Genetic transformation of the frog-killing chytrid fungus Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis, is decimating amphibian populations around the world. Bd belongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids, Bd develops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to study Bd cell biology, development, and pathogenicity have been limited by the lack of genetic tools with which to test hypotheses about underlying molecular mechanisms. Here, we report the development of a transient genetic transformation system for Bd. We used electroporation to deliver exogenous DNA into Bd cells and detected transgene expression for up to three generations under both heterologous and native promoters. We also adapted the transformation protocol for selection using an antibiotic resistance marker. Finally, we used this system to express fluorescent protein fusions and, as a proof of concept, expressed a genetically encoded probe for the actin cytoskeleton. Using live-cell imaging, we visualized the distribution and dynamics of polymerized actin at each stage of the Bd life cycle, as well as during key developmental transitions. This transformation system enables direct testing of key hypotheses regarding mechanisms of Bd pathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology.

Growth hormone-releasing hormone receptor antagonist MIA-602 attenuates cardiopulmonary injury induced by BSL-2 rVSV-SARS-CoV-2 in hACE2 mice.

COVID-19 pneumonia causes acute lung injury and acute respiratory distress syndrome (ALI/ARDS) characterized by early pulmonary endothelial and epithelial injuries with altered pulmonary diffusing capacity and obstructive or restrictive physiology. Growth hormone-releasing hormone receptor (GHRH-R) is expressed in the lung and heart. GHRH-R antagonist, MIA-602, has been reported to modulate immune responses to bleomycin lung injury and inflammation in granulomatous sarcoidosis. We hypothesized that MIA-602 would attenuate rVSV-SARS-CoV-2-induced pulmonary dysfunction and heart injury in a BSL-2 mouse model. Male and female K18-hACE2tg mice were inoculated with SARS-CoV-2/USA-WA1/2020, BSL-2-compliant recombinant VSV-eGFP-SARS-CoV-2-Spike (rVSV-SARS-CoV-2), or PBS, and lung viral load, weight loss, histopathology, and gene expression were compared. K18-hACE2tg mice infected with rVSV-SARS-CoV-2 were treated daily with subcutaneous MIA-602 or vehicle and conscious, unrestrained plethysmography performed on days 0, 3, and 5 (n = 7 to 8). Five days after infection mice were killed, and blood and tissues collected for histopathology and protein/gene expression. Both native SARS-CoV-2 and rVSV-SARS-CoV-2 presented similar patterns of weight loss, infectivity (~60%), and histopathologic changes. Daily treatment with MIA-602 conferred weight recovery, reduced lung perivascular inflammation/pneumonia, and decreased lung/heart ICAM-1 expression compared to vehicle. MIA-602 rescued altered respiratory rate, increased expiratory parameters (Te, PEF, EEP), and normalized airflow parameters (Penh and Rpef) compared to vehicle, consistent with decreased airway inflammation. RNASeq followed by protein analysis revealed heightened levels of inflammation and end-stage necroptosis markers, including ZBP1 and pMLKL induced by rVSV-SARS-CoV-2, that were normalized by MIA-602 treatment, consistent with an anti-inflammatory and pro-survival mechanism of action in this preclinical model of COVID-19 pneumonia.

Gibbs-Helmholtz Graph Neural Network for the Prediction of Activity Coefficients of Polymer Solutions at Infinite Dilution.

Machine learning models have gained prominence for predicting pure-component properties, yet their application to mixture property prediction remains relatively limited. However, the significance of mixtures in our daily lives is undeniable, particularly in industries such as polymer processing. This study presents a modification of the Gibbs-Helmholtz graph neural network (GH-GNN) model for predicting weight-based activity coefficients at infinite dilution (Ωij∞) in polymer solutions. We evaluate various polymer representations ranging from monomer, repeating unit, periodic unit, and oligomer and observe that, in data-scarce scenarios of polymer-solvent mixtures, polymer representation specifics have a reduced impact compared to data-rich environments. Leveraging transfer learning, we harness richer activity coefficient data from small-size systems, enhancing model accuracy and reducing prediction variability. The modified GH-GNN model achieves remarkable prediction results in mixture interpolation and solvent extrapolation tasks having an overall mean absolute error of 0.15, showcasing the potential of graph-neural-network-based models for property prediction of polymer solutions. Comparative analysis with the established models UNIFAC-ZM and Entropic-FV suggests a promising avenue for future research on the use of data-driven models for the prediction of the thermodynamic properties of polymer solutions.

Genome analysis of Pseudomonas sp. 14A reveals metabolic capabilities to support epiphytic behavior.

The surface of aboveground plant parts, known as the phyllosphere, is a habitat for various microorganisms called epiphytes establishing biotrophic interactions with their hosts. However, these communities can be affected by environmental and anthropogenic variations such as the application of agrochemicals. Thus, epiphytes have the capacity to survive in such environments. In this study, we obtained the genome of Pseudomonas sp. 14A, an epiphyte isolated from the pepper phyllosphere. The phylogenomic analyses suggested that Pseudomonas sp. 14A may be novel species closely related to P. moraviensis R28-S. Notably, the metabolic pathways proposed consistent with epiphytic lifestyle in Pseudomonas sp. 14A, were shared with other species displaying a different degree of phylogenetic relatedness. Furthermore, variations in configuration of metabolic gene clusters were observed, that could expand microbial metabolic diversity in close relatedness species, highlighting the relevance of microbial diversity associated with plants.

Conservation and divergence of C-terminal domain structure in the retinoblastoma protein family.

The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD-CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein-E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.

Gene duplication and co-evolution of G1/S transcription factor specificity in fungi are essential for optimizing cell fitness.

Transcriptional regulatory networks play a central role in optimizing cell survival. How DNA binding domains and cis-regulatory DNA binding sequences have co-evolved to allow the expansion of transcriptional networks and how this contributes to cellular fitness remains unclear. Here we experimentally explore how the complex G1/S transcriptional network evolved in the budding yeast Saccharomyces cerevisiae by examining different chimeric transcription factor (TF) complexes. Over 200 G1/S genes are regulated by either one of the two TF complexes, SBF and MBF, which bind to specific DNA binding sequences, SCB and MCB, respectively. The difference in size and complexity of the G1/S transcriptional network across yeast species makes it well suited to investigate how TF paralogs (SBF and MBF) and DNA binding sequences (SCB and MCB) co-evolved after gene duplication to rewire and expand the network of G1/S target genes. Our data suggests that whilst SBF is the likely ancestral regulatory complex, the ancestral DNA binding element is more MCB-like. G1/S network expansion took place by both cis- and trans- co-evolutionary changes in closely related but distinct regulatory sequences. Replacement of the endogenous SBF DNA-binding domain (DBD) with that from more distantly related fungi leads to a contraction of the SBF-regulated G1/S network in budding yeast, which also correlates with increased defects in cell growth, cell size, and proliferation.

The evolution of a G1/S transcriptional network in yeasts.

The G1-to-S cell cycle transition is promoted by the periodic expression of a large set of genes. In Saccharomyces cerevisiae G1/S gene expression is regulated by two transcription factor (TF) complexes, the MBF and SBF, which bind to specific DNA sequences, the MCB and SCB, respectively. Despite extensive research little is known regarding the evolution of the G1/S transcription regulation including the co-evolution of the DNA binding domains with their respective DNA binding sequences. We have recently examined the co-evolution of the G1/S TF specificity through the systematic generation and examination of chimeric Mbp1/Swi4 TFs containing different orthologue DNA binding domains in S. cerevisiae (Hendler et al. in PLoS Genet 13:e1006778. doi: 10.1371/journal.pgen.1006778 , 2017). Here, we review the co-evolution of G1/S transcriptional network and discuss the evolutionary dynamics and specificity of the MBF-MCB and SBF-SCB interactions in different fungal species.

Complex history of the amphibian-killing chytrid fungus revealed with genome resequencing data.

Understanding the evolutionary history of microbial pathogens is critical for mitigating the impacts of emerging infectious diseases on economically and ecologically important host species. We used a genome resequencing approach to resolve the evolutionary history of an important microbial pathogen, the chytrid Batrachochytrium dendrobatidis (Bd), which has been implicated in amphibian declines worldwide. We sequenced the genomes of 29 isolates of Bd from around the world, with an emphasis on North, Central, and South America because of the devastating effect that Bd has had on amphibian populations in the New World. We found a substantial amount of evolutionary complexity in Bd with deep phylogenetic diversity that predates observed global amphibian declines. By investigating the entire genome, we found that even the most recently evolved Bd clade (termed the global panzootic lineage) contained more genetic variation than previously reported. We also found dramatic differences among isolates and among genomic regions in chromosomal copy number and patterns of heterozygosity, suggesting complex and heterogeneous genome dynamics. Finally, we report evidence for selection acting on the Bd genome, supporting the hypothesis that protease genes are important in evolutionary transitions in this group. Bd is considered an emerging pathogen because of its recent effects on amphibians, but our data indicate that it has a complex evolutionary history that predates recent disease outbreaks. Therefore, it is important to consider the contemporary effects of Bd in a broader evolutionary context and identify specific mechanisms that may have led to shifts in virulence in this system.

[Characterization of hemodynamic ex vivo model to study endothelial activation by TNF-α in prefunded human veins]. / Caracterización de un modelo hemodinámico ex vivo para el estudio de la activación endotelial por el factor de necrosis tumoral α (TNF-α) en venas humanas perfundidas.

Inflammation is recognized as part of the etiology of numerous diseases. The interaction among cells of the immunological system with local cells and molecules, such as cytokines and chemokines, allows cellular activation and response amplification. The importance of several physicochemical factors like frictional force, vascular flow, shear stress, and pressure is now recognized because they are known to modulate genetic expression and endothelial activation; however, there are very few studies that recreate such cellular microenvironments. Hence, it is of paramount importance to develop new models that will mimic physiological conditions. Our aim was to improve a human vein ex vivo model that would allow endothelial activation in flow conditions, to study the molecular components during adhesion, taking into consideration physicochemical parameters such as flow and shear stress. Endothelial umbilical human vein was used and activated with TNF-a in order to determine U937 monocytic cells adhesion, as well as cytokines secretion and ICAM-1 expression. This model will allow leukocyte adhesion studies, using different inflammatory stimulus, along with the signaling pathways involved in several pathologies.

Tools for live-cell imaging of cytoskeletal and nuclear behavior in the unconventional yeast, Aureobasidium pullulans.

Aureobasidium pullulans is a ubiquitous fungus with a wide variety of morphologies and growth modes including "typical" single-budding yeast, and interestingly, larger multinucleate yeast than can make multiple buds in a single cell cycle. The study of A. pullulans promises to uncover novel cell biology, but currently tools are lacking to achieve this goal. Here, we describe initial components of a cell biology toolkit for A. pullulans, which is used to express and image fluorescent probes for nuclei as well as components of the cytoskeleton. These tools allowed live-cell imaging of the multinucleate and multibudding cycles, revealing highly synchronous mitoses in multinucleate yeast that occur in a semiopen manner with an intact but permeable nuclear envelope. These findings open the door to using this ubiquitous polyextremotolerant fungus as a model for evolutionary cell biology.

A guide to Agrobacterium-mediated transformation of the chytrid fungus Spizellomyces punctatus.

Chytrid fungi play key ecological roles in aquatic ecosystems, and some species cause a devastating skin disease in frogs and salamanders. Additionally, chytrids occupy a unique phylogenetic position- sister to the well-studied Dikarya (the group including yeasts, sac fungi, and mushrooms) and related to animals- making chytrids useful for answering important evolutionary questions. Despite their importance, little is known about the basic cell biology of chytrids. A major barrier to understanding chytrid biology has been a lack of genetic tools with which to test molecular hypotheses. Medina and colleagues recently developed a protocol for Agrobacterium -mediated transformation of Spizellomyces punctatus. In this manuscript, we describe the general procedure including planning steps and expected results. We also provide in-depth, step-by-step protocols and video guides for performing the entirety of this transformation procedure on protocols.io (dx.doi.org/10.17504/protocols.io.x54v9dd1pg3e/v1).

Preventive Measures among Healthcare Workers (HCWs) during the COVID-19 Pandemic.

COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), placed health systems worldwide under immense pressure, and healthcare workers (HCWs) were at the front lines. The Puerto Rico Department of Health confirmed the first case of COVID-19 in March 2020. We aimed to assess whether COVID-19 preventive measures implemented by HCWs were effective in a work scenario before vaccine availability. We conducted a descriptive cross-sectional study from July to December 2020 to evaluate the use of personal protective equipment (PPE), hygiene guidelines, and other measures taken by HCWs to prevent the spread of SARS-CoV-2. We collected nasopharyngeal specimens for molecular testing at the beginning of the study and follow-up. We recruited 62 participants aged 30-59 (79% women). Participants recruited from hospitals, clinical laboratories, and private practice included medical technologists (33%), nurses (28%), respiratory therapists (2%), physicians (11%), and others (26%). Among our participants, nurses were at higher risk (p < 0.05) of infection. We identified that 87% of participants followed the hygiene recommendation guidelines. In addition, all participants practiced handwashing or disinfection before or after caring for each patient. All participants tested negative for SARS-CoV-2 during the study period. On follow-up, all study participants reported being vaccinated against COVID-19. The implementation of PPE and hygiene measures showed high efficacy as a prevention method against SARS-CoV-2 infection when vaccines and treatment were not widely available in Puerto Rico.

Reconstructing the fungal tree of life using phylogenomics and a preliminary investigation of the distribution of yeast prion-like proteins in the fungal kingdom.

We have used three independent phylogenomic approaches (concatenated alignments, single-, and multi-gene supertrees) to reconstruct the fungal tree of life (FTOL) using publicly available fungal genomes. This is the first time multi-gene families have been used in fungal supertree reconstruction and permits us to use up to 66% of the 1,001,217 genes in our fungal database. Our analyses show that different phylogenomic datasets derived from varying clustering criteria and alignment orientation do not have a major effect on phylogenomic supertree reconstruction. Overall the resultant phylogenomic trees are relatively congruent with one another and successfully recover the major fungal phyla, subphyla and classes. We find that where incongruences do occur, the inferences are usually poorly supported. Within the Ascomycota phylum, our phylogenies reconstruct monophyletic Saccharomycotina and Pezizomycotina subphyla clades and infer a sister group relationship between these to the exclusion of the Taphrinomycotina. Within the Pezizomycotina subphylum, all three phylogenies infer a sister group relationship between the Leotiomycetes and Sordariomycetes classes. However, there is conflict regarding the relationships with the Dothideomycetes and Eurotiomycetes classes. Within the Basidiomycota phylum, supertrees derived from single- and multi-gene families infer a sister group relationship between the Pucciniomycotina and Agaricomycotina subphyla while the concatenated phylogeny infers a poorly supported relationship between the Agaricomycotina and Ustilagomycotina. The reconstruction of a robust FTOL is important for future fungal comparative analyses. We illustrate this point by performing a preliminary investigation into the phyletic distribution of yeast prion-like proteins in the fungal kingdom.

Targeted RNA-Seq Reveals the M. tuberculosis Transcriptome from an In Vivo Infection Model.

The study of host-pathogen interactions using in vivo models with intracellular pathogens like Mycobacterium tuberculosis (Mtb) entails technical limitations, such as: (i) Selecting an efficient differential lysis system to enrich the pathogen cells; (ii) obtaining sufficient high-quality RNA; and (iii) achieving an efficient rRNA depletion. Thus, some authors had used flow cytometers to separate infected cells or significantly increase the sequencing depth of host-pathogen RNA libraries to observe the pathogens' gene expression. However, these options carry additional expenses in specialized equipment typically not available for all laboratories. Here, we propose an experimental protocol involving differential cell lysis and a probe-based ribosomal depletion to determine the gene expression of Mtb and its host during in vivo infection. This method increased the number of observed pathogen-expressed genes from 13 using the traditional RNA-seq approach to 702. After eliminating rRNA reads, we observed that 61.59% of Mtb sequences represented 702 genes, while 38.41% represented intergenic regions. Some of the most expressed genes codified for IS1081 (Rv2512c) transposase and eight PE-PGRS members, such as PGRS49 and PGRS50. As expected, a critical percent of the expressed genes codified for secreted proteins essential for infection, such as PE68, lppN, and LpqH. Moreover, three Mtb ncRNAs were highly expressed (small RNA MTS2823, transfer-messenger RNA RF00023, and ribozyme RF00010). Many of the host-expressed genes were related to the inflammation process and the expression of surfactant proteins such as the Sftpa and Sftpc, known to bind Mtb to alveolar macrophages and mi638, a microRNA with no previous associations with pulmonary diseases. The main objective of this study is to present the method, and a general catalog of the Mtb expressed genes at one point of the in vivo infection. We believe our method represents a different approach to the existing ones to study host-pathogen interactions in tuberculosis and other similar intracellular infections, without the necessity of specialized equipment.

Gut dsDNA virome shows diversity and richness alterations associated with childhood obesity and metabolic syndrome.

Changes in the human gut microbiome are associated with obesity and metabolic syndrome, but the role of the gut virome in both diseases remains largely unknown. We characterized the gut dsDNA virome of 28 school-aged children with healthy normal-weight (NW, n = 10), obesity (O, n = 10), and obesity with metabolic syndrome (OMS, n = 8), using metagenomic sequencing of virus-like particles (VLPs) from fecal samples. The virome classification confirmed the bacteriophages' dominance, mainly composed of Caudovirales. Notably, phage richness and diversity of individuals with O and OMS tended to increase, while the VLP abundance remained the same among all groups. Of the 4,611 phage contigs composing the phageome, 48 contigs were highly prevalent in ≥80% of individuals, suggesting high inter-individual phage diversity. The abundance of several contigs correlated with gut bacterial taxa; and with anthropometric and biochemical parameters altered in O and OMS. To our knowledge, this gut phageome represents one of the largest datasets and suggests disease-specific phage alterations.

Chytrid fungi.

Medina and Buchler provide an introduction to chytrid fungi, an early diverging fungal lineage exhibiting characteristics found in both animals and fungi.

Serendipity in the wrestle between Trichoderma and Metarhizium.

The fungal species Trichoderma is frequently found in soil antagonizing plant-pathogenic fungi as well as parasitizing plant-pathogenic nematodes. Metarhizium species are insect-pathogenic fungi that are used throughout the world to control agricultural insect pests. Here, we determine whether the antagonism (A) of Trichoderma atroviride to Metarhizium robertsii during growth and spore formation can impact the stress biology of M. robertsii conidia. Cultures of M. robertsii were either produced without exposure to T. atroviride (control) or in the presence of T. atroviride. M. robertsii was grown in dual culture with T. atroviride on potato dextrose agar (PDA) using the following treatments: 1) Trichoderma inoculated at the same time with Metarhizium (A0); 2) Trichoderma inoculated two days after the inoculation of Metarhizium (A2); 3) Trichoderma inoculated four days after Metarhizium (A4); 4) Trichoderma inoculated 6 d after Metarhizium (A6); 5) M. robertsii grown alone on PDA medium (control); and 6) M. robertsii grown alone on minimal medium (Czapek-Dox medium without sucrose) (MM). Germination of M. robertsii conidia from all six treatments was then assessed under osmotic, oxidative, UV-B, and thermal stress. M. robertsii conidia produced on MM were the most tolerant to all stress conditions. For all stress conditions, conidia from treatments A0 and A2 were not viable. For osmotic stress, conidia produced in treatment A4 were the most tolerant, followed by conidia from treatment A6, which were both more tolerant than the control. For oxidative stress, conidia produced in both A4 and A6 treatments were similarly tolerant and more tolerant than conidia produced in the control. For thermal stress, conidia produced in treatments A4, A6, and control (PDA) were similarly heat-tolerant. For UV-B stress, conidia produced in treatments A4 and A6 were equally tolerant and more tolerant than conidia produced in the control. The germination speed of conidia produced in all treatments, A0, A2, A4, and A6 was also tested. Conidia produced on MM germinated faster than the other treatments. Conidia produced in the A4 treatment were the second fastest, followed by conidia produced in treatment A6. Both A4 and A6 conidia germinated faster than conidia produced in the control treatment. Conidia produced in the treatments A0 and A2 did not germinate in 24 h. In summary, moderate levels of biotic stress from a fungal competitor or low-nutrient conditions can enhance the stress tolerance of M. robertsii conidia.

Utility of oxcarbazepine in the treatment of childhood and adolescent psychiatric symptoms.

The primary aims of this study were to determine if oxcarbazepine is a safely tolerated option for treatment of psychiatric symptoms in children and whether its use facilitates dose modification of other psychotropic medications. A retrospective chart review was completed using data extracted from the electronic medical record of a large outpatient child psychiatry clinic. A total of 507 of 740 children prescribed oxcarbazepine for psychiatric indications for 3 months or more had adequate data to assess clinical responses and medication outcomes. Most patients prescribed oxcarbazepine experienced clinically significant control of irritability/anger, mood stabilization, aggressive outbursts, impulsivity, or anxiety, with over 80% achieving at least maintenance symptom control. In all, 51% and 25% fully discontinued second- or third-generation antipsychotic or antidepressant medication, respectively, after starting oxcarbazepine; 8% discontinued oxcarbazepine for nonresponse, while 9% stopped oxcarbazepine because of emergent side effects. In patients fully discontinuing or reducing the second- or third-generation antipsychotic dose by 50% or more, improvements in body mass index were observed. Oxcarbazepine may prove to be an appropriate alternative to antipsychotic and antidepressant medications for treating psychiatric symptoms in children and adolescents. In particular, it may be a more metabolically neutral psychotropic medication.

Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology.

Chytrids are early-diverging fungi that share features with animals that have been lost in most other fungi. They hold promise as a system to study fungal and animal evolution, but we lack genetic tools for hypothesis testing. Here, we generated transgenic lines of the chytrid Spizellomyces punctatus, and used fluorescence microscopy to explore chytrid cell biology and development during its life cycle. We show that the chytrid undergoes multiple rounds of synchronous nuclear division, followed by cellularization, to create and release many daughter 'zoospores'. The zoospores, akin to animal cells, crawl using actin-mediated cell migration. After forming a cell wall, polymerized actin reorganizes into fungal-like cortical patches and cables that extend into hyphal-like structures. Actin perinuclear shells form each cell cycle and polygonal territories emerge during cellularization. This work makes Spizellomyces a genetically tractable model for comparative cell biology and understanding the evolution of fungi and early eukaryotes.

Evolutionary innovation, fungal cell biology, and the lateral gene transfer of a viral KilA-N domain.

Fungi are found in diverse ecological niches as primary decomposers, mutualists, or parasites of plants and animals. Although animals and fungi share a common ancestor, fungi dramatically diversified their life cycle, cell biology, and metabolism as they evolved and colonized new niches. This review focuses on a family of fungal transcription factors (Swi4/Mbp1, APSES, Xbp1, Bqt4) derived from the lateral gene transfer of a KilA-N domain commonly found in prokaryotic and eukaryotic DNA viruses. These virus-derived fungal regulators play central roles in cell cycle, morphogenesis, sexual differentiation, and quiescence. We consider the possible origins of KilA-N and how this viral DNA binding domain came to be intimately associated with fungal processes.