Biology subjects

Rodriguez, D.

Publications and source records attributed to Rodriguez, D..

30 records · Page 2Linked to original sources

bioRxiv2022

Comparative Analysis of rRNA removal methods for RNA-seq Differential Expression in Halophilic Archaea

Despite intense recent research interest in archaea, the scientific community has experienced a bottleneck in the study of genome-scale gene expression experiments by RNA-seq due to the lack of commercial and specifically designed rRNA depletion kits. The high ratio rRNA:mRNA (80-90%: [~]10%) in prokaryotes hampers global transcriptomic analysis. Insufficient ribodepletion results in low sequence coverage of mRNA and therefore requires a substantially higher number of replicate samples and/or sequencing reads to achieve statistically reliable conclusions regarding the significance of differential gene expression between case and control samples. Here we show that after the discontinuation of the previous version of RiboZero (Illumina) that was useful to partially deplete rRNA from halophilic archaea, archaeal transcriptomics studies have experienced a standstill. To overcome this limitation, here we analyze the efficiency for four different hybridization-based kits from three different commercial suppliers, each with two sets of sequence-specific probes to remove rRNA from four different species of halophilic archaea. We conclude that the key for transcriptomic success with the currently available tools is the probe-specificity for the rRNA sequence hybridization. With this paper we provide insights to the archaeal community for selecting certain reagents and strategies over others depending on the archaeal species of interest. These methods yield improved RNA-seq sensitivity and enhanced detection of low abundance transcripts.

genomics↗

bioRxiv2022

Comparative host-pathogen dynamics of Snake Fungal Disease in sympatric species of water snakes (Nerodia)

The ascomycete fungus Ophidiomyces ophiodiicola (Oo) is the causative agent of ophidiomycosis (Snake Fungal Disease), which has been detected globally. However, surveillance efforts in the central U.S., specifically Texas, have been minimal. The threatened and rare Brazos water snake (Nerodia harteri harteri) is one of the most range restricted snakes in the U.S. and is sympatric with two wide-ranging congeners, N. erythrogaster transversa and N. rhombifer, in north central Texas; thus, providing an opportunity to test comparative host-pathogen dynamics in this system. To accomplish this, we surveyed a portion of the Brazos river drainage (~400 river km) over 29 months and tested 150 Nerodia spp. for the presence of Oo via quantitative PCR and recorded any potential signs of Oo infection. We found Oo was distributed across the entire range of N. h. harteri, Oo prevalence was 46 % overall, and there was a significant association between Oo occurrence and signs of infection in our sample. Models indicated adults had a higher probability of Oo infection than juveniles and subadults, and adult N. h. harteri had a higher probability of infection than adult N. rhombifer but not higher than adult N. e. transversa. High Oo prevalence estimates (94.4%) in adult N. h. harteri has implications for their conservation and management owing to their patchy distribution, comparatively low genetic diversity, and threats from anthropogenic habitat modification.

ecology↗

bioRxiv2021

Substrate, temperature, and geographical patterns among nearly 2,000 natural yeast isolates

Yeasts have broad importance as industrially and clinically relevant microbes and as powerful models for fundamental research, but we are only beginning to understand the roles yeasts play in natural ecosystems. Yeast ecology is often more difficult to study compared to other, more abundant microbes, but growing collections of natural yeast isolates are beginning to shed light on fundamental ecological questions. Here we used environmental sampling and isolation to assemble a dataset of 1,962 isolates collected from throughout the contiguous United States of America (USA) and Alaska, which were then used to uncover geographic patterns, along with substrate and temperature associations among yeast taxa. We found some taxa, including the common yeasts Torulaspora delbrueckii and Saccharomyces paradoxus, to be repeatedly isolated from multiple sampled regions of the US, and we classify these as broadly distributed cosmopolitan yeasts. A number of yeast taxon - substrate associations were identified, some of which were novel and some of which support previously reported associations. Further, we found a strong effect of isolation temperature on the phyla of yeasts recovered, as well as for many species. We speculate that substrate and isolation temperature associations reflect the ecological diversity of and niche partitioning by yeast taxa. Take AwayO_LIAnalysis of environmental metadata of nearly 2,000 yeast isolates. C_LIO_LIIndividual yeast taxa associate with specific substrates and plant genera. C_LIO_LIOptimal yeast isolation temperature differs depending on taxonomic rank. C_LIO_LISubstrate type and isolation temperatures affect isolated yeast diversity. C_LI

ecology↗

bioRxiv2021

Apo and ligand-bound high resolution Cryo-EM structures of the human Kv3.1 reveal a novel binding site for positive modulators

Kv3 ion-channels constitute a class of functionally distinct voltage gated ion channels characterized by their ability to fire at a high frequency. Several disease relevant mutants, together with biological data, suggest the importance of this class of ion channels as drug targets for CNS disorders, and several drug discovery efforts have been reported. Despite the increasing interest for this class of ion channels, no structure of a Kv3 channel has been reported yet. We have determined the cryo-EM structure of Kv3.1 at 2.6 [A] resolution using full length wild type protein. When compared to known structures for potassium channels from other classes, a novel domain organization is observed with the cytoplasmic T1 domain, containing a well resolved Zinc site and displaying a rotation by 35{degrees}. This suggests a distinct cytoplasmic regulation mechanism for the Kv3.1 channel. A high resolution structure was obtained for Kv3.1 in complex with a novel positive modulator Lu AG00563. The structure reveals a novel ligand binding site for the Kv class of ion channels located between the voltage sensory domain and the channel pore, a region which constitutes a hotspot for disease causing mutations. The discovery of a novel binding site for a positive modulator of a voltage gated potassium channel could shed light on the mechanism of action for these small molecule potentiators. This finding could enable structure-based drug design on these targets with high therapeutic potential for the treatment of multiple CNS disorders.

biochemistry↗

bioRxiv2020

Type I Interferon acts as a major barrier to the establishment of infectious bursal disease virus (IBDV) persistent infections

Infectious bursal disease virus (IBDV), the best characterized member of the Birnaviridae family, is a highly relevant avian pathogen causing both acute and persistent infections in different avian hosts. Here, we describe the establishment of clonal, long-term, productive persistent IBDV infections in DF-1 chicken embryonic fibroblasts. Although virus yields in persistently-infected cells are exceedingly lower than those detected in acutely infected cells, the replication fitness of viruses isolated from persistently-infected cells is higher than that of the parental virus. Persistently-infected DF-1 and IBDV-cured cell lines derived from them do not respond to type I interferon (IFN). High-throughput genome sequencing revealed that this defect is due to mutations affecting the IFN/{beta} receptor subunit 2 (IFNAR2) gene resulting in the expression of IFNAR2 polypeptides harbouring large C-terminal deletions that abolish the signalling capacity of IFN/{beta} receptor complex. Ectopic expression of a recombinant chicken IFNAR2 gene efficiently rescues IFN responsiveness. IBDV-cured cell lines derived from persistently infected cells exhibit a drastically enhanced proneness to establishing new persistent IBDV infections. Additionally, experiments carried out with human HeLa cells lacking the IFNAR2 gene fully recapitulate results obtained with DF-1 cells, exhibiting a highly enhanced capacity to both survive the acute IBDV infection phase and to support the establishment of persistent IBDV infections. Results presented here show that the inactivation of the JAK-STAT signalling pathway significantly reduces the apoptotic response induced by the infection, hence facilitating the establishment and maintenance of IBDV persistent infections. IMPORTANCEMembers of the Birnaviridae family, including infectious bursal disease virus (IBDV), exhibit a dual behaviour, causing acute infections that are often followed by the establishment of life-long persistent asymptomatic infections. Indeed, persistently infected specimens might act as efficient virus reservoirs, hence potentially contributing to virus dissemination. Despite the key importance of this biological trait, information about mechanisms triggering IBDV persistency is negligible. Our report evidences the capacity of IBDV, a highly relevant avian pathogen, to establishing long-term, productive, persistent infections in both avian and human cell lines. Data presented here provide novel and direct evidence about the crucial role of type I IFNs on the fate of IBDV-infected cells and their contribution to controlling the establishment of IBDV persistent infections. The use of cell lines unable to respond to type I IFNs opens a promising venue to unveiling additional factors contributing to IBDV persistency.

microbiology↗

bioRxiv2020

Inhibition of AlgU by MucA is required for viability in Pseudomonas aeruginosa

During decades-long infections in the cystic fibrosis (CF) airway, Pseudomonas aeruginosa undergoes selection. One bacterial genetic adaptation often observed in CF isolates is mucA mutations. MucA inhibits the sigma factor AlgU. Mutations in mucA lead to AlgU misregulation, resulting in a mucoid phenotype that is associated with poor CF disease outcomes. Due to its ability to be mutated, mucA is assumed to be dispensable for bacterial viability. Here we show that, paradoxically, a portion of mucA is essential in P. aeruginosa. We demonstrate that mucA is no longer required in a strain lacking algU, that mucA alleles encoding for proteins that do not bind to AlgU are insufficient for viability, and that mucA is no longer essential in mutant strains containing AlgU variants with reduced sigma factor activity. Furthermore, we found that overexpression of algU prevents cell growth in the absence of MucA, and that this phenotype can be rescued by overproduction of RpoD, the housekeeping sigma factor. Together, these results suggest that in the absence of MucA, the inability to regulate AlgU activity results in the loss of bacterial viability. Finally, we speculate that essentiality of anti-sigma factors that regulate envelope function may be a widespread phenomenon in bacteria.

microbiology↗

bioRxiv2020

Birnavirus Ribonucleoprotein Assembly

The interaction of the structural VP3 polypeptide of infectious bursal disease virus (IBDV) with virus-encoded dsRNA is essential both for the assembly of ribonucleoprotein complexes responsible for genome transcription and replication and for the evasion of hosts antiviral responses. Surface plasmon resonance analysis allowed us to determine the kinetic constants of the VP3-dsRNA interaction as well as to map the VP3 dsRNA bipartite dsRNA binding domain (dsRBD), uncovering the specific role of the previously described Patch1 and Patch2 dsRB subdomains. Here we show that the Patch1 domain plays a primary binding function while Patch2 exerts a subordinate role stabilizing VP3-dsRNA complexes. The use of a set of VP3 mutant versions facilitated the identification of K99 and K106 within Patch1 as the essential residues for the formation of VP3-dsRNA complexes. Furthermore, replacement of either one of these two residues by aspartic acid completely thwarts both evasion from hosts sensors and virus replication. Data presented here allow us to propose a VP3-dsRNA interaction model that should help to further elucidate the mechanics of IBDV morphogenesis and genome packaging as well as to better understand how VP3 counteracts recognition of virus-encoded dsRNA by specialized hosts sensors.

microbiology↗

bioRxiv2020

Multilevel community assembly of the tadpole gut microbiome

The assembly of local communities is likely to reflect the effects of local environmental factors associated with filters that act at larger spatial scales. Dissecting these multiscale effects remains a timely challenge that is particularly important for host-associated microbiomes. We investigated the relative roles of local selection (due to host species identity) and regional effects (due to water body identity) on the community structure of bacteria in the gut of tadpoles from three biogeographic areas and used graph theory and metanetwork approaches to explore and illustrate the distribution of bacteria across different ponds. The pond of origin, which represents a regional species pool of bacteria, was in general more important in shaping the gut microbiome of tadpoles than host species identity. The resulting metanetworks are modular and indicate relatively few species of bacteria occurring in more than one pond. Thus, each pond represents a relatively distinct species pool of bacteria available for community assembly of the tadpole microbiomes. Our findings indicate that microbiome community assembly in amphibian larvae, as in many other communities, is a multiscale process with important regional effects that constrain how local (i.e. host-dependent) filters act to influence microbiome community composition.Competing Interest StatementThe authors have declared no competing interest.View Full Text

ecology↗

bioRxiv2019

Genetic and morphological variation is associated with differences in cold-water tolerance and geographic expansion among invasive snails (Melanoides tuberculata) in central Texas

Melanoides tuberculata (Thiaridae) is an old-world freshwater snail that is and now circumtropical. After being introduced in the 1960s via the aquarium trade, populations of M. tuberculata are thriving in spring systems of Texas (USA). Field surveys and experimental investigations of temperature tolerance suggest M. tuberculata is stenothermal, and thus range expansions outside of aquatic habitats with water temperatures between 18{degrees} and 32{degrees}C should be unlikely. However, in 2012 snails were detected in natural aquatic habitats with seasonal temperatures below the experimentally determined lethal thermal minimum. To test whether genetic and phenotypic variation might be associated with cold-water tolerance and range expansion, we sequenced the 16S ribosomal rRNA gene and measured qualitative conch morphology of 170 snails collected at 26 sites in three central Texas rivers. We also conducted phylogenetic analyses of M. tuberculata collected globally and in Texas to determine potential source populations and estimate the number of invasion events. Our results show snails detected in variable temperature habitats are genetically divergent and morphologically distinct from snails collected in habitats with stable temperatures. These data are consistent with at least three introduction events into Texas of M. tuberculata sensu lato that are characterized by distinct habitat preferences, physiological tolerances, and/or adaptive behaviors.

zoology↗

bioRxiv2018

Genetic characterization of chytrids isolated from larval amphibians collected in central and east Texas

Chytridiomycosis, an emerging infectious disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), has caused amphibian population declines worldwide. Bd was first described in the 1990s and there are still geographic gaps in the genetic analysis of this globally distributed pathogen. Relatively few genetic studies have focused on regions where Bd exhibits low virulence, potentially creating a bias in our current knowledge of the pathogens genetic diversity. Disease-associated declines have not been recorded in Texas (USA), yet Bd has been detected on amphibians in the state. These strains have not been isolated and characterized genetically; therefore, we isolated, cultured, and genotyped Bd from central Texas and compared isolates to a panel of previously genotyped strains distributed across the Western Hemisphere. We also isolated other chytrids not known to infect amphibians from east Texas. To identify larval amphibian hosts, we sequenced part of the COI gene. Among 37 Bd isolates from Texas, we detected 19 unique multi-locus genotypes, but found no genetic structure associated with host species, Texas localities, or across North America. Isolates from central Texas exhibit high diversity and genetically cluster with Bd-GPL isolates from the western U.S. that have caused amphibian population declines. This study genetically characterizes isolates of Bd from the south central U.S. and adds to the global knowledge of Bd genotypes.

evolutionary biology↗

bioRxiv2018

Pathogenic DDX3X mutations impair RNA metabolism and neurogenesis during fetal cortical development

De novo germline mutations in the RNA helicase DDX3X account for 1-3% of unexplained intellectual disability (ID) cases in females, and are associated with autism, brain malformations, and epilepsy. Yet, the developmental and molecular mechanisms by which DDX3X mutations impair brain function are unknown. Here we use human and mouse genetics, and cell biological and biochemical approaches to elucidate mechanisms by which pathogenic DDX3X variants disrupt brain development. We report the largest clinical cohort to date with DDX3X mutations (n=78), demonstrating a striking correlation between recurrent dominant missense mutations, polymicrogyria, and the most severe clinical outcomes. We show that Ddx3x controls cortical development by regulating neuronal generation and migration. Severe DDX3X missense mutations profoundly disrupt RNA helicase activity and induce ectopic RNA-protein granules and aberrant translation in neural progenitors and neurons. Together, our study demonstrates novel mechanisms underlying DDX3X syndrome, and highlights roles for RNA-protein aggregates in the pathogenesis of neurodevelopmental disease.

genetics↗

bioRxiv2017

Improving data availability for brain image biobanking in healthy subjects: practice-based suggestions from an international multidisciplinary working group

Brain imaging is now ubiquitous in clinical practice and research. The case for bringing together large amounts of image data from well-characterised healthy subjects and those with a range of common brain diseases across the life course is now compelling. This report follows a meeting of international experts from multiple disciplines, all interested in brain image biobanking. The meeting included neuroimaging experts (clinical and non-clinical), computer scientists, epidemiologists, clinicians, ethicists, and lawyers involved in creating brain image banks. The meeting followed a structured format to discuss current and emerging brain image banks; applications such as atlases; conceptual and statistical problems (e.g. defining normality); legal, ethical and technological issues (e.g. consents, potential for data linkage, data security, harmonisation, data storage and enabling of research data sharing). We summarise the lessons learned from the experiences of a wide range of individual image banks, and provide practical recommendations to enhance creation, use and reuse of neuroimaging data. Our aim is to maximise the benefit of the image data, provided voluntarily by research participants and funded by many organisations, for human health. Our ultimate vision is of a federated network of brain image biobanks accessible for large studies of brain structure and function.

neuroscience↗