and biomarkers may be applied to differentiate KAT6A/B and KAT7-dependent phenotypes. Finally, we benchmark the activity of PF-9363 in the NCI-60 cell line screen, providing evidence that it can inhibit the growth of cell lines that are resistant to other epigenetic inhibitors by engaging the essential MYST enzyme KAT8 at high concentrations. Collectively, our studies indicate the potential for MYST KAT inhibitors to exhibit dose-dependent target engagement reminiscent of kinase inhibitors and specify assays and biomarkers for facile monitoring of selective and hierarchical effects.

biochemistry↗

bioRxiv2025

Patient-derived liver biopsy organoids enable precision alcohol-associated liver disease modeling

Background & AimsAlcohol-associated liver disease (ALD) is a major cause of liver disease worldwide with scarce therapeutic options. Animal models poorly recapitulate advanced ALD precluding the development of new treatments. Organoids have emerged as a powerful human-based preclinical tool. However, current patient-derived liver organoids fail to recapitulate the epithelial heterogeneity and its generation requires liver surgical resections, thus limiting personalized disease modeling. Here, we report the development of organoids from liver needle biopsies (b-Orgs) from patients with ALD. Methodsb-Orgs were generated from tru-cut biopsies from patients at early (n=28) and advanced (n=34) stages of ALD. b-Orgs were characterized by immunofluorescence, bulk and single cell RNA-sequencing and compared to parental tissues. b-Orgs were used to model ALD progression, identify pathogenic drivers, induce alcohol-associated hepatitis (AH) and evaluate response to prednisolone. ResultsPhenotypic and functional analysis of b-Orgs showed hepatocyte- enriched features. Single-cell RNA-sequencing revealed a heterogeneous cell composition comprising hepatocyte, biliary and progenitor populations, mirroring the epithelial landscape found in patients with advanced ALD. Moreover, b-Orgs preserved disease-stage features and allowed to identify the association of ELF3 with cell plasticity and disease progression. Finally, stimulation of b-Orgs with drivers of ALD induced pathophysiological features of alcohol-associated hepatitis, including ROS production, lipid accumulation, inflammation and decreased cell proliferation, which were mitigated in response to prednisolone. Conclusions Overall, we provide a human-based model that recapitulates epithelial complexity and patient specific features, allowing to identify drivers of cell plasticity and expanding organoid-based liver disease modeling for personalized medicine. Impact and implications Here, we describe the generation of biopsy-derived organoids (b-Orgs) from patients with liver disease. b-Orgs reproduce the liver epithelial cell composition found in patients liver tissue and are efficiently generated from different stages of the disease, providing a platform for patient- tailored disease modeling and drug testing.

cell biology↗

bioRxiv2025

Soleus H-reflex size versus stimulation rate in the presence of background muscle activity: A methodological study

IntroductionHoffmann reflex (HR) operant conditioning has emerged as an important intervention in neurorehabilitation. During conditioning, the HR is elicited at low rates ([~]0.2 Hz) to avoid the initial reduction in HR size that can occur over repeated stimulation, i.e., rate-dependent depression (RDD), thereby maintaining reflex size. This study investigated the impact of higher stimulation rates on HR size, where a stable, low-level, background electromyographic (EMG) signal is maintained over 225 conditioning trials in each of 30 sessions. A higher rate could shorten session length and/or number. MethodsFifteen healthy participants maintained low background soleus EMG (5-18 {micro}V, [~]1-3% of the maximum stimulation evoked direct muscle (M-wave) EMG response (Mmax) while standing. Soleus HR and M-wave recruitment curves were obtained at rates of 0.2, 1, and 2 Hz, from which Mmax and Hmax were calculated. Seventy-five HR trials (HRT) were collected for each stimulation rate at a target M-wave size ([~]10-20% of Mmax). ResultsThere was no evidence of RDD at higher stimulation rates. In addition, the mean HR over trials was reliable across participants and rates. The Intraclass Correlation Coefficient (ICC) was 0.965 (95%CI:0.915, 0.987). DiscussionThis study shows that H-reflex conditioning might be performed at rates up to 2 Hz with no RDD and with consistent HR values. A faster rate could increase the number of conditioning trials per session, reduce session duration, and/or reduce the number of sessions. It could thereby accelerate the conditioning process and make the process less demanding for participants. SupportNIH Grant P41 EB018783 (Wolpaw), NYS Spinal Cord Injury Research Board C37714GG (Gupta) and C38338GG (Wolpaw), VA SPiRE NCT05880251(Brangaccio), Stratton Veterans Affairs Medical Center.

neuroscience↗

bioRxiv2025

Harnessing ALDH1A2 vulnerability in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with limited therapeutic options, particularly in the relapsed/refractory (R/R) setting. Unlike other hematological malignancies, which benefit from targeted immunotherapies, T-ALL remains reliant on chemotherapy, leading to poor outcomes in R/R cases. Identifying novel therapeutic vulnerabilities is crucial to improving the outcomes of patients. Herein, we identify aldehyde dehydrogenase 1A2 (ALDH1A2) as a T-ALL-specific enzyme essential for leukemic cell survival. Transcriptomic and epigenetic analyses reveal its selective expression, regulated by the TAL1 oncogene. Pharmacological inhibition of ALDH1A2 using Dimate demonstrates potent anti-leukemic activity across diverse T-ALL subtypes, including primary samples of relapsed disease. These findings establish ALDH1A2 as a therapeutic target in T-ALL and support ALDH inhibition as a promising strategy to overcome drug resistance and improve treatment outcomes in R/R T-ALL.

cancer biology↗

bioRxiv2025

Intraline genomic heterogeneity of the triple-negative breast cancer MDA-MB-231 cell line

Cancer cell lines are valuable models for studying tumor biology, yet their genomic evolution during culture can compromise experimental reproducibility. We conducted a detailed genomic analysis of the triple-negative breast cancer cell line MDA-MB-231, examining sublines obtained from different sources, at various time points, and across distinct passages. We introduce the concept of intraline heterogeneity (ILH) to highlight the genomic variability observed among these sublines. Our analyses revealed extensive genomic diversity, including differences in single nucleotide variants (SNVs) and copy number alterations (CNAs). In particular, CNAs exhibited remarkable heterogeneity, with pronounced chromosomal gains and losses between sublines, underscoring the impact of genomic instability on ILH. These findings suggest that ILH may influence experimental outcomes, emphasizing the importance of considering passage-specific genomic characterization to ensure consistency and reliability in cancer research.

genomics↗

bioRxiv2025

c-di-GMP is required for swarming motility in E. coli

Many bacteria use flagella to swim individually through bulk liquid, or swarm collectively over a semi-solid surface. In E. coli, c-di-GMP inhibits swimming via the effector protein YcgR. We show in this study that contrary to its effect on swimming, a certain threshold level of c-di-GMP is required for swarming. Gene expression profiles first indicated that several c-di-GMP synthases - dgcJ, dgcM, dgcO - were upregulated during swarming. Of these, we found DgcO to play a critical role. DgcO is reported to affect PGA (poly-{beta}-1,6-N-acetylglucosamine) synthesis in E. coli. We show that DgcO also promotes production of colanic acid - one of the three major exopolysaccharides in E. coli, and that colanic acid has hitherto-unknown surfactant properties that are expected to aid swarming. ImportanceIt is well-established that in bacteria, c-di-GMP inhibits flagella-driven motility at various points in the pathway. Concomitantly, elevated c-di-GMP levels induce the expression and synthesis of a variety of exopolysaccharides that enmesh the bacteria in a biofilm, thereby also interfering with flagella function. This study reports the surprising finding that in E. coli, the exopolysaccharide colanic acid is required to enable surface navigation and that the diguanylate cyclase DgcO is employed for this purpose. For surface navigation, there appears to be a sweet spot where c-di-GMP levels are just right to produce polysaccharides that can serve as surfactants and wetting agents rather than promote formation of biofilms.

microbiology↗

bioRxiv2024

The role of innate immune responses against two strains of PEDV (S INDEL and non-S INDEL) in newborn and weaned piglets inoculated by combined orogastric and intranasal routes

Porcine epidemic diarrhea (PED) is a severe gastrointestinal disease in swine caused by PED virus (PEDV), leading to significant economic losses worldwide. Newborn piglets are especially vulnerable, with nearly 100% mortality, unlike older pigs. Disease severity also varies depending on the PEDV strain, with non-S INDEL strains being more virulent than S INDEL ones. This study examined early pathogenesis and innate immunity in 1-week-old suckling and 5- week-old weaned piglets (n=8 per age group, 4 per strain) inoculated with S INDEL or non-S INDEL PEDV strains via combined orogastric and intranasal route. Age-matched negative controls (n=3 per age group) were included. Body weight, temperature, and clinical signs were monitored for 48 hours post-inoculation (hpi). PEDV RNA levels were assessed in rectal swabs (RS) at 0 and 48 hpi, while pathological analyses and viral RNA loads were measured in jejunal content and intestinal mucosa. Gene expression of 75 selected antiviral and inflammatory genes were measured in laser capture microdissection (LCM)-derived jejunal samples using microfluidic qPCR at 48 hpi. Suckling piglets showed severe clinical signs, while weaned piglets were mostly asymptomatic at 48 hpi. In general, clinical signs and lesions in suckling piglets were similar, regardless of the PEDV strain. Both viral strains produced comparable viral RNA loads in the small intestine and feces, as well as consistent villous atrophy and fusion across age groups. In LCM-derived jejunal samples, weaned piglets had higher expression of antiviral genes (type I/III interferons, ISGs) and Th1/Th17 pro-inflammatory genes, particularly with the non-S INDEL strain. Conversely, the anti-inflammatory cytokine IL-10 was overexpressed in suckling compared to weaned piglets for both strains. Overall, PEDV-induced intestinal damage, viral replication, and excretion were similar regardless of viral strain or piglet age. The reduced clinical severity in weaned piglets may result from their stronger intestinal antiviral and pro-inflammatory response. AUTHOR SUMMARYPorcine epidemic diarrhea virus (PEDV) is the causative agent of a major gastrointestinal disease in piglets worldwide, characterized by severe watery diarrhea. The disease is particularly devastating in newborn piglets, especially when caused by non-S INDEL PEDV strains, while weaned piglets demonstrate resistance regardless of the strain. In this study, during the acute infection phase (48 hpi), both highly virulent non-S INDEL and less virulent S INDEL strains caused comparable intestinal atrophy, viral replication in the intestine, and viral loads in feces in both weaned and suckling piglets. However, weaned piglets mounted a robust antiviral response involving type I and III interferons (IFNs) and the induction of Th1- and Th17-related pro-inflammatory responses in the intestinal mucosa. Additionally, interferon-stimulated genes (ISGs) were broadly upregulated across the intestinal mucosa of weaned piglets in response to both PEDV strains. In contrast, suckling piglets exhibited a weaker innate immune response, coinciding with more severe clinical signs. The observed inverse relationship between disease severity and intestinal innate immune activation highlights the potential role of IFNs, ISGs, and pro-inflammatory cytokines in mitigating PEDV severity and underscores their relevance in developing novel pharmacological prevention strategies.

immunology↗

bioRxiv2024

Engineering a Biosynthetic Pathway for the Production of (+)-Brevianamides A and B in Escherichia coli

The privileged fused-ring system comprising the bicyclo[2.2.2]diazaoctane (BDO) core is prevalent in diketopiperazine (DKP) natural products with potent and diverse biological activities, with some being explored as drug candidates. Typically, only low yields of these compounds can be extracted from native fungal producing strains and the available synthetic routes remain challenging due to their structural complexity. BDO-containing DKPs including (+)-brevianamides A and B are assembled via multi-component biosynthetic pathways incorporating non-ribosomal peptide synthetases, prenyltransferases, flavin monooxygenases, cytochrome P450s and semi-pinacolases. To simplify access to this class of alkaloids, we designed an engineered biosynthetic pathway in Escherichia coli, composed of six enzymes sourced from different kingdoms of life. The pathway includes a cyclodipeptide synthase (NascA), a cyclodipeptide oxidase (DmtD2/DmtE2), a prenyltransferase (NotF), a flavin-dependent monooxygenase (BvnB), and kinases (PhoN and IPK). Cultivated in glycerol supplemented with prenol, the engineered E. coli strain produces 5.3 mg/L of (-)-dehydrobrevianamide E (4), which undergoes a terminal, ex vivo lithium hydroxide catalyzed rearrangement reaction to yield (+)-brevianamides A and B with a 46% yield and a 92:8 diastereomeric ratio. Additionally, titers of 4 were increased eight-fold by enhancing NADPH pools in the engineered E. coli strain. Our study combines synthetic biology, biocatalysis and synthetic chemistry approaches to provide a five-step engineered biosynthetic pathway for producing complex indole alkaloids in E. coli. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/627567v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@399e92org.highwire.dtl.DTLVardef@d98faaorg.highwire.dtl.DTLVardef@1671ac1org.highwire.dtl.DTLVardef@d7bc6_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Direct high-throughput deconvolution of unnatural bases via nanopore sequencing and bootstrapped learning

The discovery of non-canonical bases (NCBs) in viruses and the development of synthetic xeno-nucleic acids (XNAs) to expand the genetic alphabet has spawned interest in many applications, from viral genomics, to synthetic biology and DNA storage. However, the inability to read non-canonical bases in a direct, high-throughput manner has been a significant limitation to its study and applicability. Here we demonstrate that XNA templates containing non-canonical bases can be directly and robustly sequenced (>2.3 million reads/flowcell, similar to DNA controls) on a MinION sequencer from Oxford Nanopore Technologies to obtain signal data that is significantly distinct from DNA controls (median fold-change >6x). To enable training of machine learning models that deconvolve these signals and basecall non-canonical and canonical bases, we developed a framework to synthesize a complex pool of 1,024 NCB-containing oligonucleotides with diverse 6-mer sequence contexts and high purity (>90% NCB-insertion on average). Bootstrapped models to assist in data preparation, and data augmentation with spliced reads to provide high context diversity, enabled learning of a generalizable model to call canonical as well as non-canonical bases with high accuracy (>80%) and specificity (99%). These results highlight the versatility of nanopore sequencing as a platform for interrogating nucleic acids for viral genomic and xenobiology applications, and the potential to transform the study of genetic material beyond those that use canonical bases.

synthetic biology↗

bioRxiv2024

Porcine Nasal Organoids as a model to study the interactions between the swine nasal microbiota and the host

Interactions between the nasal epithelium, commensal nasal microbiota, and respiratory pathogens play a key role in respiratory infections. Currently there is a lack of experimental models to study such interactions under defined in vitro conditions. Here, we developed a Porcine Nasal Organoid (PNO) system from nasal tissue of pigs as well as from cytological brushes. PNOs exhibited similar structure and cell types than the nasal mucosa, as evaluated by immunostaining. PNOs were inoculated with porcine commensal strains of Moraxella pluranimalium, Rothia nasimurium and the pathobiont Glaesserella parasuis for examining host-commensal-pathogen interactions. All strains adhered to the PNOs, although at different levels. M. pluranimalium and G. parasuis strains stimulated the production of proinflammatory cytokines, whereas R. nasimurium induced the production of IFN{gamma} and diminished the proinflammatory effect of the other strains. Overall, PNOs mimic the in vivo nasal mucosa and can be useful to perform host-microbe interaction studies. In briefInteractions between nasal epithelium, commensal nasal microbiota, and respiratory pathogens play a key role in respiratory infections. We developed porcine nasal organoids to mimic the nasal mucosa and use as a model to study those interactions. Additionally, this development supports the reduction of the number of animals for animal experimentation. HighlightsO_LIFirst generation of Porcine Nasal Organoids (PNOs) from nasal turbinates and swabs C_LIO_LIPNOs recapitulate features of in vivo tissue and maintain self-renewal capacity C_LIO_LIHost-microbe interactions can be studied using the PNO system C_LIO_LIRothia nasimurium inhibits the inflammation induced by other bacteria C_LI

microbiology↗