Biology subjects

Anderson, J. C.

Publications and source records attributed to Anderson, J. C..

13 recordsLinked to original sources

bioRxiv2026

AGROtools: A comprehensive modular cloning toolset for Agrobacterium strain engineering

Agrobacterium-mediated transformation is a cornerstone of plant genetic engineering, yet many crops remain recalcitrant to transformation and its efficiency is highly species- and genotype-dependent. The limited diversity of disarmed agrobacterial strains available for biotechnology, and the technically challenging workflows typically required for strain modification, constrain efforts to develop improved plant transformation platforms. Here, we describe the development of AGROtools-a versatile, expandable, and user-friendly modular cloning plasmid collection comprising more than 75 individual components, that is designed to simplify genetic engineering of agrobacteria. Constructs assembled with this toolkit couple visual reporters with selectable and counterselectable markers to facilitate identification of recombinant clones, thereby reducing reliance on PCR-based screening. We constructed and tested reporter cassettes that are functional across diverse agrobacterial strains and genomic contexts. By linking a visual reporter combinatorially with various antibiotic resistance markers and orthogonal site-specific recombinase recognition sequences, AGROtools can enable marker removal and recycling for up to four successive genomic modifications. We demonstrate the utility of AGROtools by disarming 10 novel wild-type strains, and accomplishing serial modifications within individual strain backgrounds. In addition to conventional recA-mediated homologous recombination, the collection incorporates components for applying contemporary strain engineering approaches, including CRISPR/dCas9 base editing, INTEGRATE-mediated targeted insertion, and phage-derived recombineering systems. Constructs can be assembled and deployed rapidly, enabling isolation of strains carrying an intended modification in less than two weeks. AGROtools provides a broadly applicable resource for accelerating strain development and genetic studies in agrobacteria, with potential to advance plant biotechnology by aiding improvement of disarmed strains.

plant biology↗

bioRxiv2026

ST6GAL1 sialyltransferase promotes acinar cell survival and tissue regeneration during pancreatitis

The role of aberrant glycosylation in pancreatitis remains an under-investigated area of research. Here, we determined that the ST6GAL1 sialyltransferase, which adds 2-6-linked sialic acids to N-glycoproteins, was upregulated in pancreatic tissues from patients with acute (AP) and chronic (CP) pancreatitis, and in mice with experimental AP. Within these tissues, ST6GAL1 was selectively expressed in acinar cells undergoing acinar to ductal metaplasia (ADM), a process whereby injured acinar cells de-differentiate and re-enter the cell cycle to enable tissue repair. To study the functional role of ST6GAL1 in pancreatitis, we used HPNE pancreatic epithelial cells with modulated ST6GAL1 expression, along with pancreatic organoids from mice with transgenic expression of ST6GAL1 (SC mice). In these models, ST6GAL1 activity promoted the activation of EGFR (a well-known ADM-driver), ERK, and AKT. ST6GAL1-expressing cells also displayed an ERK-dependent upregulation of the anti-apoptotic proteins, Mcl-1 and Bcl-xL, and impaired apoptotic signaling by the TNFR1 and Fas death receptors. Unbiased kinomics profiling revealed that ST6GAL1 induced the activation of many receptor tyrosine kinases associated with cell survival and proliferation (e.g., EGFR, PDGFR, MET). Furthermore, ST6GAL1 enhanced the survival and proliferation of cells exposed to stress-inducing conditions such as serum withdrawal. Based on these findings, we hypothesized that the pro-survival phenotype imparted by ST6GAL1 would facilitate tissue healing following a bout of pancreatitis. Accordingly, we induced AP in wild type and SC mice via L-arginine injection and found that SC mice had more rapid and efficient tissue repair, evidenced by accelerated restoration of the acini, diminished acinar apoptosis, and reduced immune cell infiltration. Together, these findings highlight a novel glycosylation-dependent mechanism that drives cell survival, positioning ST6GAL1 as a key adaptive mediator of pancreatic regeneration.

cell biology↗

bioRxiv2026

O-GlcNAc transferase regulates H2O2 production via p38 MAPK

i.Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by augmented transforming growth factor-{beta} (TGF-{beta}) signaling leading to excessive extracellular matrix (ECM) deposition. The fibroblast-to-myofibroblast-transition (FMT) and metabolic reprogramming of lung fibroblasts (HLFs) are essential to IPF pathogenesis, yet the connection between nutrient metabolism and fibrogenesis remains poorly defined. The O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a nutrient-sensitive enzyme that adds O-GlcNAc moieties to substrates. We previously showed that loss of OGT reverses bleomycin-induced pulmonary fibrosis in mice. Here, using unbiased kinomics, we show that pharmacologic inhibition of OGT suppressed non-canonical TGF-{beta}-induced mitogen-activated protein kinase (MAPK) signaling. Molecular confirmation revealed that TGF-{beta}-induced phosphorylation of p38, but not ERK or JNK, was reduced by OGT blockade. Furthermore, p38 itself was O-GlcNAc-modified, which enhanced its phosphorylation and promoted downstream phosphorylation of the NADPH oxidase subunit, p47phox. Inhibition of OGT, p38, or p47phox reduced reactive oxygen species (ROS) in HLFs, revealing a previously unknown role of OGT-p38-p47phox signaling in ROS production. Collectively, this work establishes that O-GlcNAc-modified p38 enhances p47phox-dependent H2O2 production. HighlightsO_LIUsing PamChip STK arrays, we show that OGT inhibition causes broad kinomic remodeling, including suppression of non-canonical TGF-{beta} MAPKs and multiple CDKs. C_LIO_LIOGT blockade selectively attenuates p38 phosphorylation, despite TGF-{beta}-induced substrate redundancy with ERK and JNK. C_LIO_LIWe provide evidence that p38 MAPK undergoes O-GlcNAcylation in human lung fibroblasts, a modification not previously reported. C_LIO_LIThe study identifies a new signaling axis where O-GlcNAc modification of p38 modulates the phosphorylation of p47phox, therefore regulating NOX-dependent H2O2 production. C_LIO_LIBlocking OGT or inhibiting p38/p47phox dramatically reduces TGF-{beta}-driven H2O2 production in human lung fibroblasts. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/728188v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@114210eorg.highwire.dtl.DTLVardef@8647b8org.highwire.dtl.DTLVardef@1ce814corg.highwire.dtl.DTLVardef@8addec_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Growth and molecular responses of potato to lunar regolith simulants

BackgroundOn-site food production will be required to achieve NASAs goal of a sustainable Lunar habitat. Toward this end, the use of fine, soil-like material on the Lunar surface, known as regolith, has been proposed as a plant growth substrate. However, how this substrate may affect plant growth is not well understood. Lunar regolith is devoid of the organic materials that make soils on earth fertile for plant growth, and has been weathered by solar winds, cosmic rays, and micrometeorite impacts. Additionally, regolith at certain lunar sites may contain heavy metals. These metal ions may leech, thus posing challenges with accumulation in plant material. To address and verify the efficacy of regolith-based crop production, we used lunar regolith simulants (LRS). We investigated the effects of LRS on potato (Solanum tuberosum cv Modoc) plant and tuber development, gene expression, and nutrition profiles. ResultsGrowth in LRS negatively impacted the potato plant size and tuber yield. While the degree of impact differed between simulants, all plants grown in LRS were statistically significantly shorter in height than plants grown in control soil. Further experiments with the lunar mare simulant 1E (LMS-1E) show that these effects can be ameliorated through the addition of vermicompost, an organic component, with a 70:30 v/v ratio of LMS-1E to compost being virtually indistinguishable from controls. Changes in gene expression profiles also differed between simulants, with genes related to photosynthesis, biotic and abiotic stress responses, signaling, and terpenes and flavonoids metabolism being commonly altered. Despite these observed differences in transcription, broad changes in metabolite profiles were not observed. ConclusionsLRS are clearly stressful on plants. However, amendment of the substrate with composted materials appears to be a viable strategy to alleviate stress. Given these observations, regolith-based agriculture may not be viable for very early food production when organic matter content is low. However, this would improve over time with continual incorporation of organic matter to regolith. As such, we believe regolith-based agriculture is a viable long-term strategy.

plant biology↗

bioRxiv2025

EVODEX: A Mechanistic Framework for Extracting, Structuring, and Predicting Enzymatic Reactivity

Accurately modeling enzymatic reactivity is essential for synthesis planning, mass spectrometry interpretation, and biochemical knowledge extraction, but existing reaction datasets are often incomplete or inconsistently annotated. EVODEX introduces two abstractions to address this: partial reactions, which isolate atom-mapped substrate-product transformations from complex biochemical reactions, and Electronic Reaction Operators (EROs), which encode the bonding and orbital environment of atoms undergoing change. From 349,458 curated reactions in EnzymeMap, EVODEX constructs over 186,000 partial reactions and mines a core set of 1,404 EROs, each supported by at least 10 examples. Together, these explain 91 percent of test reactions at the formula level and 62 percent at the mechanistic level. A further filtered synthesis subset of 436 one-to-one operators that exclude ubiquitous metabolites provides a compact and generalizable vocabulary of single-step enzymatic transformations. EVODEX operators are auditable, reusable, and available as part of an open-source Python package with datasets, Jupyter notebooks, and a browsable website.

bioinformatics↗

bioRxiv2025

Interpreting biochemical text with language models:a machine learning framework for reaction extraction and cheminformatic validation

Recent advancements in large language models (LLMs) offer new opportunities for automating the manual curation of biochemical reaction databases from scientific literature. In this study, we present an integrated pipeline that enhances LLM-based extraction of enzymatic reactions with machine learning and cheminformatics-informed validation. Using BRENDA-linked PubMed articles, we evaluate GPT-4s ability to extract reactions and infer missing chemical entities in textual descriptions of enzymatic reactions. Extracted reactions are converted to SMILES and InChI notations before being encoded into molecular fingerprint similarity scores and atom mapping metrics. These cheminformatics metrics are then used to train machine learning classifiers that validate GPT extractions. We employ a Positive-Unlabeled learning approach with synthetic invalid reactions to train various classifiers and assess model performances. The best classifier is then benchmarked on GPT extractions. Our findings show that GPT can accurately infer incomplete reactions and cheminformatics tools can serve as effective predictors of reaction validity. This work demonstrates a scalable framework for automated and reliable curation of enzymatic reaction databases, highlighting the potential of combining LLMs with cheminformatics and machine learning for reliable scientific knowledge extraction. Author SummaryCurating databases of biochemical reactions is a time-consuming and manual task, yet it plays a vital role in advancing research in biology and chemistry. Many scientific articles describe important enzymatic reactions, but often do so in incomplete ways--such as mentioning only the starting molecule or the enzyme, and leaving out the rest. In this work, we explore how recent advancements in artificial intelligence, specifically large language models like GPT, can help extract such information automatically from scientific literature. We show that these models can not only find reactions in text, but also infer missing parts of reactions based on the surrounding context. To make sure these inferred reactions are chemically plausible, we use computational chemistry tools that analyze the structure of the molecules involved. We then train a machine learning model to help us automatically detect which reactions are likely to be valid. This combination of tools offers a new way to speed up and improve how biochemical knowledge is extracted from the growing body of scientific literature. Our study suggests that this kind of automation could help scientists keep biological databases up to date and reduce the burden of manual data entry.

bioinformatics↗

bioRxiv2024

Thiamin priming to control early blight in potato: investigation of its effectiveness and molecular mechanisms

BackgroundPrevious reports in several plant species have shown that thiamin applied on foliage primes plant immunity and is effective in controlling fungal, bacterial, and viral diseases. However, the effectiveness of thiamin against potato (Solanum tuberosum) pathogens has seldom been investigated. Additionally, the transcriptomics and metabolomics of immune priming by thiamin have not previously been investigated. Here, we tested the effect of thiamin application against Alternaria solani, a necrotrophic fungus that causes early blight disease on potato foliage, and identified associated changes in gene expression and metabolite content. ResultsFoliar applications of thiamin reduced lesion size by approximately 33% when applied at an optimal concentration of 10 mM. However, the effect of thiamin on preventing lesion growth was temporally limited, as we observed a reduction of lesion size when leaves were inoculated 4 h, but not 24 h, following thiamin treatment. Additionally, we found that the effect of thiamin on lesion size was restricted to the site of application and was not systemic. Gene expression analysis via RNA-seq showed that thiamin induced the expression of genes involved in the synthesis of salicylic acid (SA) and phenylpropanoids to higher levels than the pathogen alone, as well as fatty acid metabolism genes that may be related to jasmonic acid biosynthesis. Thiamin also delayed the downregulation of photosynthesis-associated genes in plants inoculated with A. solani, which is a typical plant response to pathogens, but could also induce a similar repression of primary metabolic pathways in non-infected leaves. Metabolite analyses revealed that thiamin treatment in the absence of pathogen decreased the amounts of several organic compounds involved in the citric acid cycle as well as sugars, sugar alcohols, and amino acids. ConclusionsOur study indicates that thiamin priming of plant defenses may occur through perturbation of primary metabolic pathways and a re-allocation of energy resources towards defense activities.

plant biology↗

bioRxiv2023

Transporter-mediated depletion of extracellular proline directly contributes to plant pattern-triggered immunity against a bacterial pathogen

Plants possess cell surface-localized immune receptors that detect pathogen-associated molecular patterns (PAMPs) and initiate defenses that provide effective resistance against microbial pathogens. Many PAMP-induced signaling pathways and cellular responses are known, yet how pattern-triggered immunity (PTI) limits pathogen growth in plants is poorly understood. Through a combined metabolomics and genetics approach, we discovered that plant-exuded proline is a virulence-inducing signal and nutrient for the bacterial pathogen Pseudomonas syringae, and that PAMP-induced depletion of proline from the extracellular spaces of Arabidopsis leaves directly contributes to PTI against P. syringae. We further show that PAMP-induced depletion of extracellular proline requires the amino acid transporter Lysine Histidine Transporter 1 (LHT1). This study demonstrates that depletion of a single extracellular metabolite is an effective component of plant innate immunity. Given the important role for amino acids as nutrients for microbial growth, their depletion at sites of infection may be a broadly effective means for defense against many pathogens.

plant biology↗

bioRxiv2023

Enabling AI in Synthetic Biology through Construction File Specification

The Construction File (CF) specification establishes a standardized interface for molecular biology operations, laying a foundation for automation and enhanced efficiency in experiment design. It is implemented across three distinct software projects: PyDNA_CF_Simulator, a Python project featuring a ChatGPT plugin for interactive parsing and simulating experiments; ConstructionFileSimulator, a field-tested Java project that showcases Experiment objects expressed as flat files; and C6-Tools, a JavaScript project integrated with Google Sheets via Apps Script, providing a user-friendly interface for authoring and simulation of CF. The CF specification not only standardizes and modularizes molecular biology operations but also promotes collaboration, automation, and reuse, significantly reducing potential errors. The potential integration of CF with artificial intelligence, particularly GPT-4, suggests innovative automation strategies for synthetic biology. While challenges such as token limits, data storage, and biosecurity remain, proposed solutions promise a way forward in harnessing AI for experiment design. This shift from human-driven design to AI-assisted workflows, steered by high-level objectives, charts a potential future path in synthetic biology, envisioning an environment where complexities are managed more effectively.

synthetic biology↗

bioRxiv2021

Cardiac fibroblast GSK-3α mediates adverse myocardial fibrosis via IL-11 and ERK pathway

BackgroundHeart failure is the leading cause of mortality, morbidity, and healthcare expenditures worldwide. Numerous studies have implicated Glycogen Synthase Kinase-3 (GSK-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms appear to play overlapping, unique, and even opposing functions in the heart. Recently our group has identified cardiac fibroblast (CF) GSK-3{beta} as a negative regulator of fibrotic remodeling in the ischemic heart. However, the role of CF-GSK-3 in myocardial fibrosis is unknown. Methods and ResultsHerein, we employed two entirely novel conditional fibroblast-specific and tamoxifen-inducible mouse models to define the role of CF-GSK-3 in fibroblast activation and myocardial fibrosis. Specifically, GSK-3 was deleted from cardiac fibroblasts or myofibroblasts with tamoxifen-inducible Tcf21- or periostin-promoter-driven Cre recombinase. At 2 months of age, WT and KO mice were subjected to cardiac injury, and heart functions were monitored by serial echocardiography. Histological analysis and morphometric studies were performed at 8 weeks post-injury. In both settings, GSK-3 deletion restricted fibrotic remodeling and improved cardiac function. To investigate underlying mechanisms, we examined the effect of GSK-3 deletion on myofibroblast transformation and pro-fibrotic TGF{beta}1-SMAD3 signaling in vitro. A significant reduction in cell migration, collagen gel contraction, and -SMA expression in TGF{beta}1 treated GSK-3 KO MEFs confirmed that GSK-3 is required for myofibroblast transformation. Surprisingly, GSK-3 deletion did not affect SMAD3 activation, indicating the pro-fibrotic role of GSK-3 is SMAD3 independent. To further delineate the underlying mechanism, total proteins were isolated from CFs of WT and KO animals at 4 weeks post-injury, and kinome profiling was performed by utilizing PamStation(R)12 high throughput microarray platform. The kinome analysis identified the downregulation of RAF family kinase activity in GSK3-KO-CFs. Moreover, mapping of significantly altered kinases against literature annotated interactions generated ERK-centric networks. Importantly, flow cytometric analysis of CFs confirmed a significant decrease in pERK levels in KO mice. Additionally, our in vitro studies demonstrated that GSK-3 deletion prevented TGF{beta}1 induced ERK activation thereby validating our findings from kinome analysis. Interestingly, IL-11, a fibroblast specific downstream effector of TGF{beta}1, was very low in GSK-3 KO MEFs as compared to WT and ERK inhibition further reduced IL-11 expression in them. All these results indicate that GSK-3 mediates pro-fibrotic response in the injured heart through IL-11 and ERK pathway. ConclusionCF-GSK-3 plays a causal role in myocardial fibrosis that could be therapeutically targeted for future clinical applications.

molecular biology↗

bioRxiv2020

The antimicrobial peptide DGL13K is active against resistant gram-negative bacteria and subinhibitory concentrations stimulate bacterial growth without causing resistance

Antimicrobial peptides may be alternatives to traditional antibiotics with reduced bacterial resistance. The antimicrobial peptide GL13K was derived from the salivary protein BPIFA2. This study determined the relative activity of the L-and D-enantiomers of GL13K to wild-type and drug-resistant strains of three gram-negative species and against Pseudomonas aeruginosa biofilms. DGL13K displayed in vitro activity against ESBL and KPC-producing Klebsiella pneumoniae (MICs 16-32 g/ml), MDR and XDR P. aeruginosa, and XDR Acinetobacter baumannii carrying metallo-beta-lactamases (MICs 8-32 g/ml). P. aeruginosa showed low inherent resistance to DGL13K and the increased metabolic activity and growth caused by sub-MIC concentrations of GL13K peptides did not result in acquired bacterial resistance. Daily dosing for approximately two weeks did not increase the MIC of DGL13K or cause cross-resistance between LGL13K and DGL13K. These data suggest that DGL13K is a promising candidate antimicrobial peptide for further development.

microbiology↗

bioRxiv2019

Re-evaluation of a Tn5::gacA mutant of Pseudomonas syringae pv. tomato DC3000 uncovers roles for uvrC and anmK in promoting virulence

Pseudomonas syringae is a taxon of plant pathogenic bacteria that can colonize and proliferate within the interior space of leaf tissue. This process requires P. syringae to rapidly upregulate the production of virulence factors including a type III secretion system (T3SS) that suppress host defenses. GacS/A is a two-component system that regulates virulence of many plant and animal pathogenic bacteria including P. syringae. We recently investigated the virulence defect of strain AC811, a Tn5::gacA mutant of P. syringae pv. tomato DC3000 that is less virulent on Arabidopsis. We discovered that decreased virulence of AC811 is not caused by loss of GacA function. Here, we report the molecular basis of the virulence defect of AC811. We show that AC811 possesses a nonsense mutation in anmK, a gene predicted to encode a 1,6-anhydromuramic acid kinase involved in cell wall recycling. Expression of a wild-type allele of anmK partially increased growth of AC811 in Arabidopsis leaves. In addition to the defective anmK allele, we also show that the Tn5 insertion in gacA exerts a polar effect on uvrC, a downstream gene encoding a regulator of DNA damage repair. Expression of the wild-type anmK allele together with increased expression of uvrC fully restored the virulence of AC811 during infection of Arabidopsis. These results demonstrate that defects in anmK and uvrC are together sufficient to account for the decreased virulence of AC811, and suggest caution is warranted in assigning phenotypes to GacA function based on insertional mutagenesis of the gacA-uvrC locus.

microbiology↗

bioRxiv2019

Therapeutically actionable PAK4 is amplified, overexpressed and involved in bladder cancer progression

Muscle-invasive bladder carcinomas (MIBCs) are aggressive genitourinary malignancies. Disease incidence and survival rates vary based on aggressiveness and treatment options. Metastatic urothelial carcinoma of the bladder is generally incurable by current chemotherapy and leads to early mortality. For a minority ([~]20%) of patients, T-cell checkpoint inhibitors provide durable benefits following prior platinum therapy. Recent studies have identified molecular subtypes of MIBCs with different sensitivities to frontline therapy, suggesting heterogeneity in these tumors and pointing to the importance of molecular characterization of MIBCs to provide effective treatment. We have performed multi-omic profiling of the kinome to identify therapeutic targets that are overexpressed in a subset of BLCAs. Our analyses revealed amplification and overexpression of P21 (RAC1) activated kinase 4 (PAK4) in a subset of BLCAs. For these tumors, multiplex kinase assay profiling identified corresponding PAK4 target substrates. By performing experiments using cultured bladder cancer cells, we confirmed the role of PAK4 in BLCA cell proliferation and invasion. Furthermore, our studies showed that a PAK4 inhibitor was effective in curtailing growth of BLCA cells. Transcriptomic analyses identified elevated expression of another kinase, Protein Tyrosine Kinase 6 (PTK6), upon treatment with a PAK4 inhibitor. Similarly, RNA interference of PAK4 led to elevated expression of PTK6. Treatment with a combination of kinase inhibitors (vandetanib and dasatinib) showed enhanced sensitivity compared to either drug alone. Thus, PAK4 may be therapeutically actionable for a subset of MIBC patients with amplified and/or overexpressed PAK4 in their tumors. Our results also indicate that combined inhibition of PAK4 and PTK6 may overcome resistance to PAK4. These observations warrant clinical investigations with selected BLCA patients.

cancer biology↗