Biology subjects

Adhikari, A.

Publications and source records attributed to Adhikari, A..

At least 19 recordsLinked to original sources

bioRxiv2026

A targetable opioid/cancer associated fibroblast axis drives extracellular matrix remodeling and tumor aggressiveness in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is an intractable disease with few effective treatment options. PDAC is characterized by a dense, fibro-inflammatory tumor microenvironment (TME) consisting mainly of cancer-associated fibroblasts (CAFs) and a CAF-generated collagen-rich extracellular matrix (ECM). As the ECM has profound impacts on tumor progression and therapy response, it is critical that we understand the mechanisms underlying ECM deposition and remodeling. In addition to a highly fibrotic and reactive TME, a hallmark of PDAC is pain. 93% of PDAC patients experience pain, and [~]70% are prescribed opioids for pain management during the course of their cancer treatment. Despite epidemiological evidence linking opioid use with diminished patient survival, how opioids impact tumor biology remains largely unknown. We now provide evidence that both endogenous and exogenous opioids drive ECM remodeling in the PDAC TME. We find that the commonly prescribed opioid morphine promotes the development of poorly differentiated tumors and increases collagen bundling and maturation in a mouse model of PDAC. Accordingly, RNA sequencing reveals that morphine induces significant upregulation of ECM genes and collagen modifying enzymes. We developed a morphine-induced gene signature which correlates significantly with the basal/mesenchymal subtypes of human PDAC and predicts worse overall survival in PDAC and other tumor types. Mechanistically, pharmacological inhibition and genetic knockdown of the mu opioid receptor (OPRM1) in CAFs attenuates expression of type 1a and type 3a collagens, and the myofibroblastic CAF marker alpha-SMA, demonstrating that opioid signaling is a direct regulator of CAF biology. Additionally, we provide the first evidence that CAFs produce endogenous opioids capable of activating OPRM1 and driving collagen expression. Finally, treatment with the FDA-approved peripherally restricted OPRM1 antagonist methylnaltrexone (MNTX) reduces desmoplasia, tumor weight, and ascites burden in a mouse model of PDAC. Therefore, we have identified a novel opioid-mediated signaling axis driving PDAC desmoplasia and reveal MNTX as a potential therapeutic to inhibit both exogenous and endogenous opioid-induced ECM remodeling and tumor aggressiveness.

cancer biology↗

bioRxiv2026

Mycobacteriophage TM4 requires XylR for successful infection in Mycobacterium smegmatis mc2155

Mycobacteriophages are viruses that infect mycobacteria, including Mycobacterium tuberculosis, and have emerged as promising alternatives to antibiotics in the face of increasing antimicrobial resistance. However, evolution of phage-resistance remains a major challenge to the clinical implementation of phage therapy. We describe a TM4 phage resistant mutant in M. smegmatis harboring an in-frame deletion in the xylR, transcriptional regulator implicated in lipid metabolism, and cell envelope homeostasis. Using spontaneous mutagenesis, transcriptomics and biochemical approaches, we identify a previously uncharacterized resistance mechanism mediated by cell-envelope remodeling that impedes productive phage infection. The xylR mutation disrupted phage DNA injection through enhanced recruitment of lipooligosaccharides to the cell surface, without inhibiting phage adsorption, genome replication, and virion assembly. Remodeling of the cell envelope was further enhanced by the induction of lipooligosaccharide biosynthesis upon TM4 infection, however, the phenotype can be reverted through chemical treatment, restoring phage sensitivity. Our study expands the paradigm of innate mechanisms underlying broad-spectrum phage resistance in mycobacteria.

microbiology↗

bioRxiv2026

Phytophthora cinnamomi populations collected from avocado in the United States exhibit high adaptive capacity to climate and disease control methods

Phytophthora cinnamomi, the causal agent of Phytophthora root rot (PRR), poses a persistent threat to the United States avocado industry, the top domestic producer and consumer. Avocado growers are facing clonal A2 P. cinnamomi populations challenging their current PRR control methods. In this study, we characterized 125 isolates collected from orchards in California, Florida, Hawaii, Texas, and Puerto Rico for radial growth per day, optimal growth temperature, in vitro fungicide sensitivity, and virulence on DAnjou pear fruit and UC2001 avocado seedlings. Across all isolates, optimal growth occurred most frequently at a range from 22 to 25{degrees}C; however, a subset of isolates from Hawaii, Florida, and California exhibited higher optimal growth temperatures (28{degrees}C and 30{degrees}C) suggesting thermal adaptation in warmer regions. Potassium phosphite EC50 values spanned from 4.61 to 763.13 {micro}g/ml, with significantly higher insensitivity in isolates from California and Florida, reflecting the continued overuse of this fungicide in these major production states. In contrast, baseline sensitivities to ethaboxam, mandipropamid, mefenoxam, fluopicolide, and oxathiapiprolin were uniformly high, with narrow, unimodal EC50 distributions across states. Finally, a wide range of virulence among isolates was detected using avocado seedlings and DAnjou pear fruits with isolates from California and Puerto Rico being the most virulent. Together, this data documents extensive phenotypic diversity within clonal A2 P. cinnamomi populations including heat-adapted and phosphite-insensitive lineages, establishes multi-state fungicide sensitivity baselines, and underscores the need for continued surveillance, integrated fungicide stewardship (especially phosphonates), and rootstock screening against phenotypically diverse populations to sustain avocado PRR management and ensure the United States avocado industry sustainability and profitability.

microbiology↗

bioRxiv2025

Profiling a large HIV-1 elite neutralizer cohort reveals remarkable CD4bs bNAb for HIV-1 prevention and therapy

Administration of HIV-1 neutralizing antibodies can suppress viremia and prevent infection in vivo. However, clinical use is challenged by broad envelope sequence diversity and rapid emergence of viral escape1-9. Here, we performed single B cell profiling of 32 top HIV-1 elite neutralizers to identify broadly neutralizing antibodies (bNAbs) with highest potency and breadth for clinical application. From 831 expressed monoclonal antibodies, we identified 04_A06, a new VH1-2-encoded CD4 binding site bNAb with remarkable breadth and potency against extended multiclade pseudovirus panels (GeoMean IC50 = 0.059 {micro}g/ml, breadth = 98.5%, 332 virus strains). Moreover, 04_A06 was not susceptible to classic viral CD4bs escape variants and maintained full viral suppression in HIV-1-infected humanized mice. Structural analyses revealed that antiviral activity is mediated by an unusually long 11-amino acid heavy chain insertion. This insertion facilitates inter-protomer contacts and interactions with highly conserved residues on the adjacent gp120 protomer. Finally, 04_A06 demonstrated high activity against contemporaneously circulating viruses from the Antibody Mediated Prevention (AMP) trials (GeoMean IC50 = 0.082 {micro}g/ml, breadth = 98.4%, 191 virus strains) and in silico modeling for 04_A06LS predicted HIV-1 prevention efficacy of >93%. Thus, 04_A06 will provide unique opportunities for effective treatment and prevention strategies of HIV-1 infection.

immunology↗

bioRxiv2025

Genome editing of papaya using both Cas9 and Cas12a

Papaya (Carica papaya L.) is an economically important tropical crop that produces papain and highly nutritious fruit, which are used in the grocery, cosmetic, pharmaceutical, and food processing industries. However, various destructive pathogens severely threaten its production. Furthermore, limited natural genetic variation restricts breeding efforts for crop improvement. Therefore, we turned to gene editing as a tool to address these problems. We utilized two CRISPR systems (Cas9 and Cas12a) and two papaya genes, CpPDS (phytoene desaturase) and CpMLO6 (Mildew Locus O 6), to establish efficient genome editing systems of papaya. The systems were delivered by an optimized protocol of Agrobacterium-mediated transformation (AMT) of embryogenic callus suspension cultures derived from hypocotyls. Accordingly, we transformed papaya with five plasmid constructs, each of which expressed one or two guide RNAs (gRNAs) for gene editing using either Cas9 or Cas12a. All except two T0 transgenic plants tested produced mutations with the majority containing indels of over 90%. Furthermore, successful mutation of the CpPDS gene using both Cas9 and Cas12a produced albino phenotypes as expected for disrupting a gene for carotenoid biosynthesis. Successful mutagenesis was achieved with seven out of eight gRNAs. Homozygous and/or biallelic mutants were generated from transformation using all five constructs, suggesting the feasibility of obtaining transgene-free homozygous segregating mutants by selfing in the second generation. Taken together, a robust and reliable papaya genome editing system was established, which enables genetic modification in various genomic environments to meet the diverse needs of basic scientific research and tropical crop improvement.

plant biology↗

bioRxiv2025

End-product inhibition of the LRRK2-counteracting PPM1H phosphatase

PPM1H phosphatase reverses Parkinsons disease-associated, Leucine Rich Repeat Kinase 2-mediated, Rab GTPase phosphorylation. We showed previously that PPM1H relies on an N-terminal amphipathic helix for Golgi membrane localization and this helix enables PPM1H to associate with liposomes in vitro; binding to highly curved liposomes activates PPM1Hs phosphatase activity. We show here that PPM1H also contains an allosteric binding site for its non-phosphorylated reaction products, Rab8A and Rab10. Microscale thermophoresis revealed that PPM1H binds thio-phosphorylated Rab8A at the active site with a KD of [~]1{micro}M; binding of Rab8A and Rab10 to an alternative site is of similar affinity and is not detected for another LRRK2 substrate, Rab12. Non-phosphorylated Rab8A or Rab10 inhibit PPM1H phosphatase reactions at concentrations consistent with their measured binding affinities and fail to inhibit PPM1H L66R phosphatase reactions. Independent confirmation of non-phosphorylated Rab binding to PPM1H was obtained by sucrose gradient co-flotation of non-phosphorylated Rabs with liposome-bound PPM1H. Finally, Rab8A or Rab10 binding also requires PPM1Hs amphipathic helix, without which the interaction affinity is decreased about 6-fold. These experiments indicate that Golgi associated Rab proteins contribute to the localization of PPM1H and non-phosphorylated Rabs regulate PPM1H phosphatase activity via an allosteric site. Targeting this site could represent a strategy to enhance PPM1H-mediated dephosphorylation of LRRK2 substrates, offering a potential therapeutic approach to counteract LRRK2-driven Parkinsons disease.

biochemistry↗

bioRxiv2025

PPM1M, a LRRK2-counteracting, phosphoRab12-preferring phosphatase with potential link to Parkinsons disease

Leucine-rich repeat kinase 2 (LRRK2) phosphorylates a subset of Rab GTPases that regulate receptor trafficking; activating mutations in LRRK2 are linked to Parkinsons disease. Rab phosphorylation is a transient event that can be reversed by phosphatases, including PPM1H, that acts on phosphoRab8A and phosphoRab10. Here we report a phosphatome-wide siRNA screen that identified PPM1M as a phosphoRab12-preferring phosphatase that also acts on phosphoRab8A and phosphoRab10. Upon knockout from cells or mice, PPM1M displays selectivity for phosphoRab12. As shown previously for mice harboring LRRK2 pathway mutations, knockout of Ppm1m leads to primary cilia loss in striatal cholinergic interneurons. We have also identified a rare PPM1M mutation in patients with Parkinsons disease that is catalytically inactive when tested in vitro and in cells. These findings identify PPM1M as a key player in the LRRK2 signaling pathway and provide a new therapeutic target for the possible benefit of patients with Parkinsons disease. TeaserParkinsons linked Rab phosphorylation is reversed by PPM1M; the inactive D440N variant is implicated in rare patient cases.

cell biology↗

bioRxiv2025

Lysosomal Glucocerebrosidase is needed for ciliary Hedgehog signaling: A convergent pathway to Parkinsons disease

Mutations in LRRK2 and GBA1 are the most common genetic causes of familial Parkinsons disease. Previously, we showed that pathogenic LRRK2 mutations inhibit primary cilia formation in rare interneurons and astrocytes of the mouse and human dorsal striatum. This blocks Hedgehog signaling and reduces synthesis of neuroprotective GDNF and NRTN, which support neurons vulnerable in PD. Here we show that GBA1 mutations also impair Hedgehog signaling through a distinct mechanism. Loss of GBA1 activity decreases accessible cholesterol in primary cilia of cultured cells, thereby disrupting Hedgehog signaling. In the mouse striatum, Gba1 mutations result in reduced Hedgehog-induced Gdnf RNA expression in cholinergic interneurons, despite having no detectable impact on cilia formation. Also, both Lrrk2 and Gba1 mutations suppress Hedgehog-induced Bdnf expression in striatal astrocytes. These findings underscore the role of Hedgehog signaling in the nigrostriatal circuit and reveal a convergent mechanism by which distinct mutations may contribute to PD pathogenesis.

neuroscience↗

bioRxiv2025

Pathogen and pest communities in food security crops across climate gradients: Anticipating future challenges in the Great Lakes region of Africa

CONTEXTTropical cropping systems must adapt to the current and future geographic distribution of pathogen and pest communities. An important research gap is how climate change may shift the distribution of pathogens and pests in tropical lowlands and highlands. OBJECTIVEWe evaluated the current geographic risk of 27 pathogens and pests in the production of four food security crops (banana, cassava, potato, and sweetpotato) in the Great Lakes region of Africa, and the potential future risk under climate change. Models for each pathogen and pest indicate the potential for changes in geographic distribution, with model fit indicating the potential for decision support systems to facilitate management. METHODSFirst, cropland connectivity analysis identified locations likely important in the spread of crop-specific pathogens and pests, such as locations in Rwanda and Burundi. Second, we surveyed the 27 economically important pathogens and pests in Rwanda and Burundi, mapping the distribution of each across climate gradients and quantifying patterns of association. Third, we used machine learning to develop models of each species as a function of environmental variables, including host landscape variables. We also evaluated the increase in temperature across altitudes under future climate change scenarios in this region. RESULTS AND CONCLUSIONSAmong the ten machine-learning algorithms evaluated, random forests and support vector machines generally performed best for predicting severity and infestation. Host landscape variables were useful predictors for some species. Based on climate matching, 44% of the pathogens and pests could become more common with warmer temperatures at higher altitudes, while 17% may become less common. SIGNIFICANCEThis study indicates adaptation priorities for crop health in a region with multiple challenges to agricultural sustainability. The models developed here also indicate which species may have more potential and relevance for future development of pathogen and pest forecasts.

ecology↗

bioRxiv2024

Genome-guided, field-deployable loop-mediated isothermal amplification (LAMP) assay for specific detection of Dickeya dadantii

Potatoes, among the most economically significant crops worldwide, are susceptible to various plant pathogens that significantly impact their propagation, production, storage, and distribution. Soft rot disease, caused primarily by Dickeya and Pectobacterium, results in substantial economic losses to the agricultural industry annually. In this study, we developed a rapid, reliable, and field-deployable loop-mediated isothermal amplification (LAMP) assay for detecting D. dadantii, a common soft rot causing bacteria. The D. dadantii-specific LAMP primers were designed targeting a highly conserved genomic region within D. dadantii, the TetR/AcrR family transcriptional regulator CDS and its flanking sequences. This assay was thoroughly validated with the members of inclusivity (nine strains of D. dadantii) and exclusivity panels (85 strains, including all Dickeya species, related taxa, and host DNA), detecting no false positives or negatives. The limit of detection (LOD) was established by performing assays with 10-fold serially diluted pure gDNA of D. dadantii and gDNA spiked with host crude extract; the assay detected the target pathogen down to 1 pg (188 copies) without being adversely affected by the host crude extract. The developed LAMP assay specifically detected the target pathogen in infected plant materials. Additional multi-operator blind and multi-instrument tests were conducted to assess the assays robustness and applicability, consistently yielding accurate results without false positives or negatives. These findings demonstrate the assays potential utility for biosecurity, routine diagnostics, and epidemiological studies.

microbiology↗

bioRxiv2024

In silico exploration of potent flavonoids for dengue therapeutics

Dengue poses a persistent and widespread threat with no effective antiviral drug available till now. Several inhibitors have been developed by targeting the viral non-structural proteins including methyl transferase (NS5) of the dengue virus with possible therapeutic values. In this work, virtual screening, molecular docking, molecular dynamics simulations (200 ns), and assessments of free energy changes to identify potential candidates from a database of flavonoids (ca. 2000) that may have good curative potential from the disease. The binding affinity of flavonoids, namely Genistein-7-glucoside (FLD1), 6-O-Acetylgenistin (FLD2), 5,6-dihydroxy-2-(4-hydroxyphenyl)-7-[3,4,5-trihydroxy-6-(hydroxymethyl)oxane-2-yl]oxychromen-4-one (FLD3), Glucoliquiritigenin (FLD4), and Chrysin-7-O-glucoronide (FLD5) showed the binding affinities of -10.2, -10.2, -10.1, -10.1, -9.9 kcal/mol, respectively, and possessed better values than that of the native ligand with showed (-7.6 kcal/mol) and diclofenac sodium (-7.3 kcal/mol). Drug-likeness of these compounds was acceptable and no toxicity was hinted by ADMET predictions. The stability of the protein-ligand complexes was accessed from 200 ns molecular dynamics simulation in terms of various geometrical parameters; RMSD, RMSF of residues, Rg, SASA, and H-bond of the protein-ligand complexes. The binding free energy changes of these compounds were calculated by the MM-PBSA solvation model with negative values (less than -38.01{+/-}7.53 kcal/mol) indicating the spontaneity of the forward reaction and favorability of the product formation. The geometrical and thermodynamic parameters infer that the flavonoid binds at the orthosteric site of the target protein of DENV-2 and could inhibit its functioning resulting in the prevention of the disease. Overall, this study highlights the anti-DENV activity of five flavonoids, positioning them as promising candidates for further development as antiviral agents against dengue infection.

bioinformatics↗

bioRxiv2023

The Natural Products Discovery Center: Release of the First 8490 Sequenced Strains for Exploring Actinobacteria Biosynthetic Diversity

Actinobacteria, the bacterial phylum most renowned for natural product discovery, has been established as a valuable source for drug discovery and biotechnology but is underrepresented within accessible genome and strain collections. Herein, we introduce the Natural Products Discovery Center (NPDC), featuring 122,449 strains assembled over eight decades, the genomes of the first 8490 NPDC strains (7142 Actinobacteria), and the online NPDC Portal making both strains and genomes publicly available. A comparative survey of RefSeq and NPDC Actinobacteria highlights the taxonomic and biosynthetic diversity within the NPDC collection, including three new genera, hundreds of new species, and [~]7000 new gene cluster families. Selected examples demonstrate how the NPDC Portals strain metadata, genomes, and biosynthetic gene clusters can be leveraged using genome mining approaches. Our findings underscore the ongoing significance of Actinobacteria in natural product discovery, and the NPDC serves as an unparalleled resource for both Actinobacteria strains and genomes.

microbiology↗

bioRxiv2023

A dual-pathway architecture enables chronic stress to promote habit formation

Chronic stress can change how we learn and, thus, how we make decisions. Here we investigated the neuronal circuit mechanisms that enable this. Using a multifaceted systems neuroscience approach in male and female mice, we reveal a dual pathway, amygdala-striatal neuronal circuit architecture by which a recent history of chronic stress disrupts the action-outcome learning underlying adaptive agency and promotes the formation of inflexible habits. We found that the basolateral amygdala projection to the dorsomedial striatum is activated by rewarding events to support the action-outcome learning needed for flexible, goal-directed decision making. Chronic stress attenuates this to disrupt action-outcome learning and, therefore, agency. Conversely, the central amygdala projection to the dorsomedial striatum mediates habit formation. Following stress this pathway is progressively recruited to learning to promote the premature formation of inflexible habits. Thus, stress exerts opposing effects on two amygdala-striatal pathways to disrupt agency and promote habit. These data provide neuronal circuit insights into how chronic stress shapes learning and decision making, and help understand how stress can lead to the disrupted decision making and pathological habits that characterize substance use disorders and mental health conditions.

neuroscience↗

bioRxiv2023

Hyperexcitability and translational phenotypes in a preclinical model of SYNGAP1 mutations

SYNGAP1 is a critical gene for neuronal development, synaptic structure, and function. Although rare, the disruption of SYNGAP1 directly causes a genetically identifiable neurodevelopmental disorder (NDD) called SYNGAP1-related intellectual disability. Without functional SynGAP1 protein, patients present with intellectual disability, motor impairments, and epilepsy. Previous work using mouse models with a variety of germline and conditional mutations has helped delineate SynGAP1s critical roles in neuronal structure and function, as well as key biochemical signaling pathways essential to synapse integrity. Homozygous loss of SYNGAP1 is embryonically lethal. Heterozygous mutations of SynGAP1 result in a broad range of phenotypes including increased locomotor activity, impaired working spatial memory, impaired cued fear memory, and increased stereotypic behavior. Our in vivo functional data, using the original germline mutation mouse line from the Huganir laboratory, corroborated robust hyperactivity and learning and memory deficits. Here, we describe impairments in the translational biomarker domain of sleep, characterized using neurophysiological data collected with wireless telemetric electroencephalography (EEG). We discovered Syngap1+/- mice exhibited elevated spike trains in both number and duration, in addition to elevated power, most notably in the delta power band. Primary neurons from Syngap1+/- mice displayed increased network firing activity, greater spikes per burst, and shorter inter-burst intervals between peaks using high density micro-electrode arrays (HD-MEA). This work is translational, innovative, and highly significant as it outlines functional impairments in Syngap1 mutant mice. Simultaneously, the work utilized untethered, wireless neurophysiology that can discover potential biomarkers of Syngap1R-ID, for clinical trials, as it has done with other NDDs. Our work is substantial forward progress toward translational work for SynGAP1R-ID as it bridges in-vitro electrophysiological neuronal activity and function with in vivo neurophysiological brain activity and function. These data elucidate multiple quantitative, translational biomarkers in vivo and in vitro for the development of treatments for SYNGAP1-related intellectual disability.

neuroscience↗

bioRxiv2023

Localization of PPM1H phosphatase tunes Parkinsons disease-linked LRRK2 kinase-mediated Rab GTPase phosphorylation and ciliogenesis

PPM1H phosphatase reverses Parkinsons disease-associated, LRRK2-mediated Rab GTPase phosphorylation. We show here that PPM1H relies on an N-terminal amphipathic helix for Golgi localization. The amphipathic helix enables PPM1H to bind to liposomes in vitro, and small, highly curved liposomes stimulate PPM1H activity. We artificially anchored PPM1H to the Golgi, mitochondria, or mother centriole. Our data show that regulation of Rab10 GTPase phosphorylation requires PPM1H access to Rab10 at or near the mother centriole. Moreover, poor co-localization of Rab12 explains in part why it is a poor substrate for PPM1H in cells but not in vitro. These data support a model in which localization drives PPM1H substrate selection and centriolar PPM1H is critical for regulation of Rab GTPase-regulated ciliogenesis. Moreover, Golgi localized PPM1H maintains active Rab GTPases on the Golgi to carry out their non-ciliogenesis-related functions in membrane trafficking. Significance StatementPathogenic, hyperactive LRRK2 kinase is strongly linked to Parkinsons disease and LRRK2 phosphorylates a subset of Rab GTPases that are master regulators of membrane trafficking. PPM1H phosphatase specifically dephosphorylates Rab8A and Rab10, the major LRRK2 substrates. Here we provide novel cell biological and biochemical insight related to the localization and activation of PPM1H phosphatase. Understanding how PPM1H modulates LRRK2 activity is of fundamental interest and also important, as activators of PPM1H may eventually benefit Parkinsons disease patients.

cell biology↗

bioRxiv2023

The landscape of tolerated genetic variation in humans and primates

Personalized genome sequencing has revealed millions of genetic differences between individuals, but our understanding of their clinical relevance remains largely incomplete. To systematically decipher the effects of human genetic variants, we obtained whole genome sequencing data for 809 individuals from 233 primate species, and identified 4.3 million common protein-altering variants with orthologs in human. We show that these variants can be inferred to have non-deleterious effects in human based on their presence at high allele frequencies in other primate populations. We use this resource to classify 6% of all possible human protein-altering variants as likely benign and impute the pathogenicity of the remaining 94% of variants with deep learning, achieving state-of-the-art accuracy for diagnosing pathogenic variants in patients with genetic diseases. One Sentence SummaryDeep learning classifier trained on 4.3 million common primate missense variants predicts variant pathogenicity in humans.

genomics↗

bioRxiv2023

Integrated Constraint-Based Modeling of E. coli Cell-Free Protein Synthesis

Cell-free protein expression has become a widely used research tool in systems and synthetic biology and a promising technology for protein biomanufacturing. Cell-free protein synthesis relies on in-vitro transcription and translation processes to produce a protein of interest. However, transcription and translation depend upon the operation of complex metabolic pathways for precursor and energy regeneration. Toward understanding the role of metabolism in a cell-free system, we developed a dynamic constraint-based simulation of protein production in the myTXTL E. coli cell-free system with and without electron transport chain inhibitors. Time-resolved absolute metabolite measurements for [M] = 63 metabolites, along with absolute concentration measurements of the mRNA and protein abundance and measurements of enzyme activity, were integrated with kinetic and enzyme abundance information to simulate the time evolution of metabolic flux and protein production with and without inhibitors. The metabolic flux distribution estimated by the model, along with the experimental metabolite and enzyme activity data, suggested that the myTXTL cell-free system has an active central carbon metabolism with glutamate powering the TCA cycle. Further, the electron transport chain inhibitor studies suggested the presence of oxidative phosphorylation activity in the myTXTL cell-free system; the oxidative phosphorylation inhibitors provided biochemical evidence that myTXTL relied, at least partially, on oxidative phosphorylation to generate the energy required to sustain transcription and translation for a 16-hour batch reaction.

synthetic biology↗

bioRxiv2023

Modeling and Analysis of a Cell-Free Gluconate Responsive Biosensor

Cell-free synthetic systems are composed of the parts required for transcription and translation processes in a buffered solution. Thus, unlike living cells, cell-free systems are amenable to rapid adjustment of the reaction composition and easy sampling. Further, because cellular growth and maintenance requirements are absent, all resources can go toward synthesizing the product of interest. Recent improvement in key performance metrics, such as yield, reaction duration, and portability, has increased the space of possible applications open to cell-free systems and lowered the time required to design-build-test new circuitry. One promising application area is biosensing. This study describes developing and modeling a D-gluconate biosensor circuit operating in a reconstituted cell-free system. Model parameters were estimated using time-resolved measurements of the mRNA and protein concentration with and without the addition of D-gluconate. Sensor performance was predicted using the model for D-gluconate concentrations not used in model training. The model predicted the transcription and translation kinetics and the dose response of the circuit over several orders of magnitude of D-gluconate concentration. Global sensitivity analysis of the model parameters gave detailed insight into the operation of the sensor circuit. Taken together, this study reported an in-depth, systems-level analysis of a D-gluconate biosensor circuit operating in a reconstituted cell-free system. This circuit could be used directly to estimate D-gluconate or as a subsystem in a more extensive synthetic gene expression program.

synthetic biology↗