Biology subjects

Jimenez, M.

Publications and source records attributed to Jimenez, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Passive Cell-Flux Partitioning Enables Single-Cell-Constrained Impedance Sensing From High-Density Microbial Cultures

Fermentation is a process through which microorganisms are used to transform basic molecules into high value substances in various applications such as biopharma, food production, and biofuel generation. In such processes, monitoring the health of microbial cells is essential, as it is a key factor in the final product yield. Traditional methods of assessing the health of fermenter populations have involved manually extracting samples from the fermenter for benchtop testing, which lacked the accuracy and efficacy of real-time continuous measurements. This has opened the door for inline sensors to be integrated into fermenters for continuous measurement; including microfluidic based Electrochemical Impedance Spectroscopy (EIS). EIS utilizes a range of frequencies to gain insight into the characteristics of microbial populations, such as cell size and membrane capacitance. EIS cytometry, in particular, has emerged as a relevant tool, as it enables single-cell level detection in real-time. However, current EIS cytometry devices cannot be deployed directly within fermenters due to the high cell density conditions. This has limited EIS analysis in fermenters to bulk measurements averaged across a population. Here, we present a microfluidic chip that uses passive cell-flux-partitioning geometry to direct cells from high-density culture through a single-cell-constrained flow path containing integrated impedance-sensing electrodes. The proposed chip combines geometric channel design and embedded electrodes to enable continuous impedance measurements from cell-containing flow while operating at input cell densities on the scale of 10^9 CFU/mL. This work demonstrates a first step toward integrating single-cell-constrained microfluidic handling with impedance sensing for inline monitoring in industrial biomanufacturing.

bioengineering↗

bioRxiv2026

Functional connectivity scaffolds the emergence of the visual word form area

Complex cognitive tasks, like reading, require coordinated computations across distant cortical regions. Connectivity among regions is therefore key for understanding the neurobiological basis of behavior. It remains an open question whether connectivity serves as an innate organizational blueprint that guides functional specialization, or if it evolves with learning. Here, we address this fundamental question through the prism of learning to read: We measured functional connectivity at five timepoints over a year in children with dyslexia who participated in an intensive reading intervention. We found that specific patterns of functional connectivity of the reading circuitry were in place prior to the intervention, and remained stable over time. Further, functional connectivity before the intervention predicted the location of text-selective regions a year later, showing that connectivity precedes and predicts functional specialization that emerge with learning. These findings support a view of functional connectivity as a stable organizational principle of high-level cortex.

neuroscience↗

bioRxiv2026

Anatomy of the Visual Word Form Area in Dyslexia

The Visual Word Form Area (VWFA), located in ventral occipitotemporal cortex, plays a critical role in skilled reading. Researchers have theorized that the VWFA develops in its specific anatomical location due to the convergence of major white matter tracts and proximity to functionally similar regions. This suggests that precise anatomical positioning may be crucial for optimal VWFA function. Previous research has identified several functional differences in this region between typical and struggling readers (i.e. dyslexia): struggling readers show weaker text-selective responses and often exhibit a smaller or even absent VWFA. However, it remains unexplored whether the precise anatomical location of this region also differs between typical and struggling readers. We tested whether VWFA anatomy differs between children with and without dyslexia (N=87). Participants completed a functional localizer, which we used to manually define the VWFA in each individual's native anatomy. We examined whether: (1) VWFA anatomical location relates to reading ability, (2) children with dyslexia show greater variability in VWFA location compared to typical readers, and (3) VWFA location with respect to white matter tracts relates to reading ability. Results reveal that, despite being smaller in children with dyslexia, there is no relationship between VWFA location and reading ability. Specifically, individual VWFA location relative to anatomy, relative to other's VWFAs, and relative to white matter tracts, is not related to reading ability. These findings suggest that while the VWFA's general anatomy may be facilitated by development, its precise location remains stable and unrelated to reading proficiency.

neuroscience↗

bioRxiv2026

C1qa⁺ muscularis macrophages maintain enteric synaptic homeostasis to regulate gastrointestinal motility

The enteric nervous system (ENS) is a complex peripheral neural network that coordinates gastrointestinal motility through highly organized synaptic communication. Although tissue-resident muscularis macrophages (MMs) closely associate with enteric neurons, whether they regulate enteric synaptic organization remains unknown. In the central nervous system (CNS), microglia sculpt neural circuits through complement-dependent synaptic remodeling, raising the possibility that analogous neuroimmune mechanisms operate in the gut. Here, we identify a previously unrecognized role for C1qa{square} MMs in regulating enteric synaptic homeostasis and gastrointestinal motility. Using macrophage-specific constitutive and inducible C1qa deletion models, single-cell RNA sequencing, enteric synaptosome proteomics, physiology, and advanced imaging, we demonstrate that loss of MMs-derived C1qa increases enteric synaptic density without altering neuronal numbers. C1qa deficiency induced broad transcriptional changes in enteric neurons and macrophages, including altered synapse-associated, lysosomal, and endocytic programs. Proteomic analysis revealed that enteric synapses share a conserved molecular architecture with brain synapses while exhibiting distinct gastrointestinal-specific complement-associated synaptic networks enriched for structural and receptor-localization pathways. Functionally, macrophage-specific C1qa deletion altered excitatory and inhibitory enteric neurotransmission, enhanced cholinergic signaling, reduced nitrergic responses, and accelerated gastrointestinal transit, while smooth muscle responsiveness remained preserved. C1qa{square} MMs displayed transcriptional and functional features consistent with a phagocytic synapse-remodeling phenotype, including enrichment of complement, lysosomal, and engulfment pathways. Loss of C1qa impaired macrophage phagocytic activity both in vitro and in vivo and was associated with synapse accumulation and altered macrophage morphology. Importantly, inducible deletion of C1qa in adulthood recapitulated the synaptic and motility phenotypes, demonstrating that C1qa{square} MMs continuously regulate enteric synaptic organization beyond development. Together, these findings identify a complement-dependent neuroimmune mechanism that regulates enteric circuit organization and gut motility, establishing MMs as active modulators of adult ENS synaptic homeostasis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/729640v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@16aadd8org.highwire.dtl.DTLVardef@bb74f3org.highwire.dtl.DTLVardef@fb040corg.highwire.dtl.DTLVardef@1c6e4a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2026

Dissetangling the Vine: Phylogenomics and Historical Biogeography of Vanilla (Orchidaceae)

Renowned for its aromatic fruits and economic importance, the genus Vanilla poses longstanding taxonomic and phylogenetic challenges. Despite recent molecular studies, a comprehensive species tree is lacking, and the evolutionary processes and historical patterns shaping the genus remain poorly understood. We present a new, comprehensive phylogenomic framework for Vanilla, based on 349 low-copy nuclear genes and 76 plastid loci from the Angiosperms353 probe set, which we used to infer evolutionary relationships, assess cyto-nuclear and gene-species tree discordance, and thoroughly investigate its historical distribution and diversification. Sampling 43% of the genus, our framework resolves phylogenetic uncertainties, clarifies major clades, confirms prior hypotheses, and reveals novel placements, including V. planifolia and Vanilla subg. Gondwana. Discordances are primarily driven by incomplete lineage sorting, particularly in the vanillin-producing clade, with evidence of both ancient and recent hybridization, including a natural hybrid from the Yucatan Peninsula. Biogeographic analyses indicate a Guiana Shield origin ([~]30 Mya), Amazonia as a major diversification source, the Andes as a permeable barrier, and Central America as the main diversification sink. This study provides a robust evolutionary framework for Vanilla, supporting taxonomic revisions, comparative trait analyses, and a deeper understanding of the processes shaping this economically and biologically important orchid genus.

evolutionary biology↗

bioRxiv2026

Serum Proteomic Profiling Implicates a Dysregulated Neurohormonal-Inflammatory Axis in Post-Fontan Tachycardia

BackgroundPost-operative tachycardia is a common and poorly understood complication following the Fontan procedure. Post-operative factors such as surgical scarring and venous hypertension can contribute to tachycardia risk, but the specific molecular signaling cascades triggering acute tachycardia remain uncharacterized, limiting therapeutic innovation and leaving clinicians with only reactive management strategies. Here, we present a retrospective translational study leveraging serum proteomics and machine learning to identify the molecular drivers of post-operative Fontan tachycardia. MethodsWe integrated a clinically relevant ovine animal model of the Fontan circulation with continuous telemetric heart rate monitoring and human patient data. Serum proteomics coupled with machine learning algorithms (LASSO and Boruta) were employed to identify protein panels predictive of post-operative tachycardia. Cross-species validation was performed by comparing proteomic signatures from sheep and pediatric patients undergoing Glenn or Fontan surgery. ResultsOvine Fontan animals demonstrated significant heart rate elevation beginning on post-operative day (POD) 1, peaking at POD 3 (159.4 {+/-} 11.7 bpm vs. pre-operative 105.3 {+/-} 10.5 bpm, p<0.0001), before trending toward baseline by POD 10. This pattern was mirrored in human pediatric patients, though with a more modest magnitude. Surgical controls did not exhibit tachycardia, indicating the response was specific to the Fontan procedure. Proteomic analysis identified distinct separation between pre- and post-operative serum profiles. Principal component analysis revealed that the principal components most correlated with heart rate (PC1: r=0.79, p=6.5x10-; PC8: r=0.40, p=0.04) were significantly enriched for inflammatory and neural pathways. We leveraged the Boruta algorithm to identify a seven-protein panel (ACE, ANGT, ITIH4, SELENOP, W5PHP7, PTX3, and F5) with superior predictive power (AUC=0.926). A cross-species comparison between human and sheep demonstrated that three proteins, angiotensinogen (ANGT), angiotensin-converting enzyme (ACE), and pentraxin 3 (PTX3), were similarly dysregulated in both species post-operatively. ConclusionsThis study provides the first direct molecular evidence implicating a dysregulated neurohormonal-inflammatory axis as a principal driver of acute post-operative Fontan tachycardia. The identified protein signature offers novel mechanistic insights and establishes a foundation for developing targeted diagnostics and therapeutics to predict and mitigate this significant clinical complication.

bioinformatics↗

bioRxiv2026

Are mosquito species present in Spain competent for Oropouche virus?

Oropouche virus (OROV; Orthobunyavirus) is an emerging arbovirus endemic to South America and the Caribbean, with imported cases in European countries, including Spain. Although primarily transmitted by biting midges (Culicoides spp.), OROV has been detected in several mosquito species, raising concerns about potential establishment in non-endemic regions. European populations of Aedes albopictus, and Culex pipiens, as well as the invasive Aedes aegypti, represent relevant models for assessing vector competence. Here, we evaluated vector competence of Spanish Cx. pipiens biotype molestus, Spanish Ae. albopictus, and Ae. aegypti (Liverpool strain), for the 2024 OROV outbreak strain. Female mosquitoes were orally exposed to infectious blood meals and maintained under controlled insectary conditions. In addition, an additional group received a second non-infectious blood meal. The survival of the mosquitoes was monitored, and infection, dissemination, and transmission rates were assessed at 7-, 14-, and 21-days post-infection. Vertical transmission of the virus to the progenies was also analyzed. Overall, Ae. albopictus exhibited low infection rates, with occasional dissemination and transmission events. Aedes aegypti and Cx. pipiens showed infection and occasional dissemination, but no evidence of transmission. A second non-infectious blood meal did not significantly affect infection, dissemination, or transmission rates in any species. Viral loads in bodies and legs were low and did not differ significantly between species, time points, or feeding regimens. Survival was not affected by infection or blood-feeding regime. We did not find vertical transmission of OROV to the progenies. Regardless of virus dissemination in mosquitoes, our study indicates poor vector competence of Spanish Ae. albopictus and a lack of competence in Ae. aegypti and Cx. pipiens for the circulating OROV strain. These findings suggest a low risk for local OROV establishment in Spain, although continuous surveillance and research are warranted to monitor potential vector-virus adaptation.

microbiology↗

bioRxiv2024

Impact of flagellated and elongated morphological phenotypes on the focusing behaviours of biological cells in inertial microfluidic devices

Inertial microfluidics has demonstrated tremendous potential to impact biological - and notably medical - fields, by offering a highly versatile, portable and cost-effective approach to cell focusing and sorting. While the range of applications of inertial devices spans medical diagnostics, bioprocessing or water engineering to mention a few, translation is still impeded by the lack of clear understanding of cell interactions in such devices. This often leads to bespoke designs that take years of development and characterisation for one targeted application, and limited tools for informed optimisation. A more fundamental knowledge of inertial behaviours is key to future translational works and impact, by enabling a deeper understanding of inertial forces in biological systems. Towards this goal, this paper focuses on high-throughput morphological phenotyping of the single-celled, flagellated parasite Leishmania mexicana to better understand how variations in cell body length, width and flagellated status impact the focusing patterns of highly non-spherical cells in curved inertial devices. Some of the key findings in this study include i) not all organelles, such as flagella, will alter focusing if the body shape is conserved, ii) the impact of cell shape is specific to a channel design and slight changes in e.g., cell confinement can completely change focusing patterns, iii) elongated prolate-like cells align in different orientations depending on their lateral position with a curved channel and iv) despite variabilities observed in focusing patterns for elongated versus rounder cell phenotypes, large morphological variations can be completely overcome at high Reynolds numbers so that all phenotypes tightly focus at a single position (here towards the channel outer wall). This last finding, in particular, may open new avenues for highly efficient cell enrichment processes, such as for the detection of pathogens in water.

bioengineering↗

bioRxiv2023

The use of imaging flow cytometry for rapid, high-throughput and automated analysis of the Leishmania mexicana promastigote cell cycle provides new insights into cell cycle events of short duration

Promastigote Leishmania mexicana have a complex cell division cycle characterised by the ordered replication of several single-copy organelles, a prolonged S phase and rapid G2 and cytokinesis phases, accompanied by cell cycle stage-associated morphological changes. Here we exploit these morphological changes to develop a high-throughput and semi-automated imaging flow cytometry (IFC) pipeline to analyse the cell cycle of L. mexicana in live cells. Firstly, we demonstrate that, unlike several other DNA stains, Vybrant DyeCycle Orange (DCO) is non-toxic and enables quantitative DNA imaging in live L. mexicana promastigotes. Secondly, by tagging the orphan spindle kinesin, KINF, with mNeonGreen, we describe KINFs cell cycle-dependent expression and localisation. Then, by combining manual gating of DCO DNA intensity profiles with automated masking and morphological measurements of parasite images, visual determination of the number of flagella per cell, and automated masking and analysis of mNG:KINF fluorescence, we provide a newly detailed description of L. mexicana promastigote cell cycle events that, for the first time, includes the durations of individual G2, mitosis and post-mitosis phases, and identifies G1 cells within the first 12 minutes of the new cell cycle. By applying IFC in this way, we were able, in minutes, to capture tens of thousands of high-quality brightfield and fluorescent images of live L. mexicana cells in solution, and to acquire quantitative data across multiple parameters for every image captured. Our custom-developed masking and gating scheme allowed us to identify elusive G2 cells and to demonstrate that the CDK-inhibitor, flavopiridol, arrests cells in G2 phase, rather than mitosis, providing proof-of-principle of the utility of IFC for drug mechanism-of-action studies. Further, the high-throughput nature of IFC allowed the close examination of promastigote cytokinesis, revealing considerable flexibility in both the timing of cytokinesis initiation and the direction of furrowing, in contrast to the related kinetoplastid parasite, Trypanosoma brucei. Significantly, our analysis demonstrate that the cleavage furrow can ingress unidirectionally from either pole of the cell, bidirectionally from both simultaneously or even commence internally along the anterior-posterior (A-P) axis. Our new pipeline offers many advantages over traditional methods of cell cycle analysis such as fluorescence microscopy and flow cytometry and paves the way for novel high-throughput analysis of Leishmania cell division. Author SummaryLeishmania mexicana is a single-celled parasite that is spread by sand flies and causes a spectrum of diseases called the leishmaniases in humans and animals. To cause disease, L. mexicana parasites must replicate and divide, and their cell division cycle has unusual and/or complex features, including that the parasite changes shape as it replicates. To aid analysis of the L. mexicana cell cycle, we developed a new quantitative DNA staining technique and also generated a fluorescent parasite cell line that highlighted when cells were dividing their DNA (mitosis) after replicating it. We then applied a high-throughput technique called imaging flow cytometry to capture images of tens of thousands of these parasites in just a few minutes. For each image, we were able to extract data about DNA replication, cell shape, whether the cells were in mitosis or not and how they divide. This provided new insights into how the parasites replicate and how long each stage of cell division takes as well as how the parasites split in two at the end of cell division. We were also able to use our analysis method to precisely determine the cell cycle stage at which a cell cycle inhibitor acts. More importantly, the imaging pipelines we have developed offer great advantages in terms of speed and depth over more traditional analysis techniques such as microscopy and should pave the way for increasingly detailed analyses of parasite cell biology in the future.

cell biology↗

bioRxiv2023

Crosslinking by ZapD drives the assembly of short, discontinuous FtsZ filaments into ring-like structures in solution

Bacterial cell division relies on the Z ring, a cytoskeletal structure that acts as a scaffold for the assembly of the divisome. To date, the detailed mechanisms underlying the assembly and stabilization of the Z ring remain elusive. This study highlights the role of the FtsZ-associated protein (Zap) ZapD in the assembly and stabilization of Z-ring-like structures via filament crosslinking. Using cryo-electron tomography and biochemical analysis, we show that, at equimolar concentrations of ZapD and FtsZ, ZapD induces the formation of toroidal structures composed of short, curved FtsZ filaments that are crosslinked vertically, but also laterally and diagonally. At higher concentrations of ZapD, regularly spaced ZapD dimers crosslink FtsZ filaments from above, resulting in the formation of straight bundles. Despite the simplicity of this reconstituted system, these findings provide valuable insights into the structural organization and stabilization of the Z ring by Zap proteins in bacterial cells, revealing the key role of optimal crosslinking density and geometry in enabling filament curvature and ring formation.

biochemistry↗

bioRxiv2023

Nest site selection and productivity of a critically endangered parrot, the great green macaw (Ara ambiguus) in an anthropogenic landscape

Nest site selection is the principal way secondary cavity-nesting species mitigate the negative effects of factors such as predation, parasitism and exposure on productivity. High-quality cavities could then be expected to be selected in response to the primary threat to nest success. Understanding how demographic rates are affected by anthropogenic changes to ecosystems is vital if effective conservation management strategies are to be developed and implemented. Large-bodied secondary cavity-nesting birds rely on large cavities in mature trees that are often absent or reduced in anthropogenically disturbed forests. Thus, the availability of high-quality nest sites may be limited for these species, potentially reducing productivity. The aim of this study was to investigate nest-site selection and the effect of nest-site features on productivity in the critically endangered great green macaw (Ara ambiguus) in northern Costa Rica. We show that great green macaws select nest sites according to the characteristics of the cavity and of the tree in which they are located. Moreover, productivity was a function of certain cavity features. We conclude that great green macaws are not reliant on primary forest for nest sites and typically choose cavities in remnant, isolated trees in pasture or young secondary forests.

ecology↗

bioRxiv2022

Drinkable, liquid in situ-forming and tough hydrogels for gastrointestinal therapeutics

Tablets and capsules are a cornerstone of medicine, but these solid dosage forms can be challenging to swallow for geriatric and pediatric patients. While liquid formulations are easier to ingest, these formulations lack the capacity to localize therapeutics and excipients nor act as controlled release devices. To bridge the advantages of solid and liquid dosage forms, here we describe drug formulations based on liquid in situ-forming and tough (LIFT) hydrogels. Drug-loaded LIFT hydrogels are formed directly in the stomach through the sequential ingestion of a crosslinker solution of calcium and dithiol crosslinkers, followed by the ingestion of a drug-containing polymer solution of alginate and 4-arm poly(ethylene glycol)-maleimide. We show that LIFT hydrogels are mechanically tough and able to robustly form in the presence of complex gastric fluid and in vivo in rat and porcine stomachs. LIFT hydrogels are retained within the porcine stomach for up to 24 h, biocompatible, and safely cleared. These hydrogels deliver a total dose comparable to unencapsulated drug but with delayed and lower maximum drug plasma concentrations, providing a method for controlled release that may mitigate drug toxicity. Co-encapsulation of lactase as a model biologic drug and calcium carbonate mitigated gastric-mediated deactivation of encapsulated enzyme in rat and porcine models. We also demonstrate the potential of these hydrogels to encapsulate and protect a model therapeutic bacterium, E. coli Nissle 1917, against acid. LIFT hydrogels present a biocompatible means of tough, double-network hydrogel formation in situ in the gastric cavity, and may expand medication access for patients with difficulty swallowing.

bioengineering↗

bioRxiv2022

Synthetic extremophiles: Species-specific formulations for microbial therapeutics and beyond.

Microorganisms have been used for millennia to produce food and medicine and are now being developed as products themselves to treat disease and boost crop production. However, as required for these new applications, maintaining high viability throughout manufacturing, transportation and use remains a significant challenge requiring sophisticated cold-chains and packaging. In fact, we found that commercial microbial products (probiotics) provide a poor solution to this challenge, in particular for key industrial organisms like E. coli. To overcome this technological gap, we report the development of synthetic extremophiles of industrially important gram-negative bacteria (E. coli Nissle 1917, Ensifer meliloti), gram positive bacteria (Lactobacillus plantarum) and yeast (Saccharomyces boulardii). Specifically, we developed a high throughput pipeline to define species-specific materials that allow these organisms to survive drying, elevated temperatures, organic solvents and even ionizing radiation. We enhanced the stability of E.coli Nissle 1917 by >4 orders of magnitude over commercial formulations and demonstrate the capacity to remain viable while undergoing tableting and pharmaceutical methodologies involving organic solvents. The development of synthetic materials-based enhanced stabilization stands to transform our capacity to apply micro-organisms in extreme environments including those found on Earth as well as in space. One-Sentence SummaryFragile therapeutic bacteria can be made to survive the manufacturing extremes normally reserved for small molecule drugs.

microbiology↗

bioRxiv2022

Ingestible capsule for detecting labile inflammatory biomarkers in situ

Transient molecules in the gastrointestinal (GI) tract, such as nitric oxide and hydrogen sulfide, are key signals and mediators of inflammatory bowel disease (IBD). Because these molecules are extremely short-lived in the body, they are difficult to detect. To track these reactive molecules in the GI tract, we have developed a miniaturized device that integrates genetically-engineered probiotic biosensors with a custom-designed photodetector and readout chip. Leveraging the molecular specificity of living sensors, we genetically encoded bacteria to respond to IBD-associated molecules by luminescing. Low-power electronic readout circuits (nanowatt power) integrated into the device convert the light from just 1 L of bacterial culture into a wireless signal. We demonstrate biosensor monitoring in the GI tract of small and large animal models and integration of all components into a sub-1.4 cm3 ingestible form factor capable of supporting wireless communication. The wireless detection of short-lived, disease-associated molecules could support earlier diagnosis of disease than is currently possible, more accurate tracking of disease progression, and more timely communication between patient and their care team supporting remote personalized care.

synthetic biology↗

bioRxiv2021

RANK is an independent biomarker of poor prognosis in estrogen receptor-negative breast cancer and a therapeutic target in patient-derived xenografts

Despite strong preclinical data, the therapeutic benefit of the RANKL inhibitor denosumab in BC patients, beyond its bone-related effects, is unclear. Here, we investigated the prognostic value of RANK expression and its functionality in human BC. We analyzed RANK and RANKL expression in more than 1500 BC cases (777 being estrogen receptor-negative (ER-)) from four independent cohorts. We confirmed that RANK is more frequently expressed in ER- tumors, but it is also found in a subset of ER+ tumors. In ER- BC, RANK expression was independently associated with poor outcome, especially in postmenopausal patients and those who received adjuvant chemotherapy. Gene expression analyses unraveled distinct biology associated with RANK in relation to ER expression and menopause, and evidenced enhanced RANK activation in ER- postmenopausal tumors, together with regulation of metabolic pathways. Functional studies and transcriptomic analyses in ER- RANK+ patients-derived orthoxenografts demonstrated that activation of RANK signaling pathway promotes tumor cell proliferation and stemness, and regulates multiple biological processes including tumor immune surveillance and metabolism. Our results demonstrate that RANK expression is an independent poor prognosis biomarker in postmenopausal ER- BC patients and support the rational of using RANK pathway inhibitors in combination with chemotherapy in ER- BC.

cancer biology↗

bioRxiv2021

FRAME-tags: genetically encoded fluorescent markers for multiplexed barcoding and time-resolved tracking of live cells

Cellular barcodes offer critical tools for tracking cellular identity in biological systems. Although genetically encoded fluorescent barcodes are ideal for real-time tracking, their scalability is constrained by the broad, overlapping emission spectra characteristic of fluorescent proteins (FPs). Here, we describe a palette of genetically encoded fluorescent barcodes called FRAME-tags, which break this scalability barrier by encoding barcode identity as unique FP expression ratios. FRAME-tags use -1 programmed ribosomal frameshifting RNA motifs to precisely control the translational output of multiple FPs from a single mRNA, leading to extremely narrow and resolvable ratios of the corresponding cellular fluorescence distributions. With this platform, we constructed 20 resolvable FRAME-tags in yeast using just two FPs, and further demonstrated that 100 or more distinguishable FRAME-tags could be made by the addition of a third FP. We used FRAME-tags to map the dynamic fitness landscape of yeast co-cultures, and to characterize the expression pattern of 20 yeast promoters in multiplex across diverse conditions. FRAME-tags offer a valuable new tool for cellular barcoding that enables time-resolved characterization of complex biological systems using widely available fluorescence detection techniques and a minimal number of spectral channels.

synthetic biology↗

bioRxiv2020

Clock proteins regulate spatiotemporal organization of clock genes to control circadian rhythms

Circadian clocks regulate [~]24 hour oscillations in gene expression, behavior, and physiology. While the molecular and neural mechanisms of circadian rhythms are well characterized, how cellular organization of clock components controls circadian clock regulation remains poorly understood. Here, we elucidate how clock proteins regulate circadian rhythms by controlling the spatiotemporal organization of clock genes. Using high-resolution live imaging techniques we demonstrate that Drosophila clock proteins are concentrated in a few discrete foci and are organized at the nuclear envelope; these results are in contrast to longstanding expectations that clock proteins are diffusely distributed in the nucleus. We also show that clock protein foci are highly dynamic and change in number, size, and localization over the circadian cycle. Further, we demonstrate that clock genes are positioned at the nuclear periphery by the clock proteins precisely during the circadian repression phase, suggesting that subnuclear localization of clock genes plays an important role in the control of rhythmic gene expression. Finally, we show that Lamin B receptor, a nuclear envelope protein, is required for peripheral localization of clock protein foci and clock genes and for normal circadian rhythms. These results reveal that clock proteins form dynamic nuclear foci and play a hitherto unexpected role in the subnuclear reorganization of clock genes to control circadian rhythms, identifying a novel mechanism of circadian regulation. Our results further suggest a new role for clock protein foci in the clustering of clock-regulated genes during the repression phase to control gene co-regulation and circadian rhythms. SIGNIFICANCEAlmost all living organisms have evolved circadian clocks to tell time. Circadian clocks regulate [~]24-hour oscillations in gene expression, behavior and physiology. Here, we reveal the surprisingly sophisticated spatiotemporal organization of clock proteins and clock genes and its critical role in circadian clock function. We show, in contrast to current expectations, that clock proteins are concentrated in a few discrete, dynamic nuclear foci at the nuclear envelope during the repression phase. Further, we uncovered several unexpected features of clock protein foci, including their role in positioning the clock genes at the nuclear envelope precisely during the repression phase to enable circadian rhythms. These studies provide fundamental new insights into the cellular mechanisms of circadian rhythms and establish direct links between nuclear organization and circadian clocks.

neuroscience↗

bioRxiv2020

Phylodynamics of SARS-CoV-2 transmission in Spain

ObjectivesSARS-CoV-2 whole-genome analysis has identified three large clades spreading worldwide, designated G, V and S. This study aims to analyze the diffusion of SARS-CoV-2 in Spain/Europe. MethodsMaximum likelihood phylogenetic and Bayesian phylodynamic analyses have been performed to estimate the most probable temporal and geographic origin of different phylogenetic clusters and the diffusion pathways of SARS-CoV-2. ResultsPhylogenetic analyses of the first 28 SARS-CoV-2 whole genome sequences obtained from patients in Spain revealed that most of them are distributed in G and S clades (13 sequences in each) with the remaining two sequences branching in the V clade. Eleven of the Spanish viruses of the S clade and six of the G clade grouped in two different monophyletic clusters (S-Spain and G-Spain, respectively), with the S-Spain cluster also comprising 8 sequences from 6 other countries from Europe and the Americas. The most recent common ancestor (MRCA) of the SARS-CoV-2 pandemic was estimated in the city of Wuhan, China, around November 24, 2019, with a 95% highest posterior density (HPD) interval from October 30-December 17, 2019. The origin of S-Spain and G-Spain clusters were estimated in Spain around February 14 and 18, 2020, respectively, with a possible ancestry of S-Spain in Shanghai. ConclusionsMultiple SARS-CoV-2 introductions have been detected in Spain and at least two resulted in the emergence of locally transmitted clusters, with further dissemination of one of them to at least 6 other countries. These results highlight the extraordinary potential of SARS-CoV-2 for rapid and widespread geographic dissemination.

microbiology↗