Biology subjects

Zuniga, A.

Publications and source records attributed to Zuniga, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Operando Failure Diagnosis and Performance Dynamics in Microbial Fuel Cells Treating Mine Waste

Artisanal gold-mining tailings carry dissolved mercury, copper and lead at concentrations requiring treatment before discharge. Bench-scale microbial fuel cells (MFCs) offer passive treatment of such streams, but exhibit operational variability that obscures whether removal is biologically driven. This study applies an integrated diagnostic framework to two bench-scale systems treating tailings from two artisanal gold-mining districts in Ecuador. System 1 evaluated Micractinium inermum bio-augmentation, while System 2 compared Psychrobacter alimentarius- and Trichococcus patagoniensis-dominated anodic consortia. The framework combines paired time-series statistical modeling, an adaptive percentile-floor change-point detector, equivalent-circuit modeling, and baseline-corrected XRD/FTIR spectroscopy. In System 1, treatment reduced mercury from 0.042 to 0.0027-0.0148 mg L^-1 and copper from 2.79 to 0.130-1.837 mg L^-1. Against the applicable discharge limits, copper complied in six of eight reactor--phase combinations, whereas mercury exceeded its limit throughout, identifying it as the limiting contaminant. Removal was governed by system-level physicochemical processes rather than algal-specific biocatalysis, with the non-algal control matching or exceeding the augmented reactors. Paired per-day testing showed that algal bio-augmentation conferred no voltage advantage under stable operation (+0.17%) but a +27.54% gain under diurnal perturbation, periodicity analysis attributed these shifts to the 24-h chamber photoperiod. An adaptive change-point detector distinguished recoverable excursions from terminal structural collapse in both systems, and equivalent-circuit fitting attributed System 2 consortium power difference to internal resistance and open-circuit voltage. The framework separates biological treatment effects from mechanical failure, supporting automated health monitoring in bio-electrochemical wastewater treatment.

bioengineering↗

bioRxiv2024

Aniline Dioxygenase in Rhodococcus ruber R1: Insights into Skatole Degradation

Skatole is an aromatic heterocyclic compound with a strong offensive odor, produced by microorganisms during the anaerobic breakdown of tryptophan. Skatole accumulation is linked to environmental and health issues. Despite its persistence and harmful effects, skatoles biodegradation by microorganisms is poorly understood. We have recently isolated a gram-positive bacterium, Rhodococcus ruber R1, which uses skatole as its sole carbon and energy source. Here we report an operon consisting of 14 genes encoding aromatic oxygenase systems involved in skatole degradation in Rhodococcus ruber R1. Cells growing on skatole accumulate aniline transiently, indicating its role as an intermediate in the degradation pathway. We characterize six genes in this cluster that encode for an aniline dioxygenase, which converts aniline to catechol and is only activated in the presence of skatole. This gene cluster was successfully introduced into a heterologous strain enabling the full degradation of aniline and its derivatives. Phylogenetic analysis of aniline dioxygenase present in R1 strain reveals a widespread distribution of this system among bacteria, in contrast to the full skatole cluster, which is restricted to a few genera. These findings advance our understanding of the skatole degradation pathway and highlight R1s potential for bioremediation of skatole, aniline, and related contaminants.

microbiology↗

bioRxiv2024

WNT signaling coordinately controls mouse limb bud outgrowth and establishment of the digit-interdigit pattern

Self-organization, such as the emergence of a pattern from a homogenous state, is a fascinating property of biological systems. Early limb bud outgrowth and patterning in mice are controlled by a robust and self-regulatory signaling system, and initiation of the periodic digit-interdigit pattern appears under control of a self-regulatory Turing system. Previous studies established the requirement of WNT and BMP signaling for both early limb bud and digit-interdigit morphogenesis, but the molecular changes underlying the transition from early limb bud signaling to the digit-interdigit patterning system remained unknown. Here, we use small molecule inhibitors to rapidly but transiently block WNT signaling to identify the early transcriptional targets that are altered during disruption and recovery of limb bud and digit development. Together, this study highlights the overarching role of WNT signaling in controlling early limb bud outgrowth and patterning, and establishment of the periodic digit-interdigit pattern. Finally, the transient WNT signaling disruption approach reveals the plasticity and robustness of these self-organizing limb bud and digit patterning systems.

developmental biology↗

bioRxiv2024

Single-cell profiling of penta- and tetradactyl mouse limb buds identifies mesenchymal progenitors controlling digit numbers and identities

The cellular mechanisms controlling digit numbers and identities have remained elusive. Profiling of wild-type (pentadactyl) and Grem1 tetradactyl mouse limb buds identifies cellular changes affecting two limb bud mesenchymal progenitor (LMP) populations. In mutant limb buds, the anteriorly biased distribution of peripheral LMPs (pLMPs) is lost and the population expanded, while the distal-posterior LMP (dLMP) population is reduced from early stages onward. Analysis of LMP signature genes in wildtype and mutant mouse limb buds with digit loss or gain establishes that pLMPs are positively regulated by BMP signaling, while dLMPs require GREM1-mediated BMP antagonism. dLMPs encompass digit progenitors and altering their population size prefigures changes in digit numbers. The anteriorly biased pLMP distribution is linked to digit asymmetry as loss of this bias in tetradactyl mouse and pig limb buds underlies middle digit symmetry and identity loss. This study indicates that variable spatial Grem1 expression in mutant and evolutionary diversified limb buds tunes BMP activity, impacting both LMP populations in a complementary manner.

developmental biology↗

bioRxiv2024

Split minireporters facilitate monitoring of gene expression and peptide production in linear cell-free transcription-translation systems

Cell-free transcription-translation (TXTL) systems expressing genes from linear dsDNA enable rapid prototyping of genetic devices while avoiding cloning steps. However, repetitive inclusion of a reporter gene is an incompressible cost and sometimes accounts for most of the synthesized DNA length. Here we present minireporters based on split-GFP systems that reassemble into functional fluorescent proteins and can be used to monitor gene expression in E. coli TXTL. The 135 bp GFP10-11 fragment produces a fluorescent signal comparable to its full-length GFP counterpart when reassembling with its complementary protein synthesized from the 535 bp fragment expressed in TXTL. We show that minireporters can be used to characterize promoter libraries, with data qualitatively comparable to full-length GFP, and matching with in vivo expression measurements. We also use minireporters as small fusion tags to measure TXTL protein and peptide production yield. Finally, we generalize our concept by providing a luminescent minireporter based on split-nanoluciferase. The [~]80% gene sequence length reduction afforded by minireporters lowers synthesis costs and liberates space for testing larger devices while producing a reliable output. In the peptide production context, the small size of minireporters compared to full-length GFP is less likely to bias peptide solubility assays. We anticipate that minireporters will facilitate rapid and cost-efficient genetic device prototyping, protein production, and interaction assays.

synthetic biology↗

bioRxiv2024

TBX3 is essential to establish the posterior boundary of anterior genes and up-regulate posterior genes with HAND2 during onset of limb bud development

During limb bud formation, axes polarities are established as evidenced by the spatially restricted expression of key regulator genes. In particular, the mutually antagonistic interaction between the GLI3 repressor and HAND2 results in the distinct and non-overlapping anterior-distal Gli3 and posterior Hand2 expression domains. This hallmarks establishment of antero-posterior (AP) limb axis polarity together with spatially restricted expression of homeodomain and other transcriptional regulators. Here, we identify TBX3 as the transcription factor to initiate AP axis polarity in mouse limb buds. ChIP-seq and differential gene expression analysis of wildtype and mutant limb buds identifies the TBX3-specific and shared TBX3-HAND2 target genes. High sensitivity fluorescent whole mount in situ hybridisation shows that the posterior expression boundaries of anterior genes are positioned by TBX3-mediated repression, which excludes anterior genes such as Gli3, Alx4, Hand1 and Irx3/5 from the posterior limb bud mesenchyme. This exclusion delineates the posterior mesenchymal territory competent to establish the Shh-expressing limb bud organiser. In turn, HAND2 is required for Shh activation and cooperates with TBX3 to up-regulate shared posterior identity target genes in early limb buds.

developmental biology↗

bioRxiv2024

Extinction Training Suppresses Activity of Fear Memory Ensembles Across the Hippocampus and Alters Transcriptomes of Fear-Encoding Cells

Contextual fear conditioning has been shown to activate a set of "fear ensemble" cells in the hippocampal dentate gyrus (DG) whose reactivation is necessary and sufficient for expression of contextual fear. We previously demonstrated that extinction learning suppresses reactivation of these fear ensemble cells and activates a competing set of DG cells - the "extinction ensemble." Here, we tested whether extinction was sufficient to suppress reactivation in other regions and used single nucleus RNA sequencing (snRNA-seq) of cells in the dorsal dentate gyrus to examine how extinction affects the transcriptomic activity of fear ensemble and fear recall-activated cells. Our results confirm the suppressive effects of extinction in the dorsal and ventral dentate gyrus and demonstrate that this same effect extends to fear ensemble cells located in the dorsal CA1. Interestingly, the extinction-induced suppression of fear ensemble activity was not detected in ventral CA1. Our snRNA-seq analysis demonstrates that extinction training markedly changes transcription patterns in fear ensemble cells and that cells activated during recall of fear and recall of extinction have distinct transcriptomic profiles. Together, our results indicate that extinction training suppresses a broad portion of the fear ensemble in the hippocampus, and this suppression is accompanied by changes in the transcriptomes of fear ensemble cells and the emergence of a transcriptionally unique extinction ensemble.

neuroscience↗

bioRxiv2022

An Optimized Electrotransformation Protocol For Lactobacillus Jensenii.

Engineered bacteria are promising candidates for in situ detection and treatment of diseases. The female uro-genital tract presents several pathologies, such as sexually transmitted diseases or genital cancer, that could benefit from such technology. While bacteria from the gut microbiome are increasingly engineered, the use of chassis isolated from the female uro-genital resident flora has been limited. A major hurdle to implement the experimental throughput required for efficient engineering in these non-model bacteria is their low transformability. Here we report an optimized electrotransformation protocol for Lactobacillus jensenii, one the most widespread species across vaginal microflora. Starting from classical conditions, we optimized buffers, electric field parameters, cuvette type and DNA quantity to achieve an 80-fold improvement in transformation efficiency, with up to 3.5{middle dot}103 CFUs/{micro}g of DNA in L. jensenii ATCC 25258. We also identify several plasmids that are maintained and support reporter gene expression in L. jensenii. Finally, we demonstrate that our protocol provides increased transformability in three independent clinical isolates of L. jensenii. This work will facilitate the genetic engineering of L. jensenii and enable its use for addressing challenges in gynecological healthcare.

microbiology↗

bioRxiv2022

PeroxiHUB: a modular cell-free biosensing platform using H2O2 as signal integrator

Cell-free systems have great potential for delivering robust, cheap, and field-deployable biosensors. Many cell-free biosensors rely on transcription factors responding to small molecules, but their discovery and implementation still remain challenging. Here we report the engineering of PeroxiHUB, an optimized H2O2-centered sensing platform supporting cell-free detection of different metabolites. H2O2 is a central metabolite and a by-product of numerous enzymatic reactions. PeroxiHUB uses enzymatic transducers to convert metabolites of interest into H2O2, enabling rapid reprogramming of sensor specificity using alternative transducers. We first screen several transcription factors and optimize OxyR for the transcriptional response to H2O2 in cell-free, highlighting the need for pre-incubation steps to obtain suitable signal-to-noise ratios. We then demonstrate modular detection of metabolites of clinical interest - lactate, sarcosine, and choline - using different transducers mined via a custom retro-synthesis workflow publicly available on the SynBioCAD Galaxy portal. We find that expressing the transducer during the pre-incubation step is crucial for optimal sensor operation. Finally, we show that different reporters can be connected to PeroxiHUB, providing high adaptability for various applications. Given the wide range of enzymatic reactions producing H2O2, the PeroxiHUB platform will support cell-free detection of a large number of metabolites in a modular and scalable fashion.

synthetic biology↗

bioRxiv2022

A spatio-temporally constrained gene regulatory network directed by PBX1/2 acquires limb patterning specificity via HAND2

During development cell fates are specified by tightly controlled gene expression programs. PBX TALE transcription factors control gene regulatory networks (GRN) that direct vertebrate tissue patterning and organ morphogenesis. How PBX1/2 proteins acquire context-specific functions, despite widespread embryonic expression of Pbx1/2, remains elusive. In mouse limb buds, mesenchymal-specific loss of PBX1/2 or of the transcriptional regulator HAND2 results in similar phenotypes, suggesting that PBX1/2- and HAND2-dependent programs converge to control limb development. To investigate this scenario, we combined tissue-specific and temporally-controlled mutagenesis with multi-omics approaches using the murine hindlimb bud as a model. We reconstructed a GRN collaboratively directed by PBX1/2 and HAND2, demonstrating that Pbx1-Hand2 genetically interact in vivo during hindlimb patterning, with PBX1 concomitantly acting as an upstream regulator of Hand2. At organismal-level resolution the GRN is active within restricted subsets of posterior-proximal hindlimb mesenchymal cells, wherein Pbx1/2 and Hand2 are co-expressed with their target genes. Genome-wide profiling of PBX1 binding across multiple tissues further revealed that HAND2 selects a subset of PBX-bound regions to impart limb patterning functionality. This research elucidates mechanisms underlying limb bud-specific functions by PBX1/2, while informing general principles by which promiscuous transcription factors cooperate with select cofactors to instruct distinct developmental programs.

developmental biology↗

bioRxiv2022

Rapid prototyping of metabolites detection by bacterial biosensors in human fecal samples.

Gut metabolites are pivotal mediators of host-microbiome interactions and provide an important window on human physiology and disease. However, current methods to monitor gut metabolites rely on heavy and expensive technologies such as liquid chromatography-mass spectrometry (LCMS). In that context, robust, fast, field-deployable, and cost-effective strategies for monitoring fecal metabolites would support large-scale functional studies and routine monitoring of metabolites biomarkers associated with pathological conditions. Living cells are an attractive option to engineer biosensors due to their ability to detect and process many environmental signals and their self-replicating nature. Here we optimized a protocol for feces processing and gut metabolites detection using bacterial biosensors (bactosensors), enabling rapid evaluation of their operational capacity in these samples. We show that a simple filtration step is enough to remove host microbes and reproducibly obtain a physiological-derived media retaining important characteristics of human feces, such as matrix effects and endogenous metabolites. We measured how fecal samples affect the performance of biosensors for benzoate, lactate, anhydrotetracycline, and bile acids, and found that bactosensors are highly sensitive to fecal matrices. Sensitivity to the matrix is biosensor-dependent but also varies between individuals, highlighting the need for case-by-case optimization for bactosensors operation in feces. Finally, by detecting endogenous bile acids, we demonstrate that bactosensor can be used for metabolites monitoring in feces. This work lays the foundation for the optimization and use of bacterial biosensors for fecal metabolites monitoring. In the future, our method could also allow rapid pre-prototyping of engineered bacteria designed to operate in the gut, with applications to in situ diagnostics and therapeutics.

synthetic biology↗

bioRxiv2021

SMAD4 target genes are part of a transcriptional network that integrates the response to BMP and SHH signaling during early limb bud patterning

SMAD4 regulates gene expression in response to BMP and TGF{beta} signal transduction and is required for diverse morphogenetic processes, but its target genes have remained largely elusive. Here, we use an epitope-tagged Smad4 allele for ChIP-seq analysis together with transcriptome analysis of wild-type and mouse forelimb buds lacking Smad4 in the mesenchyme. This analysis identifies the SMAD4 target genes during establishment of the feedback signaling system and establishes that SMAD4 predominantly mediates BMP signal-transduction during early limb bud development. Unexpectedly, the initial analysis reveals that the expression of cholesterol biosynthesis enzymes is precociously down-regulated and intracellular cholesterol levels reduced in Smad4-deficient limb bud mesenchymal progenitors. The SMAD4 target GRNs includes genes, whose expression in the anterior limb bud is up-regulated by interactions of SMAD4 complexes with enhancers active in the anterior mesenchyme. This reveals a predominant function of SMAD4 in up-regulating target gene expression in the anterior limb bud mesenchyme. Analysis of differentially expressed genes that are shared between Smad4- and Shh-deficient limb buds corroborates the positive role of SMAD4 in transcriptional regulation of anterior genes and reveals a repressive effect on posterior genes that are positively regulated by SHH signaling. This analysis uncovers the overall opposing effects of SMAD4-mediated BMP and SHH signalling on transcriptional regulation during early limb bud development. In summary, this analysis indicates that during early digit patterning and limb bud outgrowth, the anterior/proximal and proximo/distal expression dynamics of co-regulated genes are controlled by distinct and contrasting trans-regulatory inputs from SHH and SMAD4-mediated BMP signal transduction.

developmental biology↗

bioRxiv2021

The water-soluble chitosan derivative, N-methylene phosphonic chitosan, is an effective fungicide against the phytopathogen Fusarium eumartii

Chitosan has been considered an environmental-friendly polymer. However, its use in agriculture has not been extended yet due to its relatively low solubility in water. In an attempt to improve such chemical characteristics, a chitosan-derivative prepared by adding a phosphonic group to chitosan N-methylene phosphonic chitosan, NMPC, was obtained from shrimp fishing industry waste from Argentinean Patagonia. This study showed that NMPC had a fungicidal effect on the phytopathogenic fungus Fusarium solani f. sp. eumartii (F. eumartii). NMPC inhibited F. eumartti mycelial growth and spore germination with low IC50 values. In vivo studies showed that NMPC affected fungal membrane permeability, ROS production, and cell death. NMPC also exerted antifungal effects against two other phytopathogens, Botrytis cinerea, and Phytophthora infestans. NMPC did not affect tomato cell viability at the same doses applied to these phytopathogens. Furthermore, the selective cytotoxicity of NMPC could give it added value in its application as an antimicrobial agent in agriculture.

microbiology↗

bioRxiv2021

A synthetic receptor platform enables rapid and portable monitoring of liver dysfunction via engineered bacteria.

Bacterial biosensors, or bactosensors, are promising field-deployable agents for medical and environmental diagnostics. However, the lack of scalable frameworks to systematically program ligand detection limits their applications. Here we present a synthetic receptor platform, termed EMeRALD (Engineered Modularized Receptors Activated via Ligand-induced Dimerization) which supports the modular assembly of sensing modules onto a high-performance, generic signaling scaffold controlling gene expression in E. coli. We applied EMeRALD to detect bile salts, a biomarker of liver dysfunction, by repurposing sensing modules from enteropathogenic Vibrio species. We improved the sensitivity and lowered the limit-of-detection of the sensing module by directed evolution. We then engineered a colorimetric bactosensor detecting pathological bile salt levels in serum from patients having undergone liver transplant, providing an output detectable by the naked-eye. The EMeRALD technology enables functional exploration of natural sensing modules and rapid engineering of synthetic receptors for diagnostics, environmental monitoring, and control of therapeutic microbes.

synthetic biology↗

bioRxiv2021

An engineered lactate responding promoter system operating in glucose-rich and anaerobic environments.

Bacteria equipped with genetically-encoded lactate biosensors would support several applications in biopharmaceutical production, diagnosis, or therapeutics. However, many applications involve glucose-rich and anaerobic environments, in which current whole-cell lactate biosensors have low performance. Here we engineered a synthetic lactate biosensor system by repurposing the natural LldPRD promoter regulated by the LldR transcriptional regulator. We removed glucose catabolite repression by designing a hybrid promoter, containing LldR operators and tuned both regulator and reporter gene expression to optimize biosensor signal-to-noise ratio. The resulting lactate biosensor, termed ALPaGA (A Lactate Promoter Operating in Glucose and Anaerobia) can operate in glucose rich, aerobic and anaerobic conditions. Our work provides a versatile lactate biosensing platform suitable for many environmental conditions.

synthetic biology↗

bioRxiv2020

Arabidopsis thaliana interaction with Ensifer meliloti can support plant growth under N-deficiency

Nitrogen (N) is an essential macronutrient for plants. Some plant species obtain this nutrient by interacting with N-fixing bacteria. These beneficial interactions are well described in legumes but have also been observed in non-legume plant species that are unable to form root nodules. We studied the expanding role of beneficial plant-bacteria interactions for N-nutrition in the widely used model plant Arabidopsis thaliana. We found that the bacteria Ensifer meliloti enhanced A. thaliana growth under severe N-deficiency conditions, allowing plants to complete their life cycle. Our results showed that bacteria colonize the rhizosphere associated with the epidermis of the plant root. We also demonstrated that A. thaliana possesses genes that are critical for this beneficial interaction and are required for plant-growth promotion by E. meliloti under N-deficiency. This work shows association between A. thaliana and E. meliloti for plant nutrition under severe N-deficiency, and suggests that plants have conserved-molecular mechanisms to interact with N-fixing bacteria to procure N and escape adverse conditions. Under these circumstances, the supply of N via N-fixation is critical for survival, allowing the plant to complete its life cycle. Our findings provide a new framework and an experimental model system that expand our understanding of plant-rhizobia interactions for plant N-nutrition.

plant biology↗

bioRxiv2020

Gli3 utilizes Hand2 to synergistically regulate tissue-specific transcriptional networks

Despite a common understanding that Gli TFs are utilized to reiterate a Hh morphogen gradient, genetic analyses suggest craniofacial development does not completely fit this paradigm. We demonstrated that rather than being driven by a Hh threshold, robust Gli3 transcriptional activity during skeletal and glossal development required interaction with the bHLH TF Hand2. Not only did genetic and expression data support a co-factorial relationship, but genomic analysis further revealed that Gli3 and Hand2 were enriched at regulatory elements for genes essential for mandibular patterning and development. Interestingly, motif analysis at sites co-occupied by Gli3 and Hand2 uncovered mandibular-specific, low-affinity, divergent Gli binding motifs (dGBMs). Functional validation revealed these dGBMs conveyed synergistic activation of Gli targets essential for mandibular patterning and development. In summary, this work elucidates a novel, sequence-dependent mechanism for Gli transcriptional activity within the craniofacial complex that is independent of a graded Hh signal.

developmental biology↗

bioRxiv2019

Rational programming of history-dependent logic in cellular populations.

Genetic programs operating in an history-dependent fashion are ubiquitous in nature and govern sophisticated processes such as development and differentiation. The ability to systematically and predictably encode such programs would advance the engineering of synthetic organisms and ecosystems with rich signal processing abilities. Here we implement robust, scalable history-dependent programs by distributing the computational labor across a cellular population. Our design is based on recombinase-driven DNA scaffolds expressing different genes according to the order of occurrence of inputs. These multicellular computing systems are highly modular and any program can be built by differential composition of strains containing well-characterized logic scaffolds. We developed an automated workflow that researchers can use to streamline program design and optimization. We anticipate that the history-dependent programs presented here will support many applications using cellular populations for material engineering, biomanufacturing and healthcare.\n\nOne Sentence SummarySystematic and automated frameworks for implementing robust history-dependent genetic programs in cellular populations.

synthetic biology↗