Biology subjects

Flores, D.

Publications and source records attributed to Flores, D..

11 recordsLinked to original sources

bioRxiv2026

High-Throughput Morphological Profiling of Patient-Derived Human Liver Organoids for Hepatotoxicity Hazard Identification and Compound Prioritization

Drug-induced liver injury (DILI) remains a major challenge in drug development because existing in vitro models poorly predict clinical hepatotoxicity. We coupled patient-derived human liver organoids (HLOs) with high-content imaging and automated machine learning (ML) to detect hepatotoxicity-associated phenotypes by morphological profiling. HLOs from six DILI patients and one healthy donor (n = 7) retained donor-specific baseline phenotypes in hepatocyte maturity, metabolism, and morphology. ML models trained per line on MAD-robustized, well-level-aggregated median features achieved compound-level AUROCs of 0.88 (95% CI 0.86-0.90) to 0.91 (95% CI 0.89-0.93) and balanced accuracies of 0.81-0.84 in distinguishing 54 hepatotoxicants from 20 non-hepatotoxic controls. Averaging predictions across all seven lines at an optimized threshold gave 94.4% sensitivity (95% CI 84.6-98.8%) and 85.0% specificity (95% CI 62.1-96.8%), substantially outperforming HLO cytotoxicity assays and cancer cell line profiling. Lines were moderately-to-highly concordant (Pearson r = 0.66-0.85), and benchmark concentration modeling revealed reproducible inter-line differences in phenotypic sensitivity (median 4.0-fold, several compounds exceeding 20-fold). Exposure anchoring of benchmark concentrations to human-equivalent plasma concentrations, using in vitro mass-balance dosimetry and curated plasma free fractions, placed organoid points of departure within 100-fold of clinical Cmax for 60 of 66 compounds (23-fold residual offset, 95% CI 12-45), and placed two of the three misclassified negative controls an order of magnitude above clinical Cmax. Sulindac injury signatures were significantly enhanced by TNF co-treatment, demonstrating capacity to detect pro-inflammatory-mediated DILI. This platform preserves inter-line variability while enabling reproducible, scalable multi-line consensus hazard classification; because compounds were tested over a common nominal concentration range, the readout defines hepatocellular hazard, which must be combined with exposure as an independent input to estimate clinical DILI risk.

pharmacology and toxicology↗

bioRxiv2026

Structural and Functional Plasticity of the Staphylococcus aureus Virulence-Associated Amyloid Peptide PSMα1

Phenol-soluble modulin 1 (PSM1) is a cytolytic peptide secreted by Staphylococcus aureus that contributes to host-cell damage and biofilm stability, yet the relationship between its assembly behavior and function remains incompletely understood. Here, we combine cellular assays, molecular spectroscopy, and high-resolution structural approaches to elucidate how environmental conditions govern PSM1 activity and supramolecular organization. Live-cell imaging and cytotoxicity assays show that PSM1 accumulates at the plasma membrane of human cells prior to membrane permeabilization, linking membrane association to cytotoxic outcomes. This process is strongly attenuated by epigallocatechin gallate (EGCG). Cryogenic electron microscopy (cryo-EM) reveals two polymorphic canonical amyloid fibril architectures that share a conserved hydrophobic core and protofilament interface. In parallel, we identify pH as a key determinant of PSM1 assembly pathways, driving a bifurcation between cross-{beta} amyloid fibrils at extreme acidic and alkaline conditions and heterogeneous, long-lived, thermally stable -helical nanotubular assemblies at acidic, near-neutral, and slightly alkaline conditions, which act as transient intermediates under highly acidic conditions. Together, these findings demonstrate that PSM1 is not a single amyloid structure but a condition-dependent structural system in which environmental cues dictate assembly, membrane interaction, and cytotoxic function. This work provides a framework for understanding how polymorphic assembly of bacterial virulence peptides interfaces with host-cell interactions and suggests new avenues for targeting PSM-mediated pathogenicity. Statement of significanceStaphylococcus aureus causes severe infections and uses the peptide PSM1 to damage host cells and strengthen protective biofilms. Like many disease-associated proteins, PSM1 self-assembles into amyloid fibrils, though their role in virulence remains unclear. We show that PSM1 does not adopt a single architecture. Instead, environmental changes, such as those at infection sites, drive the peptide into distinct assemblies, including cross-{beta} amyloid fibrils and unexpectedly stable nanotubes with -helical features. Live-cell imaging shows PSM1 accumulates at the plasma membrane before cell death, and that epigallocatechin gallate reduces membrane association and toxicity. These findings show that bacterial virulence can be regulated through environmentally controlled transitions between protein assemblies, identifying membrane accumulation as a promising anti-virulence target.

biophysics↗

bioRxiv2025

Pharmacokinetics and In vivo Efficacy of Epetraborole against Burkholderia pseudomallei

Melioidosis, caused by Burkholderia pseudomallei, remains a major therapeutic challenge due to high relapse rates and intrinsic antibiotic resistance. Epetraborole (EBO), a leucyl-tRNA synthetase inhibitor, represents a novel therapeutic approach with a distinct mechanism of action compared to standard-of-care antibiotics. Preclinical studies included MIC determination, pharmacokinetic (PK) profiling, dose range and fractionation studies, and efficacy assessments in a 24-hour post-bacterial challenge model of a murine B. pseudomallei lung infection. EBO demonstrated a clear dose-dependent reduction in lung bacterial burden. Doses [≥]200 mg/kg (achieving AUC-2 [~]110 {micro}g{middle dot}h/mL) produced >1.6 log CFU decreases from the start-of-therapy baseline across all ten B. pseudomallei strains. Notably, an AUC-2 of [~]110 {micro}g{middle dot}h/mL was achieved in humans with a 2000 mg IV dose in a phase 1 clincial trial where doses up to 4000 mg per day for 14 days were well tolerated with no serious adverse events or dose-limiting adverse events. When EBO doses of 600, 300 and 100 mg/kg SC were fractionated by once, twice and three times a day against the B. pseudomallei strain NCTC7383, which represents the MIC100 strain, the efficacy indicated that the pharmacokinetics-pharmacodynamics (PK/PD) driver of epetraborole is total drug exposure (AUC) rather than peak concentration (Cmax) or time above MIC. The inhibition of leucyl-tRNA synthetase represents a unique molecular target, reducing cross-resistance potential with existing {beta}-lactam antibiotics and enabling combination therapy strategies. These findings substantiate EBO as a promising therapeutic option for clinical melioidosis to improve treatment outcomes. Notably, this study represents the first demonstration of in vivo efficacy against a panel of ten genetically and geographically diverse B. pseudomallei strains in a murine model. This unprecedented breadth of strain coverage provides strong evidence of EBOs robust and strain-independent therapeutic potential. Author SummaryMelioidosis is a life-threatening disease caused by Burkholderia pseudomallei, a bacterium found in soil and water in tropical regions. It is challenging to treat because it is often resistant to standard antibiotics, requires long courses of therapy, and can relapse even after treatment. New treatment options are urgently needed. Epetraborole is an experimental antibiotic with a novel mechanism that blocks bacterial protein synthesis. In this study, we tested how well epetraborole works in mice infected with B. pseudomallei, using ten different strains of the bacteria collected from geographically diverse regions. This is the first study to test a melioidosis treatment against such a broad panel of B. pseudomallei strains in an animal model. We found that epetraborole significantly reduced bacterial counts in the lungs of infected mice. The antibiotic remained effective even against strains with higher resistance and high bacterial loads 24 hours after infection, showing promise for severe clinical infections. Our findings suggest that epetraborole could be a valuable addition to existing melioidosis treatments, especially in regions with limited therapeutic options.

microbiology↗

bioRxiv2024

A parasite-inclusive food web for the California rocky intertidal zone

We present a highly resolved, species-rich food web, including parasitic interactions, for the California rocky intertidal zone. The food web is comprised of 1809 nodes, representing 1845 taxa, and 13,222 links representing trophic interactions between nodes. While only 670 links represent parasitic interactions, we have assembled possibly the most speciose parasite-inclusive food web ever published. The inclusion of all nodes and links are justified using multiple lines of evidence which are built into the dataset. In addition, metadata, including trophic strategy, taxonomic information, habitat, and other ecological attributes allow the data user to filter the food web to their specifications. The food web is a powerful and flexible tool for researchers with questions about large network properties, ecological dynamics of rocky shores, and the role of parasites in ecosystems. Our food web can be used to predict how complex ecosystems like the California rocky intertidal will respond to anthropogenic change and management strategies.

ecology↗

bioRxiv2023

Imaging the dynamics of uterine contractions in early pregnancy

The myometrium or smooth muscle of the uterus contracts throughout the life of the organ. Uterine muscle contractility is essential for reproductive processes including sperm and embryo transport, and during the uterine cycle to remove menstrual effluent or estrus debris. Even still, uterine contractions have primarily only been studied in the context of preterm labor. This is partly due to a lack of methods for studying the contractile characteristics of the uterine muscle in the intact organ. Here, we describe an imaging-based method to evaluate the contractility of both the longitudinal and circular muscles of the uterus in the cycling stages and in early pregnancy. By transforming the image-based data into 3D spatiotemporal contractility maps, we calculate waveform characteristics of muscle contractions, including amplitude, frequency, wavelength, and velocity. We report that the native organ is highly contractile during the progesterone-dominant diestrus stage of the cycle when compared to the estrogen-dominant proestrus and estrus stages. We also observed correlations between contractility during pre-implantation stages of pregnancy and observed embryo movement patterns. During the first phase of embryo movement when clustered embryos move towards the middle of the uterine horn, uterine contractions are dynamic and non-uniform between different segments of the uterine horn. In the second phase of embryo movement, contractions are more uniform and rhythmic throughout the uterine horn. Finally, when our method is applied to Lpar3 mutant uteri that display faster embryo movement, we observe global and regional increases in contractility. Our method provides a means to understand the wave characteristics of uterine smooth muscle in response to modulators and in genetic mutants. Better understanding uterine contractility in the early pregnancy stages is critical for the advancement of artificial reproductive technologies and a possibility of modulating embryo movement during clinical embryo transfers.

developmental biology↗

bioRxiv2022

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis.

We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection both in vitro, and in the Syrian golden hamster model in vivo. In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants. ONE SENTENCE SUMMARYSARS-CoV-2 ORF6 subverts bidirectional nucleo-cytoplasmic trafficking to inhibit host gene expression and contribute to viral pathogenesis.

microbiology↗

bioRxiv2022

Performance of novel antibodies for lipoarabinomannan to develop diagnostic tests for Mycobacterium tuberculosis

Lipoarabinomannan (LAM), a component of the Mycobacterium tuberculosis (MTB) cell wall, is detectable in the urine of MTB infected patients with active tuberculosis (TB). LAM-specific antibodies (Igs) have been developed by a variety of traditional and recombinant methods for potential use in a rapid diagnostic test (RDT). We evaluated the analytical performance of the TB LAM Igs to identify pairs that offer superior performance over existing urine LAM tests. We assessed 25 new and 4 existing Igs in a matrixed format using a multiplex electrochemiluminescence-based liquid immunoassay. A total of 841 paired Ig combinations were challenged with in vitro cultured LAM (cLAM) derived from MTB strains representing diverse phylogenetic lineages, alongside urinary LAM (uLAM) from the urine of adults with active pulmonary TB. Analytical sensitivity of down-selected Ig pairs was determined using MTB Aoyama-B cLAM, while diagnostic accuracy was determined using clinical samples. When testing cLAM, the reactivity of Ig pairs was similar across MTB lineages 1-4 but lineage 5:6 had significantly more reactivity among Ig pairs. Overall, 41 Ig pairs had a strong binding affinity to cLAM, as compared to the reference pair of S4-20/A194-01, and 28 Ig pairs therein exhibited a strong affinity for both cLAM and uLAM. Retrospective testing on clinical urine specimens demonstrated varying sensitivities (12-80%) and specificities (14-100%). The five top pairs had a similar analytical limit of detection to the reference pair but in four instances, the sensitivity and specificity with clinical uLAM samples was poor. Overall, epitopes presented by uLAM are different from cLAM, which may affect antibody performance when testing uLAM in patient samples. Several new Ig pairs had similar ranges of high sensitivity to cLAM but overall, there were no new candidate Ig pairs identified in this round of screening with increased performance with uLAM as compared to an existing optimal pair.

microbiology↗

bioRxiv2021

Pre-implantation mouse embryo movement under hormonally altered conditions

Precise regulation of embryo movement is crucial to successful implantation, but the role of ovarian hormones in this process is not understood. We ascertain the effects of altered hormonal environment on embryo movement using two delayed implantation models: Natural lactational Diapause (ND), a naturally occurring alternate model of pregnancy, and Artificially induced Diapause (AD), a laboratory version of ND. Our previous work suggests that embryos in a natural pregnancy (NP) first display unidirectional clustered embryo movement, followed by bidirectional scattering and spacing movement. In contrast, in the ND model, embryos are present as clusters near the oviductal-uterine junction for ~24-hours longer than NP, followed by locations consistent with a unidirectional scattering and spacing movement. Intriguingly, the AD model closely resembles embryo location in NP and not ND. Further, unlike the popular paradigm of reduced estrogen (E2) levels in diapause E2 levels are comparable across NP, ND, and AD, while progesterone (P4) levels are reduced in ND and highly increased in AD when compared to NP. Exogenous administration of E2 or P4 modifies the unidirectional clustered embryo movement, while E2 treatment causes a reduction in P4 and affects the bidirectional phase of embryo movement. Taken together, our data suggest embryo movement can be modulated by both P4 and E2. Understanding natural hormonal adaptation in diapause provides an opportunity to determine key players regulating embryo movement and implantation success. This knowledge can be leveraged to understand pregnancy survival and implantation success in hormonally altered conditions in the clinic.

developmental biology↗

bioRxiv2021

L-WNK1 is required for BK channel activation in intercalated cells

BK channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K+ secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high K+ diet. In cell culture, the long isoform of the kinase WNK1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high K+ diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K+. We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high K+ diet. Mice with an IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high K+ (5% K+ as KCl) diet for at least 10 days. IC-L-WNK1-KO mice exhibited higher blood K+ concentrations ([K+]) than controls. BK channel-dependent whole-cell currents in ICs from cortical collecting ducts of high K+ fed IC-L-WNK1-KO mice were reduced compared to controls. Six-hour urinary K+ excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice have higher blood [K+] and reduced IC BK channel currents are consistent with impaired urinary K+ secretion, and suggest that IC L-WNK1 has a role in the renal adaptation to a high K+ diet.

physiology↗

bioRxiv2020

Characterization of a novel endopeptidase Cwl0971 that hydrolyzes peptidoglycan of Clostridioides difficile involved in pleiotropic cellular processes

Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Although the production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, the mechanism of TcdA and TcdB release from cell remains unclear. In this study, we identified and characterized a new cell wall hydrolase Cwl0971 (CDR20291_0971) from C. difficile R20291, which is involved in bacterial autolysis. The gene 0971 deletion mutant (R20291{Delta}0971) generated with CRISPR-AsCpfI exhibited significantly delayed cell autolysis and increased cell viability compared to R20291, and the purified Cwl0971 exhibited hydrolase activity for Bacillus subtilis cell wall. Meanwhile, 0971 gene deletion impaired TcdA and TcdB release due to the decreased cell autolysis in the stationary / late phase of cell growth. Moreover, sporulation of the mutant strain decreased significantly compared to the wild type strain. In vivo, the defect of Cwl0971 decreased fitness over the parent strain in a mouse infection model. Collectively, Cwl0971 is involved in cell wall lysis and cell viability, which affects toxin release, sporulation, germination, and pathogenicity of R20291, indicating that Cwl0971 could be an attractive target for C. difficile infection therapeutics and prophylactics.

microbiology↗

bioRxiv2020

Mechanical and signaling mechanisms that guide pre-implantation embryo movement.

How a mammalian embryo determines and arrives at its site of attachment is a mystery that has puzzled researchers for decades. Additionally, in multiparous species, embryos face a unique challenge of achieving adequate spacing to avoid competition for maternal resources. Using our enhanced confocal imaging and 3D image reconstruction technology, we evaluate murine embryo location in the uterus along the longitudinal oviductal-cervical axis. Our analysis reveals three distinct pre-implantation stages: a) Embryo entry; b) Unidirectional movement of embryo clusters; and c) Bidirectional scattering and spacing of embryos. We show that unidirectional movement of embryo clusters is facilitated by a mechanical stimulus of the embryo as a physical object and is regulated by adrenergic uterine smooth muscle contractions. Embryo scattering, on the other hand, relies on embryo-uterine communication reliant on the LPAR3 signaling pathway and is independent of adrenergic muscle contractions. We propose that the presence of embryo clusters in the uterine horn provides an opportunity for the uterus to sense and count the embryos, followed by scattering and spacing these embryos along the given length of the horn. Thus, uterine implantation sites in mice are neither random nor predetermined but are guided by the number of embryos entering the uterine lumen. These studies have implications for understanding how embryo-uterine communication is key to determining an optimal implantation site, which is necessary for the success of a pregnancy. Significance StatementIn mammals that carry multiple offspring in one gestation, embryos seemingly acquire even embryo spacing. Such even distribution would imply a guided interaction between the mother and the fetus very early on in pregnancy to allow favorable pregnancy outcomes. Thus, it is essential to understand quantitatively if and when such a uniform distribution of embryos is established. Further, uncovering the physical and biological mechanisms that allow for such equal distribution of embryos, will improve our understanding of early pregnancy events and provide for novel targets for improving pregnancy success in case of infertility and artificial reproductive technologies as well as to develop non-hormonal therapies for contraception.

developmental biology↗