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Aguilar, C.

Publications and source records attributed to Aguilar, C..

17 recordsLinked to original sources

Unconstrained dosing agar (UDA) Reduces Stress in Mouse Oral Administration

BackgroundOral gavage is the standard method for delivering drugs and other substances orally in rodent studies, but it can cause significant stress and risk injury. To improve animal welfare and reduce confounding stress effects, this study aimed to replace oral gavage by developing and testing a new voluntary ingestion method that is easy to adopt, minimizes stress in mice, and is suitable for a wide range of compounds. ResultsWe developed a soft agar formulation with an appealing scent and taste that mice readily consumed without fasting or restraint. We called this method "unconstrained dosing agar" (UDA). Analysis of fecal corticosterone levels demonstrated that the method is associated with low stress in the animals. After training, mice quickly consumed the agar units. Body weight gain was unaffected by the treatment. ConclusionsThis study introduces a simple, low-stress method for administering substances orally in mice. By encouraging voluntary consumption and removing the need for fasting or restraint, this method provides a practical alternative to oral gavage and could improve animal welfare and experimental consistency.

animal behavior and cognition↗

Statins and genetic inhibition of the mevalonate pathway activate an ATF3-STMN2 regenerative program

Loss of neuronal regenerative capacity is a common feature of neurodegenerative disease and axonal injury, yet the transcriptional programs governing this state remain poorly defined. Stathmin-2 (STMN2), a tubulin-binding protein essential for axon maintenance and repair, is profoundly depleted following loss of nuclear TDP-43 in neurodegenerative disease. Here, we identify statins as potent inducers of STMN2 expression. Pharmacological and genetic suppression of the mevalonate pathway, and subsequent prevention of protein geranylgeranylation, restored STMN2 levels in TDP-43 deficient cells and promoted neurite growth. STMN2 induction was abrogated when using a statin analogue unable to interact with HMG-CoA reductase, and through co-administration of mevalonate or geranylgeranyl diphosphate substrates. RNA-seq revealed that statins induce a coordinated pro-regenerative transcriptional response, including activation of the AP-1 transcription factor complex gene, ATF3. Loss of ATF3 attenuated STMN2 induction in vitro, and diminished injury-induced Stmn2 upregulation in spinal motor neurons in vivo. These results demonstrate statins as modulators of ATF3 and STMN2 expression and highlight their therapeutic potential in neurodegenerative disease.

neuroscience↗

Comparative analysis of NSP5/VP2-induced viroplasm-like structures in rotavirus species A to J

Rotavirus (RV) is classified into nine species, A-D and F-J, with RV species A (RVA) being the most extensively studied. While RVA infects infants and young animals, non-RVA species infect adult humans, various mammals, and birds. However, the lack of appropriate research tools has limited our understanding of non-RVA life cycles. RVA replication and assembly occur in cytosolic inclusions termed viroplasms. We recently identified viroplasm-like structures (VLS) composed of NSP5 and NSP2, designated as VLS(NSP2)i, in non-RVA. In this context, globular VLS(NSP2)i formed in RVA, RVB, RVD, RVF, RVG, and RVI, but not in RVC, RVH, and RVJ. Additionally, in RVA, VLS can also be formed through co-expression of NSP5 with VP2, referred to as VLS(VP2)i. Here, we report VLS(VP2)i formation in all non-RVA species except RVB, with notable VLS formation in RVH and RVJ. Moreover, NSP2 RVH or RVJ are recruited into VLS(VP2)i. The NSP5 C-terminal region in non-RVA is required for association with VP2 and forming VLS(VP2)i. Mutation of conserved VP2-L141 in RVA to alanine disrupts viroplasm formation, impairing RV replication. Equivalent residues within the same predicted VP2 region disrupt VLS formation across non-RVA. We also observed interspecies VLS formation, particularly between closely related RVA and RVC, RVH and RVJ, and RVD and RVF. Interestingly, substituting the N-terminal region of VP2 RVB with that of the closely related VP2 RVG restores its ability to form VLS with NSP5 RVB. Elucidating the formation of viroplasms is essential for developing strategies to halt infection across RV species A to J. ImportanceRotaviruses (RV) are a group of viruses classified into species A through J, with species A being the best understood. Other RV species infecting animals and humans are less studied due to limited research tools. In RVA, the virus replicates in specialized compartments called viroplasms formed in the cytoplasm by viral proteins, including NSP5, NSP2, and VP2. In this study, we explored how similar structures, termed viroplasm-like structures (VLS), are formed by proteins of other RV species. We found that in most species, NSP5 and VP2 form VLSs. One exception was RVB, where VLS formation was observed only when the VP2 protein was mutated to resemble that of a related species. We also identified key regions in the VP2 protein that are essential for forming these structures. Understanding how viroplasms form across different RV species may help develop new strategies to block infection in humans and animals.

microbiology↗

Prostate-specific membrane receptor PPAP facilitates E. coli invasion of luminal prostate cells via FimH binding

Bacterial prostatitis caused by uropathogenic Escherichia coli (UPEC) strains is a highly prevalent and recurrent infection responsible for significant morbidity in men. However, the molecular pathogenesis of prostatitis remains poorly understood, partly due to the lack of comprehensive in-vitro models. In this study, we introduce a murine prostate organoid model that replicates the cellular heterogeneity of the prostate epithelium with a cell composition and transcriptional signature comparable to the native prostate tissue. Using this model, we uncovered that UPEC preferentially attaches to, invades, and replicates within luminal prostate cells. This selective interaction is mediated by the binding of the bacterial adhesin FimH to the prostate- specific membrane protein PAPP, which is exclusively expressed on luminal prostate cells. Altogether, we identified a new mechanism by which UPEC infects the prostate epithelium, highlighting FimHs adaptability in engaging host receptors and its potential for targeted therapeutic strategies.

molecular biology↗

KLHDC7B, a novel gene associated with age-related hearing loss in humans, is required for the maintenance of hearing in mice.

BackgroundAge-related hearing loss (ARHL) is the most common sensory loss in older adults, but the underlying pathological mechanisms remain unclear. Recent genome wide association studies (GWAS) have linked variation in a large number of genes with increased risk of ARHL for the first time. Amongst the strongest of these associations is variation in KLHDC7B, a gene of unknown function and one not previously linked to hearing. MethodsTo confirm whether KLHDC7B plays a role in hearing we investigated auditory function in two independent knockout mutant mouse models of Klhdc7b: Klhdc7bIMPC-/-and Klhdc7bRegn{Delta}/{Delta} on the C57BL/6N background and the B6.CAST-Cdh23753A>G background respectively. The B6.CAST-Cdh23753A>G background was backcrossed to correct a known age-related hearing loss (ahl) mutation in cadherin 23 present in both the C57BL/6N and C57BL/6J strains. ResultsWe showed that Klhdc7b is expressed exclusively in inner and outer sensory hair cells within the cochlea of mice at the RNA and protein level. Homozygous mutants for both knockout mouse models display a similar early-onset, progressive and severe hearing loss. Histological characterization of the two mouse models suggests that hair cells develop normally and are present in neonates. However, after the onset of hearing there is a progressive loss of outer hair cells in a gradient from base to apex of the cochlea consistent with the hearing deficit in the mice and the pattern of hearing loss in ARHL. Inner hair cells remain intact up to the latest age examined (~8 weeks). ConclusionsOur data suggests KLHDC7B is required for maintenance of auditory function rather than in its development, supporting the novel association with ARHL in humans detected in recent GWAS. To our knowledge, this is the first validation in mouse of an ARHL association in humans detected in a GWAS. Our work also provides two distinct mouse models to further investigate the role of KLHDC7B in auditory function and for use in the development of therapeutic tools to prevent or treat ARHL.

genetics↗

Characterization of Viroplasm-Like Structures by Co-Expression of NSP5 and NSP2 Across Rotavirus Species A to J

Rotaviruses (RV) are classified into nine species, A-C and D-J, with species A being the most studied. In rotavirus of species A (RVA), replication occurs in viroplasms, which are cytosolic globular inclusions primarily composed of the proteins NSP5, NSP2, and VP2. The co-expression of NSP5 with either NSP2 or VP2 leads to the formation of viroplasm-like structures (VLS). Although morphologically identical to viroplasms, VLSs cannot replicate, but they serve as excellent simplified tools for studying complex viroplasms. There is a knowledge gap regarding viroplasms of non-RVA species due to a lack of research tools, such as specific antibodies and tissue culture systems. In this study, we explored the ability of NSP5 and NSP2 from non-RVA species to form VLSs. The co-expression of these two proteins led to globular VLSs in RV species A, B, D, F, G, and I, while RVC formed filamentous VLSs. The co-expression of NSP5 and NSP2 of RV species H and J did not result in VLS formation. Interestingly, NSP5 of all RV species self-oligomerizes, with the ordered C-terminal region, termed the tail, being necessary for self-oligomerization of RV species A-C and G-J. Except for NSP5 from species J, all NSP5 bound with their respective NSP2. We also found that interspecies VLS are formed between closely related RV species B with G and D with F. Additionally, VLS from RVH and RVJ formed when the tail of NSP5 RVH and RVJ was replaced by the tail of NSP5 from RVA and co-expressed with their respective NSP2. ImportanceRotaviruses (RV) are classified into nine species, A-D and F-J, infecting mammals and birds. Due to the lack of research tools, all cumulative knowledge on RV replication is based on RV species A (RVA). The RV replication compartments are globular cytosolic structures named viroplasms, which have only been identified in RV species A. In this study, we examined the formation of viroplasm-like structures (VLS) by the expression of NSP5 with NSP2 across RV species A to J. Globular VLSs formed for RV species A, B, D, F, G, and I, while RV species C formed filamentous structures. The RV species H and J did not form VLS with NSP5 and NSP2. Similar to RVA, NSP5 self-oligomerizes in all RV species, which is a requirement for VLS formation. This study provides basic knowledge of the non-RVA replication mechanisms, which could help develop strategies to halt virus infection across RV species.

microbiology↗

Performance of Orbicella faveolata larval cohorts does not align with previously observed thermal tolerance of adult source populations

Orbicella faveolata, commonly known as the mountainous star coral, is a dominant reef-building species in the Caribbean, but populations have suffered sharp declines since the 1980s due to repeated bleaching and disease-driven mortality. Prior research has shown that inshore adult O. faveolata populations in the Florida Keys are able to maintain high coral cover and recover from bleaching faster than their offshore counterparts. However, whether this origin-specific variation in thermal resistance is heritable remains unclear. To address this knowledge gap, we produced purebred and hybrid larval crosses from O. faveolata gametes collected at two distinct reefs in the Upper Florida Keys, a nearshore site (Cheeca Rocks, CR) and an offshore site (Horseshoe Reef, HR), in two different years (2019, 2021). We then subjected these aposymbiotic larvae to severe (36 {degrees}C) and moderate (32 {degrees}C) heat challenges to quantify their thermal tolerance. Contrary to our expectation based on patterns of adult thermal tolerance, HR purebred larvae survived better and exhibited gene expression profiles that were less driven by stress response under elevated temperature compared to purebred CR and hybrid larvae. One potential explanation could be compromised reproductive output of CR adult colonies due to repeated summer bleaching events in 2018 and 2019, as gametes originating from CR in 2019 contained less storage lipids than those from HR. These findings provide an important counter-example to the current selective breeding paradigm, that more tolerant parents will yield more tolerant offspring, and highlight the importance of adopting a holistic approach when evaluating larval quality for conservation and restoration purposes.

physiology↗

Comparative phylogeography reveals widespread cryptic diversity driven by ecology in Panamanian birds

Widespread species often harbor unrecognized genetic diversity, and investigating the factors associated with such cryptic variation can help us better understand the forces driving diversification. Here, we identify potential cryptic species based on a comprehensive dataset of COI mitochondrial DNA barcodes from 2,333 individual Panamanian birds across 429 species, representing 391 (59%) of the 659 resident landbird species of the country, as well as opportunistically sampled waterbirds. We complement this dataset with additional publicly available mitochondrial loci, such as ND2 and cytochrome b, obtained from whole mitochondrial genomes from 20 taxa. Using barcode identification numbers (BINs), we find putative cryptic species in 19% of landbird species, highlighting hidden diversity in the relatively well-described avifauna of Panama. Whereas some of these mitochondrial divergence events corresponded with recognized geographic features that likely isolated populations, such as the Cordillera Central highlands, the majority (74%) of lowland splits were between eastern and western populations. The timing of these splits are not temporally coincident across taxa, suggesting that historical events, such as the formation of the Isthmus of Panama and Pleistocene climatic cycles, were not the primary drivers of cryptic diversification. Rather, we observed that forest species, understory species, insectivores, and strongly territorial species--all traits associated with lower dispersal ability--were all more likely to have multiple BINs in Panama, suggesting strong ecological associations with cryptic divergence. Additionally, hand-wing index, a proxy for dispersal capability, was significantly lower in species with multiple BINs, indicating that dispersal ability plays an important role in generating diversity in Neotropical birds. Together, these results underscore the need for evolutionary studies of tropical bird communities to consider ecological factors along with geographic explanations, and that even in areas with well-known avifauna, avian diversity may be substantially underestimated. LAY SUMMARY- What factors are common among bird species with cryptic diversity in Panama? What role do geography, ecology, phylogeographic history, and other factors play in generating bird diversity? - 19% of widely-sampled bird species form two or more distinct DNA barcode clades, suggesting widespread unrecognized diversity. - Traits associated with reduced dispersal ability, such as use of forest understory, high territoriality, low hand-wing index, and insectivory, were more common in taxa with cryptic diversity. Filogeografia comparada revela amplia diversidad criptica causada por la ecologia en las aves de Panama RESUMENEspecies extendidas frecuentemente tiene diversidad genetica no reconocida, y investigando los factores asociados con esta variacion criptica puede ayudarnos a entender las fuerzas que impulsan la diversificacion. Aqui, identificamos especies cripticas potenciales basadas en un conjunto de datos de codigos de barras de ADN mitocondrial de 2,333 individuos de aves de Panama en 429 especies, representando 391 (59%) de las 659 especies de aves terrestres residentes del pais, ademas de algunas aves acuaticas muestreada de manera oportunista. Adicionalmente, complementamos estos datos con secuencias mitocondriales disponibles publicamente de otros loci, tal como ND2 o citocroma b, obtenidos de los genomas mitocondriales completos de 20 taxones. Utilizando los numeros de identificacion de codigo de barras (en ingles: BINs), un sistema taxonomico numerico que proporcina una estimacion imparcial de la diversidad potencial a nivel de especie, encontramos especies cripticas putativas en 19% de las especies de aves terrestres, lo que destaca la diversidad oculta en la avifauna bien descrita de Panama. Aunque algunos de estos eventos de divergencia conciden con caracteristicas geograficas que probablemente aislaron las poblaciones, la mayoria (74%) de la divergencia en las tierras bajas se encuentra entre las poblaciones orientales y occidentales. El tiempo de esta divergencia no coincidio entre los taxones, sugiriendo que eventos historicos tales como la formacion del Istmo de Panama y los ciclos climaticos del pleistoceno, no fueron los principales impulsores de la especiacion. En cambio, observamos asociaciones fuertes entre las caracteristicas ecologicas y la divergencia mitocondriale: las especies del bosque, sotobosque, con una dieta insectivora, y con territorialidad fuerte mostraton multiple BINs probables. Adicionalmente, el indice mano-ala, que esta asociado a la capacidad de dispersion, fue significativamente menor en las especies con BINs multiples, sugiriendo que la capacidad de dispersion tiene un rol importamente en la generacion de la diversidad de las aves neotropicales. Estos resultos demonstran la necesidad de que estudios evolutivos de las comunidades de aves tropicales consideren los factores ecologicos en conjunto con las explicaciones geograficos. Palabras clave: biodiversidad tropical, biogeografia, codigos de barras, dispersion, especies cripticas

evolutionary biology↗

Comprehensive preclinical evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9ORF72 disease

Hexanucleotide G4C2 repeat expansions in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intracellular poly-GA and reduced aggregate formation in a poly-GA over-expressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 months was well-tolerated and led to measurable brain penetration of antibodies. Long term treatment with anti-GA antibodies produced improvement in an open field movement test in aged C9ORF72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model. SignificanceImmunotherapy has been proposed for neurodegenerative disorders including Alzheimers or Parkinsons diseases. Recent reports using antibodies against poly-GA or active immunization suggested similar immunotherapy in ALS/FTD caused by repeat expansion in the C9ORF72 gene (1, 2). Here, we systematically characterized human antibodies against multiple DPR species and tested the biological effects of antibodies targeting poly-GA in different cellular and mouse models. Target engagement was shown in three independent cellular models. Anti-GA antibodies reduced the number of intracellular poly-GA aggregates in human T98G cells but not in cultured human neurons. Whereas chronic anti-GA treatment in BAC C9ORF72450 mice did not impact poly-GA levels and modestly improved one behavioral phenotype, poly-GA levels detected by immunoassays were increased and disease progression was unaltered in AAV-(G4C2)149 mice.

neuroscience↗

Neuroplastin genetically interacts with Cadherin 23 and the encoded isoform Np55 is sufficient for cochlear hair cell function and hearing

Mammalian hearing involves the mechanoelectrical transduction (MET) of sound-induced fluid waves in the cochlea. Essential to this process are the specialised sensory cochlear cells, the inner (IHCs) and outer hair cells (OHCs). While genetic hearing loss is highly heterogeneous, understanding the requirement of each gene will lead to a better understanding of the molecular basis of hearing and also to therapeutic opportunities for deafness. The Neuroplastin (Nptn) gene, which encodes two protein isoforms Np55 and Np65, is required for hearing, and homozygous loss-of-function mutations that affect both isoforms lead to profound deafness in mice. Here we have utilised several distinct mouse models to elaborate upon the spatial, temporal, and functional requirement of Nptn for hearing. While we demonstrate that both Np55 and Np65 are present in cochlear cells, characterisation of a Np65-specific mouse knockout shows normal hearing thresholds indicating that Np65 is functionally redundant for hearing. In contrast, we find that Nptn-knockout mice have significantly reduced maximal MET currents and MET channel open probabilities in mature OHCs, with both OHCs and IHCs also failing to develop fully mature basolateral currents. Furthermore, comparing the hearing thresholds and IHC synapse structure of Nptn-knockout mice with those of mice that lack Nptn only in IHCs and OHCs shows that the majority of the auditory deficit is explained by hair cell dysfunction, with abnormal afferent synapses contributing only a small proportion of the hearing loss. Finally, we show that continued expression of NEUROPLASTIN in OHCs of adult mice is required for membrane localisation of Plasma Membrane Ca2+ ATPase 2 (PMCA2), which is essential for hearing function. Moreover, Nptn haploinsufficiency phenocopies Atp2b2 (encodes PMCA2) mutations, with heterozygous Nptn-knockout mice exhibiting hearing loss through genetic interaction with the Cdh23ahl allele. Together, our findings provide further insight to the functional requirement of Neuroplastin for mammalian hearing. Author SummarySensorineural hearing loss, caused by problems with sensory cells in the cochlea or the auditory nerve, is the most common type of hearing loss. Mutations in Neuroplastin have already been implicated in deafness in mice. We have used mutant mouse models to investigate where Neuroplastin is expressed in the cochlea and its function. When mice do not express a functioning copy of Neuroplastin they have disruptions to the primary sensory synapse. We show that although synaptic disruption contributes to the loss of hearing function it is not the primary cause. Instead, continued expression of Neuroplastin is needed to maintain the localisation of Plasma Membrane Ca2+ ATPase 2 channels which help regulate calcium flow. We have also shown that two types of NEUROPLASTIN protein (isoforms) are both expressed within the cochlea, although only one of these isoforms needs to be expressed for normal hearing. Finally, we also demonstrate that the hearing loss caused by the absence of Neuroplastin is made worse when combined with a common mutation within a gene called Cadherin 23 (Cdh23ahl). This is an important finding as although there are currently no human patients with an identified NEUROPLASTIN mutation, it may be involved in human deafness in combination with other mutations.

neuroscience↗

Drug targeting to sites of oxidative stress using the Baeyer-Villiger reaction

The antioxidant nuclear factor erythroid 2-related factor 2 (Nrf2) is a desirable therapeutic target for a broad range of pathologies, including chronic diseases of the lung and liver, and autoimmune, neurodegenerative, and cardiovascular disorders. However, current Nrf2 activators are limited by unwanted effects due to non-specificity, and systemic distribution and action. Here we report that a 1,2-dicarbonyl moiety masks the electrophilic reactivity of the Nrf2 activator monomethyl fumarate (MMF), otherwise responsible for its non-specific effects. The 1,2-dicarbonyl compound is highly susceptible to Baeyer-Villiger oxidation, with generation of MMF specifically on exposure to pathological levels of hydrogen peroxide or peroxynitrite. Oral treatment with the MMF generating 1,2-dicarbonyl compound reversed chronic neuropathic and osteoarthritis pain in mice, and selectively activated Nrf2 at sites of oxidative stress. This 1,2-dicarbonyl platform may be used to treat additional disorders of oxidative stress, and to selectively target other therapeutics to sites of redox imbalance.

pharmacology and toxicology↗

Myosin-driven Nucleation of Actin Filaments Drives Stereocilia Development Critical for Hearing

The assembly and maintenance of actin-based mechanosensitive stereocilia in the cochlea is critical for lifelong hearing. Myosin-15 (MYO15) is hypothesized to modulate stereocilia height by trafficking actin regulatory proteins to their tip compartments, where actin polymerization must be precisely controlled during development. We identified a mutation (p.D1647G) in the MYO15 motor-domain that initially maintained trafficking, but caused progressive hearing loss by stunting stereocilia growth, revealing an additional function for MYO15. Consistent with its maintenance of tip trafficking in vivo, purified p.D1647G MYO15 modestly reduced actin-stimulated ATPase activity in vitro. Using ensemble and single-filament fluorescence in vitro assays, we demonstrated that wild-type MYO15 directly accelerated actin filament polymerization by driving nucleation, whilst p.D1647G MYO15 blocked this activity. Collectively, our studies suggest direct actin nucleation by MYO15 at the stereocilia tip is necessary for elongation in vivo, and that this is a primary mechanism disrupted in DFNB3 hereditary human hearing loss.

cell biology↗

Convergent evolution of Streptomyces protease inhibitors involving a tRNA-mediated condensation-minus NRPS

Small peptide aldehydes (SPAs) with protease inhibitory activity are natural products typically synthesized by nonribosomal peptide synthetases (NRPS). SPAs are widely used in biotechnology, as therapeutic agents, they are physiologically relevant and regulate development of the natural hosts. During genome evolutionary analysis of Streptomyces lividans 66 we identified an NRPS-like biosynthetic gene cluster (BGC) that lacked a condensation (C) domain but included a tRNA-Utilizing Enzyme (tRUE) belonging to the leucyl/phenylalanyl (L/F) transferase family. This system was predicted to direct the synthesis of a novel SPA with protease inhibitory activity, called livipeptin. Following genome mining and phylogenomic analyses we confirmed the presence of tRUEs within diverse Streptomyces genomes, including fusions with a C-minus NRPS-like protein. We further demonstrate functional cooperation between these enzymes and provide the biosynthetic rules for the synthesis of livipeptin, expanding the known universe of acetyl-leu/phe-arginal SPAs. The L/F-transferase C-minus NRPS productive interaction was shown to be tRNA-dependent after semisynthetic assays in the presence of RNAse, which contrasts with leupeptin, an acetyl-leu-arginal SPA that we show to be produced by Streptomyces roseous ATCC 31245 via a tRUE-minus BGC with multiple complete NRPSs. Thus, livipeptin and leupeptin are the result of convergent evolution, which has driven the appearance of unprecedented biosynthetic logics directing the synthesis of protease inhibitors thought to be at the core of Streptomyces colony biology. Our results pave the way for understanding this Streptomyces trait, as well as for the discovery of novel natural products following evolutionary genome mining approaches. Abstract importanceConvergent evolution in microbiology is believed to be highly recurrent yet examples that have been comprehensively characterized are scarce. Proteases inhibition by small peptide aldehydes is at the core of many microbiological processes, both within the cell and during colony development, and in microbial ecology. Here we report the biosynthetic foundations of leupeptin, the main Streptomyces protease inhibitor, and of livipeptin, a protease inhibitor produced by Streptomyces lividans. Although these peptides belong to the same chemical class, here we show that their biosynthetic routes result from convergent evolution, as they involve unrelated biosynthetic mechanisms, including the recruitment of a tRNA-utilizing enzyme that functionally replaces the condensation domain of a nonribosomal peptide synthetase during livipeptin biosynthesis. Thus, these results pave the way for understanding Streptomyces protease inhibitors as a trait and provide unprecedented knowledge for genome mining of natural products and synthetic biology where proteases inhibition is desirable.

microbiology↗

Hidden biodiversity in Neotropical streams: DNA barcoding uncovers high endemicity of freshwater macroinvertebrates at small spatial scales

Aquatic macroinvertebrates play a crucial role in freshwater ecosystems, but their diversity remains poorly known, particularly in the tropics. This "taxonomic void" represents limits our understanding of biodiversity patterns and processes in freshwater ecosystems, and the scale at which they operate. We used DNA barcoding to estimate lineage diversity (and the diversity of unique haplotypes) in 224 specimens of freshwater macroinvertebrates at a small spatial scale within the Panama Canal Watershed (PCW). In addition, we compiled available barcoding data to assess macroinvertebrate diversity at a broader spatial scale spanning the Isthmus of Panama. Consistently across two species delimitation algorithms (i.e., ABGD and GMYC), we found high lineage diversity within the PCW, with ~ 100-106 molecular operational taxonomic units (MOTUs at 2% sequence divergence) across 168 unique haplotypes. We also found a high lineage diversity along the Isthmus of Panama, but this diversity peaked within the PCW. However, our rarefaction/extrapolation approach showed that this diversity remains under sampled. As expected, these results indicate that the diversity of Neotropical freshwater macroinvertebrates is higher than previously thought, with the possibility of high endemicity even at narrow spatial scales. Geographic isolation is likely a main factor shaping these patterns of diversity. However, local disturbances such as the rupture of the continental divide due to the construction of the Panama Canal might be reshaping these patterns of diversity at a local scale. Although further work is needed to better understand the processes driving diversification in freshwater macroinvertebrates, we suggest that Neotropical streams represent continental islands of diversity. Understanding these islands of diversity is crucial in the face of increasing human disturbance.

evolutionary biology↗

Aberrant synaptic release underlies sleep/wake transition deficits in a mouse Vamp2 mutant

Sleep-wake transitions are modulated through extensive subcortical networks although the precise roles of their individual components remain elusive. Using forward genetics and in vivo electrophysiology, we identified a recessive mouse mutant line characterised by a reduced propensity to transition between all sleep states while a profound loss in total REM sleep time was evident. The causative mutation, an Ile102Asn substitution in VAMP2, was associated with substantial synaptic changes while in vitro electrophysiological investigations with fluorescence imaging revealed a diminished probability of vesicular release in mutants. We conclude that the synaptic efficiency of the entire subcortical brain network determines the likelihood that an animal transitions from one vigilance state to the next.

neuroscience↗

EvoMining reveals the origin and fate of naturalproducts biosynthetic enzymes

Natural products, or specialized metabolites, are important for medicine and agriculture alike, as well as for the fitness of the organisms that produce them. Microbial genome mining aims at extracting metabolic information from genomes of microbes presumed to produce these compounds. Typically, canonical enzyme sequences from known biosynthetic systems are identified after sequence similarity searches. Despite this being an efficient process the likelihood of identifying truly novel biosynthetic systems is low. To overcome this limitation we previously introduced EvoMining, a genome mining approach that incorporates evolutionary principles. Here, we release and use our latest version of EvoMining, which includes novel visualization features and customizable databases, to analyze 42 central metabolic enzyme families conserved throughout Actinobacteria, Cyanobacteria, Pseudomonas and Archaea. We found that expansion-and-recruitment profiles of these enzyme families are lineage specific, opening a new metabolic space related to shell enzymes, which have been overlooked to date. As a case study of canonical shell enzymes, we characterized the expansion and recruitment of glutamate dehydrogenase and acetolactate synthase into scytonemin biosynthesis, and into other central metabolic pathways driving microbial adaptive evolution. By defining the origins and fates of metabolic enzymes, EvoMining not only complements traditional genome mining approaches as an unbiased and rule-independent strategy, but it opens the door to gain insights into the evolution of natural products biosynthesis. We anticipate that EvoMining will be broadly used for metabolic evolutionary studies, and to generate genome-mining predictions leading to unprecedented chemical scaffolds and new antibiotics.\n\nDATA SUMMARYDatabases have been deposited at Zenodo; DOI: 10.5281/zenodo.1162336 http://zenodo.org/deposit/1219709\n\nTrees and metadata have been deposited in MicroReact\n\nGDH Actinobacteria https://microreact.org/project/r1IhjVm6X\n\nGDH Cyanobacteria https://microreact.org/project/HyjYUN7pQ)\n\nGDH Pseudomonas https://microreact.org/project/rJPC4EQa7\n\nGDH Archaea https://microreact.org/project/ByUcvNmaX\n\nALS Cyanobacteria https://microreact.org/project/B11HkUtdm\n\nEvoMining code has been deposited in gitHub https://github/nselem/evomining\n\nDocker container in Dockerhub https://hub.docker.com/r/nselem/evomining/\n\nWe confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.\n\nIMPACT STATEMENTEvoMining allows studying expansion-and-recruitment events of enzyme families in prokaryotic lineages, with the goal of providing both evolutionary insights and a genome mining approach for the discovery of truly novel natural products biosynthetic gene clusters. Thus, by better understanding the origin and fate of gene copies within enzyme families, this work contributes towards the identification of lineage-dependent enzymes that we call shell enzymes, which are ideal beacons to unveil chemical dark matter. We show that enzyme functionality is a continuum, including transition enzymes located between central and specialized metabolism. To exemplify these evolutionary dynamics, we focused in the genes directing the synthesis of the sunscreen peptide scytonemin, as the two key enzymes of this biosynthetic pathway behave as shell enzymes and were correctly identified by EvoMining. We also show how evolutionary approaches are better suited to study unexplored lineages, such as those belonging to the Archaea domain, which is systematically mined here for novel natural products for the first time. The release of EvoMining as a stand-alone tool will allow researchers to explore its own enzyme families of interest, within their own genomic lineages of expertise, by taking into account the lessons learned from this work

bioinformatics↗

Mitochondrial genomes and phylogenetic analysis of Central American weakly-electric fishes: Apteronotus rostratus, Brachyhypopomus occidentalis and Sternopygus dariensis

Electric fishes are a diverse group of freshwater organisms with the ability to generate electric organ discharges (EODs) that are used for communication and electrolocation. Over 200 species have originated in South America, but of these, only a few have managed to colonize the Central American Isthmus. Here, we assembled two complete and one nearly complete mitochondrial genomes (mitogenomes) for three Central American Gymnotiformes: Sternopygus dariensis, Brachyhypopomus occidentalis and Apteronotus rostratus. We then explored the three species phylogenetic position in the context of South American electric fishes. Mitogenomes were organized in the standard fish mitogenome order, and presented sizes of 16,600, 16,540 and 15,940 base pairs (bp) (nearly complete) for S. dariensis, B. occidentalis and A. rostratus, respectively. We uncovered a novel 60 bp intergenic spacer (IGS) located between the COII and tRNALys genes, which appears to be unique to the Apteronotidae. Furthermore, phylogenetic relationships supported the traditional monophyly of Gymnotiformes, with the three species positioned within their respective family. In addition, the genus Apteronotus was placed as the basal taxon of the order. Finally, we found high sequence divergence (13.3%) between our B. occidentalis specimen and a sequence previously reported in GenBank, suggesting that the prior mitogenome of B. occidentalis represents a different South American species that was misidentified. Indeed, phylogenetic analyses using Cytochrome b gene across the genus placed the previously reported individual within B. bennetti. Our study provides novel mitogenome resources that will advance our understanding of the diversity and phylogenetic history of Neotropical fishes.

evolutionary biology↗