Biology subjects

Lee, D. H.

Publications and source records attributed to Lee, D. H..

18 recordsLinked to original sources

bioRxiv2026

Pirfenidone improves adipose dysfunction and obesity-driven steatohepatitis via mTORC1 signaling

Obesity-driven metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) are shaped by depot-specific adipose tissue dysfunction, including maladaptive expansion and visceral adipose tissue (VAT) fibrosis. Pirfenidone, an anti-fibrotic agent, improves experimental liver disease. However, its actions on adipose depots and adipose-liver crosstalk remain unclear. Here, we identify pirfenidone as a modulator of mechanistic target of rapamycin complex 1 (mTORC1)-dependent adipose tissue remodeling with divergent outputs in subcutaneous and visceral fat. In diet-induced obese MASH mice, pirfenidone decreased subcutaneous adipose tissue (SAT), inhibiting mTORC1-driven lipogenesis and enhancing oxidative lipid metabolism. Pirfenidone attenuated VAT fibrosis by suppressing an mTORC1-mothers against decapentaplegic homolog 3 (SMAD3)-yes-associated protein (YAP) axis and extracellular matrix gene programs. Pirfenidone also lowered hepatic triglycerides, improved steatosis and fibrosis, reduced hepatic mTORC1 activity. Conditioned medium from fibrotic adipocytes induced lipogenic, inflammatory, and pro-fibrotic programs in AML12, which effects that were blunted by pirfenidone. These data reveal adipose tissue-centered actions of pirfenidone that link mTORC1 remodeling to improved obesity-associated liver disease.

molecular biology↗

bioRxiv2026

Reconstructing multi-scale tissue spatial architecture from single-cell RNA-seq with REMAP

Understanding spatial organization of cells is critical for deciphering tissue function and disease. Single-cell RNA-sequencing (scRNA-seq) profiles transcriptomes at scale but loses spatial context, while spatial transcriptomics (ST) preserves spatial information but is constrained by cost and gene coverage. Here, we present REMAP, a deep learning framework that integrates gene expression with neighborhood-level gene-gene covariance to reconstruct multi-scale spatial organization of scRNA-seq data using one or multiple ST references. Across 2D and 3D mouse brain, human fetal cortex, and seven human cancer types, REMAP consistently outperformed existing approaches. Applied to a human multiple sclerosis atlas, REMAP resolved microglial neighborhood heterogeneity, and identified a rare pro-inflammatory microglia-astrocyte subpopulation. Across diverse cancers, REMAP recovered conserved spatially-defined cancer-associated fibroblast subtypes with known prognostic significance. By transforming cost-efficient single-cell datasets into spatially interpretable tissue maps, REMAP enables spatial hypothesis generation, microenvironment discovery, and population-scale inference of conserved and perturbed architectural principles in human disease.

genomics↗

bioRxiv2025

Major histocompatibility complex class II-expressing bone marrow megakaryocytes activate CD4+ T cells and induce regulatory T cell fate

While professional antigen-presenting cells drive adaptive immunity, atypical cell types can fulfill this role in the bone marrow. Megakaryocytes (MKs) are canonically recognized for platelet production, but recent studies indicate functional heterogeneity and immune potential. We found that ~20% of bone marrow MKs express Major Histocompatibility Complex (MHC) II and co-stimulatory receptors CD80, CD86, CD40, and CD83. These MKs process and present antigen to activate T cells ex vivo in an MHC II-dependent manner. MK/T cell interactions induced TGF-{beta}1 secretion and promoted induced Treg differentiation. Prior stimulation of MKs with LPS or Poly I:C was associated with modest Th1-associated CD4+ T cell responses, including IFN-{gamma} and TNF- production, without robust Th17 differentiation. Immunopeptidomics of the murine MK MHC II receptor confirmed occupancy by exogenous peptides, suggesting in vivo functionality. Using a murine model with MK-targeted deletion of MHC II (Pf4-MHC{Delta}/{Delta}), we observed altered TLR signaling and reduced bone marrow TGF-{beta}1. Together, these findings identify MHC II+ MKs as noncanonical antigen-presenting cells with the potential to modulate CD4 T cell responses as part of the immune regulation of the bone marrow niche.

immunology↗

bioRxiv2025

RUNX2 inhibition disrupts a PAX3::FOXO1-RUNX2 feed-forward loop and dismantles oncogenic gene programs in fusion-positive rhabdomyosarcoma

Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer of skeletal muscle lineage, with a 5-year overall survival of <30% for high-risk disease, and <8% when metastatic. The PAX3::FOXO1 fusion gene, resulting from t(2:13), is a signature driver of fusion-positive rhabdomyosarcoma, but similar to other transcription-factor based fusion genes in other cancers, not currently pharmacologically tractable. To identify novel druggable proteins in fusion-positive rhabdomyosarcoma tumor tissue and cell lines, we performed mRNA-seq of RMS patient tumors and utilizing the human FP-RMS cell lines Rh30 and Rh4, found that the RUNX2 transcription factor was the top druggable dependency. In vitro loss of function studies using genetic (RNAi) or pharmacologic (small molecule CADD522) inhibition showed that RUNX2 suppression inhibited FP-RMS cell growth, induced myogenic differentiation and apoptosis, and phenocopied PAX3::FOXO1 suppression. In vivo loss of function studies using conditional (dox-inducible) or pharmacologic (small molecule CADD522) blockade of tumor growth in a xenograft model system showed that RUNX2 suppression inhibited tumor growth. Mechanistically, we identify a PAX3::FOXO1 feed-forward loop whereby PAX3::FOXO1 binds a RUNX2 enhancer to upregulate gene expression alongside MYOD1, while RUNX2 expression supports the expression of PAX3::FOXO1 at the mRNA and protein level. SignificanceRUNX2 inhibition reduces PAX3::FOXO1 expression and signaling, which impairs fusion-positive rhabdomyosarcoma oncogenic phenotypes. In vivo treatment with CADD522 decreased tumor growth and increased survival, indicating that RUNX2 is a promising therapeutic target.

cancer biology↗

bioRxiv2025

Induction of Moderate DNA Damage Enhances Megakaryopoiesis and Platelet Production

A common side effect of poly-ADP ribose polymerase (PARP) inhibitors is low platelet counts, or thrombocytopenia, presumably mediated through platelet progenitors, megakaryocytes (MKs). MKs are large, hematopoietic cells with a polyploid, multi-lobulated nucleus. While DNA replication in MKs (endomitosis) is well studied, limited investigations have examined the impact of DNA damage and repair inhibition on megakaryopoiesis. To explore PARP inhibitor-induced thrombocytopenia, we treated mice with PARP inhibitors (niraparib and olaparib), which are approved for the treatment of solid tumors. While high-dose niraparib treatment led to thrombocytopenia, consistent with clinical observations, treatment at a lower dosage led to a significant, >1.5-fold increase in both the number of bone marrow MKs and circulating platelets. This increase was accompanied by elevated DNA damage in both MKs and MK progenitors, as measured by both {gamma}H2AX accumulation and comet assays of MKs. Notably, platelets from niraparib-treated mice were functionally normal in their response to ADP, TRAP, and collagen. Gamma-irradiation treatment similarly increased MK and platelet counts in mice, suggesting that moderate DNA damage enhances megakaryopoiesis and increases platelet counts. These data reveal a previously unknown relationship between MKs and DNA damage and present a novel target for triggering enhanced platelet production in vivo. Key PointsO_LITreatment of mice with low dose PARP inhibitors or gamma-irradiation enhances platelet counts. C_LIO_LILow dose PARP inhibitor treatment leads to increased DNA damage in MKs and MK progenitors and enhances bone marrow megakaryopoiesis. C_LI

cell biology↗

bioRxiv2024

Consolidated sustainable organic nanozyme integrated with Point-Of-Use sensing platform for dual agricultural and biological molecule detection

The newly introduced organic nanozymes offer a promising alternative to inorganic nanozymes by sustainable and cost-effective solutions. Given the potential of nanozyme-based Point-of-Care (PoC) systems for on-demand molecule sensing; these user-friendly Point-of-Use(PoU) platforms can be highly beneficial for real-world agricultural and biological applications. Herein, a novel, fully organic compound/monomer-based, sustainable organic nanozyme is introduced, that exhibits peroxidase-like enzyme-like catalytic activity. Utilizing a unique self-assembled particle fabrication process, homogenous nanozyme are fabricated within a short time (within 50 nm, D90), exhibiting decent kinetic profiles (Km= 0.046 mM, H2O2), and degradability for improved waste management after its use. OM nanozyme-incorporated colorimetric sensing platforms successfully detect popular agricultural toxic biomolecules and biological molecules while achieving decent molecule sensing performance (practical LOD, glyphosate: at least 1pgmL-1, glucose: 3.47 ngmL-1) with high analytical sensitivity and selectivity. Additionally, a smartphone image-based polynomial regression analysis system and paper-microfluidic hardware were developed to facilitate PoU applications in a lab-free environment with satisfactory analytic sensitivity (at least 1.6gmL-1, for glyphosate, and glucose). This sustainable OM nanozyme, coupled with the PoU platform, is envisioned to have broad applications in agricultural and biological systems.

bioengineering↗

bioRxiv2024

Highly multiplexed design of an allosteric transcription factor to sense novel ligands

Allosteric transcription factors (aTF), widely used as biosensors, have proven challenging to design for detecting novel molecules because mutation of ligand-binding residues often disrupts allostery. We developed Sensor-seq, a high-throughput platform to design and identify aTF biosensors that bind to non-native ligands. We screened a library of 17,737 variants of the aTF TtgR, a regulator of a multidrug exporter, against six non-native ligands of diverse chemical structures - four derivatives of the cancer therapeutic tamoxifen, the antimalarial drug quinine, and the opiate analog naltrexone - as well as two native flavonoid ligands, naringenin and phloretin. Sensor-seq identified novel biosensors for each of these ligands with high dynamic range and diverse specificity profiles. The structure of a naltrexone-bound design showed shape-complementary methionine-aromatic interactions driving ligand specificity. To demonstrate practical utility, we developed cell-free detection systems for naltrexone and quinine. Sensor-seq enables rapid, scalable design of new biosensors, overcoming constraints of natural biosensors.

synthetic biology↗

bioRxiv2023

Decoding Pain: Uncovering the Factors that Affect Performance of Neuroimaging-Based Pain Models

AO_SCPLOWBSTRACTC_SCPLOWNeuroimaging-based pain biomarkers, when combined with machine learning techniques, have demonstrated potential in decoding pain intensity and diagnosing clinical pain conditions. However, a systematic evaluation of how different modeling options affect model performance remains unexplored. This study presents results from a comprehensive literature survey and benchmarking analysis. We conducted a survey of 57 previously published articles that included neuroimaging-based predictive modeling of pain, comparing classification and prediction performance based on the following modeling variables--the levels of data, spatial scales, idiographic vs. population models, and sample sizes. The findings revealed a preference for population-level modeling with brain-wide features, aligning with the goal of clinical translation of neuroimaging biomarkers. However, a systematic evaluation of the influence of different modeling options was hindered by a limited number of independent test results. This prompted us to conduct benchmarking analyses using a locally collected functional Magnetic Resonance Imaging (fMRI) dataset (N = 124) involving an experimental thermal pain task. The results clearly demonstrated that data levels, spatial scales, and sample sizes significantly impact model performance. Specifically, incorporating more pain-related brain regions, increasing sample sizes, and using less data averaging in training while increasing it in testing enhanced performance. These findings provide a useful reference for decision-making in the development of neuroimaging-based biomarkers, highlighting the importance of the careful selection of modeling strategies and options to build better-performing neuroimaging pain biomarkers. These findings offer useful guidance for developing neuroimaging-based biomarkers, underscoring the importance of strategic selection of modeling approaches to build better-performing neuroimaging pain biomarkers.

neuroscience↗

bioRxiv2023

What does the fox (genome) say? A chromosome-level assembly for the gray fox (Urocyon cinereoargenteus)

Reference genome assemblies have been created from multiple lineages within the Canidae family, however, despite its phylogenetic relevance as a basal genus within the clade, there is currently no reference genome for the gray fox (Urocyon cinereoargenteus). Here, we present a chromosome-level assembly for the gray fox (U. cinereoargenteus), which represents the most contiguous, non-domestic canid reference genome available to date, with 90% of the genome contained in just 34 scaffolds and a contig N50 and scaffold N50 of 59.4 and 72.9 Megabases (Mb), respectively. Repeat analyses identified an increased number of simple repeats relative to other canids. Based on mitochondrial DNA, our Vermont sample clusters with other gray fox samples from the northeastern US and contains slightly lower levels of heterozygosity than gray foxes on the west coast of California. This new assembly lays the groundwork for future studies to describe past and present population dynamics, including the delineation of evolutionarily significant units of management relevance. Importantly, the phylogenetic position of Urocyon allows us to verify the loss of PRDM9 functionality in the basal canid lineage, confirming that pseudogenization occurred at least 10 million years ago.

genomics↗

bioRxiv2023

A Novel Subtype of Myeloproliferative Neoplasms Driven by a MYC-Alarmin Axis

Despite advances in understanding the genetic abnormalities in myeloproliferative neoplasms (MPNs) and the development of JAK2 inhibitors, there is an urgent need to devise new treatment strategies, particularly for triple negative myelofibrosis (MF) patients whose MPNs lack mutations in the JAK2 kinase pathway and have very poor clinical outcomes. Here we report that MYC copy number gain and increased MYC expression frequently occur in triple negative MF, and that MYC-directed activation of S100A9, an alarmin protein that plays pivotal roles in inflammation and innate immunity, is necessary and sufficient to drive development and progression of MF. Notably, the MYC-S100A9 circuit provokes a complex network of inflammatory signaling that involves various hematopoietic cell types in the bone marrow microenvironment. Accordingly, genetic ablation of S100A9 or treatment with small molecules targeting the MYC-S100A9 pathway effectively ameliorates MF phenotypes, highlighting the MYC-alarmin axis as a novel therapeutic vulnerability for this subgroup of MPNs. SIGNIFICANCEThis study establishes that MYC expression is increased in triple negative MPNs via trisomy 8, that a MYC-S100A9 circuit manifest in these cases is sufficient to provoke myelofibrosis and inflammation in diverse hematopoietic cell types in the BM niche, and that the MYC-S100A9 circuit is targetable in triple negative MPN.

cancer biology↗

bioRxiv2023

Interindividual differences in pain can be explained by fMRI, sociodemographic, and psychological factors

In a recent article, Hoeppli et al. (2022) reported that sociodemographic and psychological factors were not associated with interindividual differences in reported pain intensity. In addition, the interindividual differences in pain could not be detected by thermal pain-evoked brain activities measured by functional Magnetic Resonance Imaging (fMRI). Their comprehensive analyses provided convincing evidence for these null findings, but here we provide another look at their conclusions by analyzing their behavioral data and a large-scale fMRI dataset involving thermal pain (N = 124). Our main findings are as follows: First, a multiple regression model incorporating all available sociodemographic and psychological measures could significantly predict the interindividual differences in reported pain intensity. The key to achieving a significant prediction was including multiple individual difference measures in a single model. Second, with fMRI data from a relatively homogeneous group of 124 participants, we could identify brain regions and a multivariate pattern-based predictive model significantly correlated with the interindividual differences in reported pain intensity. Our results, along with the findings of Hoeppli et al., highlight the challenge of predicting interindividual differences in pain, but also suggest that it is not an impossible task.

neuroscience↗

bioRxiv2022

PPARγ/ETV2 Axis Regulates Endothelial-to-Mesenchymal Transition in Pulmonary Hypertension

Endothelial-to-mesenchymal transition (EndoMT) plays an important role in pulmonary hypertension (PH). Also, the molecular mechanisms regulating EndoMT in PH remain to be defined. In this study, we first showed that reduced expression of the transcription factors ETV2 (ETS variant 2) and PPAR{gamma} (Peroxisome Proliferator-Activated Receptor gamma) along with reduced endothelial markers and increased EndoMT markers were consistently observed in lungs and pulmonary artery endothelial cells (PAECs) of idiopathic pulmonary arterial hypertension (IPAH) patients, in hypoxia-exposed mouse lungs, human PAECs, and in induced EndoMT cells. Base on this observation, we aimed to investigate the function of ETV2 and PPAR{gamma} in EndoMT. We have explored the function of ETV2 and PPAR{gamma} and its mechanism in PH using in Etv2+/- mice or PPAR{gamma} KO mice. Etv2+/- mice spontaneously developed PH and right ventricular hypertrophy, associated with increased EndoMT markers and decreased EC markers. PPAR{gamma} transcriptionally activated the ETV2 promoter. Endothelial PPAR{gamma} expression in mice is positively correlated with ETV2 expression, but inversely with EndoMT markers. Overexpression of ETV2 in hypoxia-exposed rat pulmonary artery led to vascular relaxation. We conclude that PPAR{gamma}-ETV2 signaling can function as a novel pathway in PH pathogenesis by attenuating EndoMT.

physiology↗

bioRxiv2021

Dynamic Functional Brain Reconfiguration During Sustained Pain

AO_SCPLOWBSTRACTC_SCPLOWPain is constructed through complex interactions among multiple brain systems, but it remains unclear how functional brain network representations are dynamically reconfigured over time while experiencing pain. Here, we investigated the dynamic changes in the functional brain networks during 20-min capsaicin-induced sustained orofacial pain. In the early stage, the orofacial areas of the primary somatomotor cortex were separated from the other primary somatomotor cortices and integrated with subcortical and frontoparietal regions, constituting a brain-wide pain supersystem. As pain decreased over time, the subcortical and frontoparietal regions were separated from this pain supersystem and connected to multiple cerebellar regions. Machine-learning models based on these dynamic network features showed significant predictions of changes in pain experience across two independent datasets (n = 48 and 74). This study provides new insights into how multiple brain systems dynamically interact to construct and modulate pain experience, potentially advancing our mechanistic understanding of chronic pain.

neuroscience↗

bioRxiv2019

Integrative Genomic Analysis and Functional Studies Reveal GP5, GRN, MPO and MCAM as Causal Protein Biomarkers for Platelet Traits

RationaleMean platelet volume (MPV) and platelet count (PLT) are platelet measures that have been linked to cardiovascular disease (CVD) and mortality risk. Identifying protein biomarkers for these measures may yield insights into CVD mechanisms. ObjectiveWe aimed to identify causal protein biomarkers for MPV and PLT among 71 CVD-related plasma proteins measured in Framingham Heart Study (FHS) participants. Methods and ResultsWe conducted integrative analyses of genetic variants associated with PLT and MPV with protein quantitative trait locus (pQTL) variants associated with plasma proteins followed by Mendelian randomization (MR) to infer causal relations of proteins for PLT/MPV, and tested protein-PLT/MPV association in FHS participants. Utilizing induced pluripotent stem cell (iPSC)-derived megakaryocyte (MK) clones that produce functional platelets, we conducted RNA-sequencing and analyzed transcriptome-wide differences between low- and high-platelet producing clones. We then performed small interfering RNA (siRNA) gene knockdown experiments targeting genes encoding proteins with putatively causal platelet effects in MK clones to examine effects on platelet production. Protein-trait association analyses were conducted for MPV (n = 4,348) and PLT (n = 4,272). Eleven proteins were associated with MPV and 31 with PLT. MR identified four putatively causal proteins for MPV and four for PLT. Glycoprotein V (GP5), granulin (GRN), and melanoma cell adhesion molecule (MCAM) were associated with PLT in both protein-trait and MR analyses. Myeloperoxidase (MPO) showed significant association with MPV in both analyses. MK RNA-sequencing analysis results were directionally concordant with observed and MR-inferred associations for GP5, GRN, and MCAM. In siRNA gene knockdown experiments, silencing GP5, GRN, and MPO decreased platelet counts. ConclusionsBy integrating population genomics data, epidemiological data, and iPSC-derived MK experiments, we identified four proteins that are causally linked to platelet counts. These proteins and genes may be further explored for their utility in increasing platelet production in bioreactors for transfusion medicine purposes as well as their roles in the pathogenesis of CVD via a platelet/blood coagulation-based mechanism.

genomics↗

bioRxiv2019

High-phytate/low-calcium diet is a risk factor for crystal nephropathies, renal phosphate wasting, and bone loss

Phosphate overload contributes to mineral bone disorders associated with crystal nephropathies. Phytate, the major form of phosphorus in plant seeds, is known as an indigestible and negligible in humans. However, the mechanism and adverse effects of high-phytate intake on Ca2+ and phosphate absorption and homeostasis are unknown. Here we show that excessive intake of phytate with a low-Ca2+ diet fed to rats contributed to the development of crystal nephropathies, renal phosphate wasting, and bone loss through tubular dysfunction secondary to dysregulation of intestinal calcium and phosphate absorption. Moreover, Ca2+ supplementation alleviated the detrimental effects of excess dietary phytate on bone and kidney through excretion of undigested Ca2+-phytate, which prevented a vicious cycle of intestinal phosphate overload and renal phosphate wasting while improving intestinal Ca2+ bioavailability. Thus, we demonstrate that phytate is digestible without a high-Ca2+ diet and a risk factor for phosphate overloading and developing crystal nephropathies and bone disease.

physiology↗

bioRxiv2018

The dynamic association of SPO11-1 with conformational changes of meiotic axial elements in maize

Meiotic double-strand breaks (DSBs) are generated by the evolutionarily conserved SPO11 complex in the context of chromatin loops that are organized along axial elements (AEs) of chromosomes. However, how DSBs are formed with respect to chromosome axes and the SPO11 complex remains unclear in plants. Here, we confirm that DSB and bivalent formation are defective in maize spo11-1 mutants. Super-resolution microscopy demonstrates dynamic localization of SPO11-1 during recombination initiation, with variable numbers of SPO11-1 foci being distributed in nuclei but similar numbers of SPO11-1 foci being found on AEs. Notably, cytological analysis of spo11-1 meiocytes revealed an aberrant AE structure. At leptotene, AEs of wild-type and spo11-1 meiocytes were similarly curly and discontinuous. However, during early zygotene, wild-type AEs become uniform and exhibit shortened axes, whereas the elongated and curly AEs persisted in spo11-1 mutants, suggesting that loss of SPO11-1 compromised AE structural maturation. Our results reveal an interesting relationship between SPO11-1 loading onto AEs and the conformational remodeling of AEs during recombination initiation. Author SummaryMeiosis is essential during sexual reproduction to produce haploid gametes. Recombination is the most crucial step during meiotic prophase I. It enables pairing of homologous chromosomes prior to their reductional division and generates new combinations of genetic alleles for transmission to the next generation. Meiotic recombination is initiated by generating DNA double-strand breaks (DSBs) via SPO11, a topoisomerase-related enzyme. The activity, timing and location of this DSB machinery must be controlled precisely, but how this is achieved remains obscure. Here, we show dynamic localization of SPO11-1 on chromatin during meiotic initiation in maize, yet a similar number of SPO11-1 is able to load onto axial elements (AEs), which accompanies a structural change of the AEs of wild-type meiotic chromosomes. Interestingly, loss of SPO11-1 not only affects DSB formation but also impairs structural alterations of AEs, resulting in abnormally long and curly AEs during early meiosis. Our study provides new insights into SPO11-1 localization during recombination initiation and suggests an intimate relationship between DSB formation and AE structural changes.

molecular biology↗

bioRxiv2018

Human pathogenic bacterium Shigella infects Arabidopsis plants using type-III effectors that suppress conserved MAP kinase signaling

Originality-significance statementIncreased incidence of food-borne disease outbreaks caused by fresh produce contaminated with Escherichia coli O157:H7 and Salmonella spp. has prompted researchers to examine the interaction between these bacteria and various plant species under different environmental conditions. Although studies show that human enteropathogenic bacteria survive both on the surface of and inside plants, little is known about the molecular mechanism underlying plant invasion and colonization. Here, we examined the interaction between the human pathogenic bacterium Shigella and the model plant Arabidopsis. We found that four Shigella spp. strains proliferated successfully in Arabidopsis, causing symptom-like lesions in tissues. Using mutants lacking T3S effectors (i.e., noninvasive human strains), we demonstrated that effectors regulating pathogenesis of shigellosis in humans also play a central role in bacteria-plant interactions. To the best of our knowledge, this is the first study to examine Shigella-mediated virulence and host immune suppression in a plant host at a molecular level.\n\nSummaryAlthough there is debate about whether human intestinal pathogenic bacteria are also plant pathogens, it is clear that these bacteria use plants as an alternative host. Shigella, which infects primates, is reportedly transmitted by fresh vegetables; however, its molecular interactions with plants have not been extensively studied. Here, we show that four Shigella strains, S. boydii (S. b), S. sonnei (S. s), S. flexneri 2a (S. f 2a), and S. flexneri 5a (S. f 5a), proliferated at different levels in Arabidopsis thaliana. Microscopic studies revealed that these bacteria were present inside leaves and damaged plant cells. GFP-labeled S. b, S. f 2a, and S. f 5a entered plants via guard cells, and S. f 2a infiltrated root tissues and colonized roots. Using mutants lacking type III secretion systems (T3SS), we found that T3SS of Shigella that regulate the pathogenesis of shigellosis in humans also play a central role in attachment and multiplication in Arabidopsis. Furthermore, the immunosuppressive activity of two T3S effectors, OspF and OspG, were needed for the proliferation of Shigella in Arabidopsis. These findings demonstrate that Shigella-mediated virulence determinants are expressed, and pathogenic symptoms are observed, in model plants.

microbiology↗

bioRxiv2017

Measuring Biotherapeutic Viscosity and Degradation On-Chip with Particle Diffusometry

In absence of efficient ways to test drug stability and efficacy, pharmaceuticals that have been stored outside of set temperature conditions are destroyed, often at great cost. This is especially problematic for biotherapeutics, which are highly sensitive to temperature fluctuations. Current platforms for assessing the stability of protein-based biotherapeutics in high throughput and in low volumes are unavailable outside of research and development laboratories and are not efficient for use in production, quality control, distribution, or clinical settings. In these alternative environments, microanalysis platforms could provide significant advantages for the characterization of biotherapeutic degradation. Here we present particle diffusometry (PD), a new technique to study degradation of biotherapeutic solutions. PD uses a simple microfluidic chip and microscope setup to calculate the Brownian motion of particles in a quiescent solution using a variation of particle image velocimetry (PIV) fundamentals. We show that PD can be used to measure the viscosity of protein solutions to discriminate intact protein from degraded samples as well as to determine the change in viscosity as a function of therapeutic concentration. PD viscosity analysis is applied to two particularly important biotherapeutic preparations: insulin, a commonly used protein for diabetic patients, and monoclonal antibodies which are an emerging class of biotherapeutics used to treat a variety of diseases such as autoimmune disorders and cancer. PD-based characterization of solution viscosity is a new tool for biotherapeutic analysis, and owing to its easy setup could readily be implemented at key points of the pharmaceutical delivery chain and in clinical settings.

bioengineering↗