Biology subjects

Miranda, M.

Publications and source records attributed to Miranda, M..

At least 19 recordsLinked to original sources

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

Matched single-cell chromatin, transcriptome, and surface marker profiling captures in vivo epigenomic reprogramming during basal-to-luminal transition in the mammary gland

Single-cell multi-omics methods enable simultaneous mapping of chromatin states and transcriptomes, offering deep insights into gene regulation. Yet, the full potential of these approaches remains untapped for rare cell populations, as most methods require thousands of cells and are limited in their ability to capture multiple molecular layers comprehensively within the same cell. Here, we introduce OneCell CUT&Tag a user-friendly method that provides matched high-resolution epigenome, full-transcriptome, and surface marker quantification from every cell, with input as low as one cell. Using this approach, we uncovered epigenomic priming of basal cells in the mammary gland and captured the dynamics of basal-to-luminal transdifferentiation. We identified a transitional cell population with intermediate epigenomic profiles--absent in reference populations--and demonstrated a continuous epigenomic progression from basal to luminal states, while transcriptomes exhibited a binary switch. Adaptable to diverse samples and tissues, this method also revealed the role of H3K27me3 in shaping zygotic expression programs. By matching multiple layers of molecular information at single-cell resolution, OneCell CUT&Tag dissects the complementary roles of each omics layer in shaping cellular identity and function, opening new avenues to study rare and complex biological systems.

genomics↗

bioRxiv2026

4,5-dihydroxyhexanoic acid is a robust circulating and urine marker of mitochondrial disease and its severity

Management of patients with mitochondrial respiratory chain diseases is challenging, in part because of our incomplete understanding of pathogenesis and a lack of biomarkers. Unknown metabolites account for >90% of detected features in modern metabolomics experiments and hold immense untapped promise for new basic and biomedical research. We recently used mass spectrometry-based metabolomics to identify and validate 19 circulating blood-based biomarkers for patients with the mitochondrial DNA (mtDNA) m.3243A>G pathogenic variant, which is the most frequent cause of the mitochondrial disorder MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). However, the most significantly changing biomarker corresponded to an "unknown" metabolite. Here, we combine cheminformatics with analytical chemistry and identify that feature as 4,5-dihydroxyhexanoic acid (4,5-DHHA), a metabolite previously associated with inherited defects of gamma-aminobutyric acid (GABA) catabolism, but with no prior links to mitochondrial respiratory chain disorders. We validate this finding in an independent MELAS cohort and further show that 4,5-DHHA levels correlate with disease severity and are elevated in patients with other forms of mitochondrial disease and sepsis. Furthermore, brain 4,5-DHHA levels were elevated in two genetic mouse models of mitochondrial disease. In vitro and tissue culture experiments indicate that 4,5-DHHA is generated when the GABA catabolite succinic semialdehyde reacts with an intermediate of the pyruvate dehydrogenase reaction and is sensitive to mitochondrial complex I function. Our work identifies 4,5-DHHA as a robust plasma and urine marker of mitochondrial dysfunction in humans and reveals new connections between the respiratory chain and GABA metabolism. Significance StatementInborn errors of the mitochondrial respiratory chain cause severe, progressive diseases, yet effective treatments and biomarkers remain limited. Modern metabolomics detects thousands of molecules in biofluids, but the vast majority are unidentified. In this study, we investigate the most significantly altered blood metabolite in patients with the most common mitochondrial disease - MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) - and identify it as an 4,5-dihydroxyhexanoic acid (4,5-DHHA). We show that 4,5-DHHA is reproducibly elevated and correlates with severity. Levels are increased across multiple mitochondrial disorders as well as in sepsis and rise when respiratory chain function is impaired. These findings establish 4,5-DHHA as a promising biomarker of mitochondrial dysfunction and reveal a link to dysregulated GABA metabolism.

biochemistry↗

bioRxiv2025

Chromosome-Scale Genome Assembly of Australian Finger Lime and Resequencing Reveal the Hidden Diversity of Oceanian Citrus

Oceanian citrus species, including wild taxa native to Australia and Papua New Guinea, form a genetically distinct clade within the Citrus L. (1753) genus. These species remain largely underexplored, despite their adaptation to diverse environments and relevance for citrus improvement. To support their use in breeding and evolutionary studies, we generated a high-quality, Chromosome-scale genome assembly of an Australian finger lime accession (SRA 1002), a natural interspecific hybrid. The genome was assembled using long-read sequencing, optical mapping, and a high-density genetic map, resulting in nine pseudomolecules covering over 97% of the genome. Using this reference, we analyzed whole-genome resequencing data from 132 accessions representing the diversity of Asian and Oceanian citrus. Variant calling across the dataset produced a high-resolution catalogue of single nucleotide polymorphisms (SNPs) and derived database of SNP fully discriminant of 20 Citrus species (DSNPs), enabling detailed exploration of inter- and intra-specific diversity. The data reveal a strong population structure within the group with clear heterozygosity variation between ancestral species and admixed accessions, reflecting their complex evolutionary history and hybridization patterns. This study provides the first integrated genomic framework for Oceanian citrus diversity, offering essential tools for downstream applications in citrus breeding, conservation, and evolutionary genomics. The resources generated lay the groundwork for future association studies and the targeted introgression of beneficial traits into cultivated citrus.

plant biology↗

bioRxiv2025

Cytotoxic Vδ2+T cell subsets expand in response to malaria in human tonsil and spleen organoids

Vaccine effectiveness against malaria is dramatically reduced in malaria-exposed compared to malaria-naive populations, potentially due to altered immune responses in secondary lymphoid organs following repeated infection. Newly developed human tonsil and spleen organoids, which replicate key features of B and T cell immunity, provide an exciting opportunity to overcome challenges of other models and to improve our understanding of innate-adaptive interactions in lymphoid tissue. The objectives of this study were to use these organoids to investigate the impact of malaria parasites on 1) cells within lymphoid tissues and 2) responses to a heterologous antigen. When we exposed organoids from malaria-naive donors to Plasmodium falciparum-infected red blood cells (iRBC), we observed that iRBC exposure did not disrupt organoid formation and significantly increased V{delta}2+ {gamma}{delta} T cell frequencies in both tonsil and spleen organoids at multiple timepoints. Single-cell RNA/TCR sequencing revealed that iRBC-responsive V{delta}2+ T cells in organoids were clonally expanded and exhibited activated, cytotoxic phenotypes with upregulated expression of granzymes, interferon-stimulated genes, and antigen presentation machinery. TCR repertoire analysis demonstrated that malaria exposure drove clonal expansion of cytotoxic V{delta}2+ T cells, contrasting with the diverse, smaller clones observed in control conditions. To validate these findings, we analyzed tonsils from Ugandan children with asymptomatic malaria infection and found expanded V{delta}2+ T cells with enhanced cytotoxic potential compared to uninfected controls. When we tested whether malaria pre-exposure affected subsequent recall responses to influenza vaccine, malaria pre-exposure or {gamma}{delta} T cell depletion did not significantly alter cellular frequencies or influenza-specific antibody responses in most donors, though modest reductions were observed in some individuals. This work demonstrates the utility of human lymphoid organoids for studying malaria-host interactions and provides novel insights into V{delta}2+ T cell biology, including evidence for antigen-specific clonal expansion and cytotoxic differentiation in response to malaria parasites within secondary lymphoid tissues. Author SummaryMalaria vaccines are significantly less effective in populations with endemic malaria exposure compared to malaria-naive individuals. We used human tonsil and spleen organoids to investigate whether repeated malaria infections alter immune responses in secondary lymphoid organs, potentially contributing to this reduced vaccine efficacy. These organoids create a controlled system that preserves the architecture and cellular interactions of secondary lymphoid tissues. When we exposed organoids to Plasmodium falciparum-infected red blood cells, we observed dramatic expansion of the V{delta}2+ subset of {gamma}{delta} T cells. This finding was particularly noteworthy because V{delta}2+ T cells are not typically considered major participants in immune responses within secondary lymphoid organs. Single-cell analysis revealed that these expanded V{delta}2+ T cells underwent clonal expansion and acquired cytotoxic phenotypes, suggesting antigen-specific responses. Tonsil tissue from Ugandan children with asymptomatic malaria infections showed similar patterns of V{delta}2+ T cell expansion and enhanced cytotoxic potential. Surprisingly, malaria pre-exposure did not affect subsequent recall responses to influenza vaccine in most donors, although this does not discount a possible impact on immune responses to primary vaccination. Our work reveals unexpected roles for {gamma}{delta} T cells in lymphoid tissues during malaria infection and establishes organoids as valuable models for studying host-pathogen interactions.

immunology↗

bioRxiv2025

Phylogeny-driven design of broadly protective sarbecovirus receptor-binding domain nanoparticle vaccines

Vaccines against emerging SARS-CoV-2 variants and sarbecoviruses with pandemic potential must elicit a robust humoral immune response in a population imprinted with the SARS-CoV-2 spike (S) protein. Here, we designed protein nanoparticle (NP) vaccines co-displaying the SARS-CoV-2 BA.5, SARS-CoV-1, and BtKY72 receptor-binding domains (RBDs) with or without the Wuhan-Hu-1 (Wu) RBD. We show that these vaccines elicit cross-reactive and broadly neutralizing plasma antibody responses against SARS-CoV-2 variants and sarbecoviruses in naive and pre-immune animals. Immunization with multivalent RBD-NPs overcomes immune imprinting and elicits neutralizing antibodies and memory B cells specific for the BA.5, SARS-CoV-1, and BtKY72 RBDs in mRNA-1273-vaccinated non-human primates. Multivalent RBD-NPs outperform a monovalent Wu RBD-NP vaccine by providing superior protection in mice and non-human primates challenged with the vaccine-mismatched SARS-CoV-2 XBB.1.5 or the pre-emergent RsSHC014. These data support the use of multivalent RBD-NP vaccines for SARS-CoV-2 variants and sarbecoviruses in naive and pre-immune populations.

immunology↗

bioRxiv2025

Interactions Between Native Soil Microbiome and a Synthetic Microbial Community Reveals Bacteria with Persistent Traits

Synthetic microbial communities (SynComs) are curated microbial groups designed to enhance plant growth or disease resistance by augmenting soil microbiomes. Attaining SynCom stability in the presence of native soil communities remains a key challenge. This study investigated the survival, persistence, and chemical interactions of a SynCom with a native soil microbial community using a transwell system that spatially constrains bacteria while permitting chemical interactions. The SynCom, composed of six compatible Pseudomonas species identified through whole-genome sequencing, was analyzed for antagonistic interactions with native microbes over time and assessed using biomass and viability measurements. Over time, the SynCom exhibitedreduced growth in the presence of native soil microbes compared to the SynCom not exposed to the native microbes. Flow cytometry analysis showed an 81% reduction of live cells for the persistent strain in the presence of native microbes and a 78% and 99% increase in dead and unstained cells, respectively. Compared to a non-persistent strain, one persistent SynCom strain showed lower metabolic utilization across five key compound classes: polymers, carboxylic acids, amino acids, amines, and phenols when exposed to the native soil microbes. These findings underscore the importance of understanding complex SynCom-environment interactions to enhance SynCom stability and optimize in situ applications. ImportanceSynthetic Microbial Communities, or SynComs, are an emerging technology that can potentially augment plant health. Still, their application in situ depends on deciphering the complex interactions between SynCom microbes and native microbial communities. This study provides insight into several Pseudomonas strains displaying persistent characteristics, which makes these bacteria promising candidates for SynCom stability in implanted environments. Understanding the persistent traits of these bacteria is a vital advancement in SynCom technology, and an important next step toward implementing SynComs in agricultural systems.

microbiology↗

bioRxiv2025

Modulation of microRNA-502-3p significantly influences synaptic activity, dendritic spine density and mitochondrial morphology in the mice brain

Synapse dysfunction is the root cause of Alzheimers disease (AD). Uninterrupted and regulated synapse action is crucial to maintain healthy brain function. Our previous study discovered microRNA-502-3p (miR-502-3p), a synapse-specific miRNA, highly expressed at the AD synapses. Further, in vitro studies unveiled the biological relevance of miR-502-3p in modulating GABA receptor function, synaptic activity and mitochondrial morphology. Current study focuses to investigate the role of miR-502-3p in vivo using stereotaxic injection of miR-502-3p overexpression (OE) and suppression (sponge) lentivirus (LV) into the hippocampus of C57BL/6 wild-type (WT) mice. MiR-502-3p OE and sponge LV were characterized by transducing HT22 cells followed by QRT-PCR and miRNAScope analysis of miR-502-3p. MiR-502-3p OE LV showed a very high-fold upregulation and sponge LV showed significant reduction in miR-502-3p levels. MiR-502-3p OE and sponge LV were injected into three months old WT mice brain hippocampus. Overexpression and suppression effects of miR-502-3p were studied on synaptic proteins, synapse number, mitochondrial morphology and dendritic spine density at eight-weeks post-injection. Mice injected with miR-502-3p OE LV showed reduced levels of synaptic proteins, diminished synapse formation, defective mitochondrial morphology and reduced dendritic spine density relative to control LV treated mice. While mice treated with sponge LV showed elevated levels of synaptic proteins, augmented synapses, improved mitochondrial morphology and elongated dendrites and spine density. Our in vivo study unveiled translational abilities of miR-502-3p to restore synapse dysfunction in AD and other neurological disorders.

neuroscience↗

bioRxiv2024

D2-expressing neurons of the anterior paraventricular nucleus of the thalamus modulate learned aversive and safety salience

Associative learning provides a means to assign appropriate value signals to experiences that animals can later use to make appropriate value-based choices. By generalizing from prior aversive experiences and comparing with a current experience, animals can learn the relative aversive ("better or worse than") value or relative positive safety value between experiences. Yet, how such relative value signals are acquired during learning remain poorly known. The paraventricular nucleus of the thalamus (PVT) is critical for the control of salience and valence signals. It is therefore well-placed to be involved in learning relative aversive value and learned safety. Using a newly-developed conditioned place preference task, we uncover specific behavioral signatures associated with learning relative aversive value and learned safety, and related value-based choices. We then show that neurons expressing dopamine D2 receptor (D2+) in the anterior PVT (aPVT) are preferentially recruited by both relative aversive value learning and learned safety. Using a pharmacogenetic strategy, we demonstrate that aPVT D2+, but not D2-, neurons are involved in learning relative- but not absolute- aversive value. Finally, we show that aPVT D2+, but not D2-, neurons are also critical for the encoding of learned safety. Overall, our findings reveal a novel role of aPVT D2+ neurons in computing relative aversive/safety value signals between experiences during learning to promote appropriate value-based choices.

animal behavior and cognition↗

bioRxiv2024

A super-pangenome for cultivated citrus reveals evolutive features during the allopatric phase of their reticulate evolution

Most of the rich genetic diversity observed in cultivated citrus results from a reticulate evolution involving four ancestral taxa, C. medica, C. reticulata, C. maxima and C. micrantha, whose radiation occurred in allopatry. In such an evolutive context, genome diversity studies, GWAS analysis and transcriptomic studies will be significantly enhanced through pangenome approaches. We report the implementation of a super-pangenome for cultivated citrus, established with de novo assemblies in pseudochromosomes of C. medica (346 Mb), C. reticulata (332 Mb) and C. micrantha (333 Mb), released for the first time alongside a previously published chromosome-scale assembly of C. maxima. Gene annotation of these four genome assemblies revealed 28,090, 29,477, 29,258 and 30,101 genes, respectively, with a focus on pattern recognition receptors. Repetitive element annotation revealed that nearly half of each genome consisted of transposable elements or DNA-satellites. The 3 new genome assemblies display strong synteny and collinearity, while significant discrepancies are observed with the C. maxima assembly. Resequencing information (single nucleotide polymorphisms [SNPs], small indels and gene presence-absence variation [PAV]) from 55 accessions were used to explore the intra- and interspecific diversity of the four ancestral taxa and their relationships with the main horticultural groups resulting from reticulate evolution. Diagnostic SNPs of the ancestral taxa, all over the four genomes assemblies, revealed interspecific introgressions in several accessions representative of C. reticulata, C. maxima and C. medica as well as insights into the origin and phylogenomic structures of modern horticultural groups. PAV analysis revealed a gene whose absence or presence was specific to one of the ancestral taxa (dPAV). Interestingly, diagnostic PAV (dPAV) analysis uncovered a large chloroplastic introgression in chromosome 4 of C. medica, inherited in horticultural groups and recent hybrids having this species as the male parent. Implementing the super-pangenome combined the identification of orthologous genes across the four genome assemblies and the PAV information from the resequencing data. Significant inter- and intraspecific variations were highlighted, including 25,291 core, 2,431 soft-core and 5,171 dispensable genes. The analysis of the functional enrichment and species-specific adaptations in the citrus super-pangenome revealed distinct functional specializations. This highlights the evolutionary paths that have shaped each species, contributing to the diversity in the citrus super-pangenome while maintaining a shared foundation of essential biological processes. The development of this super-pangenome is a significant milestone in citrus genomics, providing a comprehensive resource that captures the extensive genetic diversity of modern cultivated citrus. Additionally, we established a dedicated Genome Hub, offering a platform for continuous genomic research and allowing for ongoing updates and future inclusion of additional ancestral species.

genomics↗

bioRxiv2024

Investigating Novel Streptomyces Bacteriophage Endolysins as Potential Antimicrobial Agents

BackgroundAs antibiotic resistance has become a major global threat; the World Health Organization (WHO) has urgently called for alternative strategies for control of bacterial infections. Endolysin, a phage-encoded protein, can degrade bacterial peptidoglycan (PG) and disrupt bacterial growth. According to the WHO, there are only three endolysin products currently in clinical phase development. In this study we explore novel endolysins from Streptomyces phages as only a few of them have been experimentally characterized. Using several bioinformatics tools, we identified nine different functional domain combinations from 250 Streptomyces phages putative endolysins. LazerLemon gp35 (CHAP; LL35lys), Nabi gp26 (amidase; Nb26lys), and Tribute gp42 (PGRP/amidase; Tb42lys) were selected for experimental studies. We hypothesized that (1) the proteins of interest will have the ability to degrade purified PG, and (2) the proteins will have potential antimicrobial activity against bacteria from families of importance in antibiotic resistance, such as ESKAPE safe relatives (Enterococcus raffinosus, Staphylococcus epidermidis, Klebsiella aerogenes, Acinetobacter baylyi, Pseudomonas putida, and Escherichia coli). ResultsLL35lys, Nb26lys, and Tb42lys exhibit PG-degrading activity on zymography and hydrolysis assay. The enzymes (100 {micro}g/mL) can reduce PG turbidity to 32-40%. The killing assay suggests that Tb42lys has a broader range (E. coli, P. putida, A. baylyi and K. aerogenes). While Nb26lys better attacks Gram-negative than Gram-positive bacteria, LL35lys can only reduce the growth of the Gram-positive ESKAPE strains but does so effectively with a low MIC90 of 2 {micro}g/mL. A higher concentration ([≥]300 {micro}g/mL) of Nb26lys is needed to inhibit P. putida and K. aerogenes. ConclusionFrom 250 putative endolysins, bioinformatic methods were used to select three putative endolysins for cloning and study: LL35lys, Nb26lys, and Tb42lys. All have shown PG-degrading activity, a critical function of endolysin. With a low MIC, LL35lys shows activity for the Gram-positive ESKAPE strains, while Nb26lys and Tb42lys are active against the Gram-negatives. Therefore, endolysins from Streptomyces phage have potential as possible antimicrobial agents against ESKAPE bacteria.

microbiology↗

bioRxiv2024

Increased mitochondrial transcription initiation does not promote oxidative phosphorylation

POLRMT is the sole RNA polymerase in human mitochondria where it generates primers for mitochondrial DNA (mtDNA) replication and transcribes the mitochondrial genome to express genes encoding essential components of the oxidative phosphorylation (OXPHOS) system. Elevated POLRMT levels are found in several cancers and in mouse models with severe mitochondrial dysfunction. Here, we generated and characterized mice over-expressing Polrmt to investigate the physiological and molecular consequences of elevated POLRMT levels. Increasing POLRMT did not result in any pathological phenotype but instead positively affected exercise capacity under stress conditions. POLRMT overexpression increased in organello transcription initiation, resulting in higher steady-state levels of the promoter-proximal L-strand transcript 7S RNA and higher mtDNA levels. Surprisingly, the abundance of mature mitochondrial RNAs was not affected by the elevated POLRMT levels. Furthermore, ubiquitous simultaneous overexpression of POLRMT and LRPPRC, which stabilizes mitochondrial messenger RNAs, did not increase steady-state levels of mitochondrial transcripts in the mouse. Our data show that POLRMT levels regulate transcription initiation, but additional regulatory steps downstream of transcription initiation and transcript stability limit OXPHOS biogenesis.

molecular biology↗

bioRxiv2023

Dichotomy of neutralizing antibody, B cell and T cell responses to SARS-CoV-2 vaccination and protection in healthy adults

Heterogeneity in SARS-CoV-2 vaccine responses is not understood. Here, we identify four patterns of live-virus neutralizing antibody responses: individuals with hybrid immunity (with confirmed prior infection); rare individuals with low responses (paucity of S1-binding antibodies); and surprisingly, two further groups with distinct serological repertoires. One group - broad responders - neutralize a range of SARS-CoV-2 variants, whereas the other - narrow responders - neutralize fewer, less divergent variants. This heterogeneity does not correlate with Ancestral S1-binding antibody, rather the quality of the serological response. Furthermore, IgDlowCD27-CD137+ B cells and CCR6+ CD4+ T cells are enriched in broad responders before dose 3. Notably, broad responders have significantly longer infection-free time after their third dose. Understanding the control and persistence of these serological profiles could allow personalized approaches to enhance serological breadth after vaccination.

immunology↗

bioRxiv2023

The potential of Senicapoc, a KCNN4 inhibitor, for the prevention and treatment of breast cancer

BackgroundGenome-wide association studies have identified a breast cancer risk locus at 19q13.31. The candidate causal variants at this locus are located in the first exon of KCNN4. KCNN4, which regulates membrane potential and Ca2+ signaling, is a good candidate for drug repositioning because its inhibitor, Senicapoc, has been shown to be well tolerated in Phase-II and -III clinical trials for asthma and sickle cell anemia. MethodsWe evaluated public mRNA expression data to determine whether the allele at 19q13.31 associated with increased breast cancer risk was associated with KCNN4 expression. We also used immunohistochemistry to evaluate the relationship between KCNN4 protein expression and breast cancer survival. We then used Senicapoc in two murine mammary tumor models to determine if it would delay tumor development. We also treated mice bearing 4T1 mammary tumors with Senicapoc, by subcutaneous injection and by oral gavage. Finally we used gene editing to make deletions within Kcnn4 in 4T1 to determine whether Senicapoc had off-target effects on tumor growth. ResultsAnalysis of the Genotype-Tissue Expression Project showed that the allele at 19q13.31 associated with increased breast cancer risk is associated with increased KCNN4 expression, suggesting that inhibiting KCNN4 might reduce breast cancer risk. Using immunohistochemistry in a large breast cancer cohort, we found that membrane and cytoplasmic expression is a marker of poor prognosis in triple negative breast cancer. We then tested the efficacy of Senicapoc to prevent and treat breast cancer. This showed that it delays the development of mammary tumors in two murine models, and slows growth of a syngeneic (4T1) model of triple negative breast cancer. Senicapoc monotherapy showed similar efficacy to anthracycline/taxane-based chemotherapy in these studies, with a stronger effect when they were combined. ConclusionsThese results provide a rationale for clinical testing of Senicapoc for treating, and even preventing, breast cancer.

cancer biology↗

bioRxiv2023

Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies

Growth factors and cytokines signal by binding to the extracellular domains of their receptors and drive association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affects signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo designed fibroblast growth-factor receptor (FGFR) binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and MAPK pathway activation. The high specificity of the designed agonists reveal distinct roles for two FGFR splice variants in driving endothelial and mesenchymal cell fates during early vascular development. The ability to incorporate receptor binding domains and repeat extensions in a modular fashion makes our designed scaffolds broadly useful for probing and manipulating cellular signaling pathways. HighlightsO_LIDe novo designed cyclic oligomers with tunable geometric properties C_LIO_LICyclic, homo-oligomeric FGFR binding modules induce geometry- and valency-dependent activity of isoform-specific FGF signaling C_LIO_LIModulation of FGFR isoform activity controls bifurcation of endothelial and mesenchymal fate during vascular development C_LIO_LIC-isoform activation favors arterial endothelial cell formation while B-isoform induces pericyte differentiation C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/532666v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@151f6eforg.highwire.dtl.DTLVardef@2ae877org.highwire.dtl.DTLVardef@103e0d1org.highwire.dtl.DTLVardef@19cd695_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Behavioral, molecular and neuronal mechanisms involved in recognition memory retrieval under degraded spatial cues in the rat hippocampus

In a constantly changing environment, organisms face the challenge of adapting their behavior by retrieving previous experiences or acquiring new information. Previous research has postulated that this balance between memory generalization and differentiation manifests in a dichotomic manner. When environmental information exceeds a given threshold, activation of a stored representation could initiate retrieval, but below this threshold, a novel event could be encoded with a concomitant remapping of the internal representation in the hippocampus. Here, we examined the hippocampal molecular and neuronal mechanisms underlying retrieval in a cue-degraded environment by combining in vivo electrophysiological recordings and pharmacological manipulations. We developed a memory recognition task that allows a graded decrease in the contextual cues present during retrieval. We found that the manipulation of the number of visual cues was consistent with the activation or not of the contextual memory trace. Retrieval of a specific context memory was reflected by the level of CA3 remapping, demonstrating a clear relationship between remapping and contextual recognition. Also, manipulation of NMDAR activity in the DG-CA3 circuit bidirectionally modulated contextual memory retrieval. The blockade of NMDAR in CA3 impaired recognition in a cue-degraded, but not in a full-cue context, while their activation has the opposite effect. Conversely, blockade of NMDAR in the DG promoted retrieval under an even more cue-degraded environment, while activation had the opposite effect. Our results provide evidence for a flexible interaction between environmental cues and information stored in the hippocampus and give new insights into the biological mechanisms that balance memory encoding and retrieval.

neuroscience↗

bioRxiv2022

Accurate Computational Design of 3D Protein Crystals

Protein crystallization plays a central role in structural biology1, with broad impact2 in pharmaceutical formulation3, drug delivery4, biosensing5, and biocatalysis6,7. Despite this importance, the process of protein crystallization remains poorly understood and highly empirical8-10, with largely unpredictable crystal contacts, lattice packing arrangements, and space group preferences, and the programming of protein crystallization through precisely engineered sidechain-sidechain interactions across multiple protein-protein interfaces is an outstanding challenge. Here we develop a general computational approach to designing three-dimensional (3D) protein crystals with pre-specified lattice architectures at atomic accuracy that hierarchically constrains the overall degree of freedoms (DOFs) of the system. We use the approach to design three pairs of oligomers that can be individually purified, and upon mixing, spontaneously self-assemble into large 3D crystals (>100 m). Small-angle X-ray scattering and X-ray crystallography show these crystals are nearly identical to the computational design models, with the design target F4132 and I432 space groups and closely corresponding overall architectures and protein-protein interfaces. The crystal unit cell dimensions can be systematically redesigned while retaining space group symmetry and overall architecture, and the crystals are both extremely porous and highly stable, enabling the robust scaffolding of inorganic nanoparticle arrays. Our approach thus enables the computational design of protein crystals with high accuracy, and since both structure and assembly are encoded in the primary sequence, provides a powerful new platform for biological material engineering.

synthetic biology↗

bioRxiv2022

A novel image-based approach for soybean seed phenotyping using machine learning techniques

Soybean is one of the most important sources of protein and vegetable oil in the world. Given its increasing demand, the increment in yield has only been possible due to investments in research and production technology, especially in breeding programs. One of the main factors influencing soybean yield is the seed morphology; however, its analyses are hampered by the lack of efficient computational approaches with not only accurate results, but also a high flexibility to user preferences and needs. In this context, the present work provides a methodological framework for: (i) seed segmentation in soybean images; (ii) seed morphological evaluation; and (iii) image-based prediction of the hundred-seed weight trait. We used genotypes from a partial diallel cross design, which aimed at obtaining genotypes with high agronomic performance. In addition to the measurement of the hundred-seed weight, we collected RGB images of seeds of each plot. For image segmentation, we created an in-house image processing pipeline, which enabled a full morphological seed evaluation. For predicting the hundred-seed weight, we compared different machine learning algorithms using as input the morphological characteristics obtained, and also features from state-of-the-art convolutional neural network (CNN) architectures. The image segmentation methodology showed to be highly efficient, as more than 98% of the seeds in the images were correctly identified. Even if the seeds were close, the segmentation strategy could separate them into independent image components. In addition to supplying a highly accurate decision support system for soybean breeders, we verified the morphological phenotyping adaptability in other plant species, fully assessing the pipeline generalization. We consider the use of this methodology highly advantageous, as the method is entirely based on widely used morphological operations, which results in an easy implementation and low computational costs. Using these morphological measures, we could estimate machine learning models for predicting the hundred-seed weight, achieving considerable predictive accuracy. The same results were observed for CNN-obtained features, showing the efficiency of the morphological measurements as feature extractors. The possibility of obtaining seed morphological characteristics provides a valuable tool for the continuous and efficient development of new soybean cultivars in breeding programs aimed at long-term genetic gain. Additionally, through a faster seed image acquisition workflow, with less chance of errors and low cost, it is also possible to make predictions of important soybean characteristics. The work conducted has the potential to help future research and the industry to develop automated phenotyping tools, incorporating the proposed analytical workflows.

plant biology↗