title: HREM, RNAseq and cell-cycle analyses reveal the role of the G2/M-regulatory protein, Wee1, on the survivability of chicken embryos during diapause.

abstract: Avian blastoderm can enter into diapause when kept at low temperatures, and successfully resume development (SRD) when re-incubated in body-temperature. These abilities, which are largely affected by the temperature and duration of the diapause, are poorly understood at the cellular and molecular level. To determine how temperature affects embryonic morphology during diapause, High-Resolution Episcopic Microscopy (HREM) analysis was utilized. While blastoderms diapausing at 12{degrees}C for 28 days presented typical cytoarchitecture, similar to non-diapaused embryos, at 18{degrees}C much thicker blastoderms with higher cell-number were observed. RNAseq was conducted to discover the genes underlying these phenotypes, revealing differentially-expressed cell-cycle regulatory genes. Amongst them, Wee1, a negative-regulator of G2/M transition, was highly expressed at 12{degrees}C compared to 18{degrees}C. This finding suggested that cells at 12{degrees}C are arrested at the G2/M phase, as supported by bromodeoxyuridine incorporation (BrdU) assay and phosho-histone-H3 (pH3) immuno-staining. Inhibition of Wee1 during diapause at 12{degrees}C resulted in cell-cycle progression beyond the G2/M and augmented tissue volume, resembling the morphology of 18{degrees}C-diapaused embryos. These findings suggest that diapause at low temperatures leads to Wee1 upregulation which arrests the cell-cycle at the G2/M phase, promoting the perseverance of embryonic cytoarchitecture and future SRD. In contrast, Wee1 is not up-regulated during diapause at higher temperature, leading to continuous proliferation and maladaptive morphology associated with poor survivability. Combining HREM-based analysis with RNAseq and molecular manipulations, we present a novel mechanism that regulates the ability of diapaused-avian embryos to maintain their cytoarchitecture via cell-cycle arrest, which enables their SRD.


title: Long read mitochondrial genome sequencing using Cas9-guided adaptor ligation

abstract: The mitochondrial genome (mtDNA) is an important source of disease-causing genetic variability, but existing sequencing methods limit understanding, precluding phased measurement of mutations and clear detection of large sporadic deletions. We adapted a method for amplification-free sequence enrichment using Cas9 cleavage to obtain full length nanopore reads of mtDNA. We then utilized the long reads to phase mutations in a patient with an mtDNA-linked syndrome and demonstrated that this method can map age-induced mtDNA deletions. We believe this method will offer deeper insight into our understanding of mtDNA variation.

category: genomics


title: Microcompartment assembly around multicomponent fluid cargoes

abstract: This article describes dynamical simulations of the assembly of an icosahedral protein shell around a bi-component fluid cargo. Our simulations are motivated by bacterial microcompartments, which are protein shells found in bacteria that assemble around a complex of enzymes and other components involved in certain metabolic processes. The simulations demonstrate that the relative interaction strengths among the different cargo species play a key role in determining the amount of each species that is encapsulated, their spatial organization, and the nature of the shell assembly pathways. However, the shell protein-protein and shell-cargo interactions that help drive assembly and encapsulation also influence cargo composition within certain parameter regimes. These behaviors are governed by combination of thermodynamic and kinetic effects. In addition to elucidating how natural microcompartments encapsulate multiple components involved within reaction cascades, these results have implications for synthetic biology efforts to colocalize alternative sets of molecules within microcompartments to accelerate reactions. More broadly, the results suggest that coupling between self-assembly and multicomponent liquid-liquid phase separation may play a role in organization of the cellular cytoplasm.

category: biophysics


title: AIMP1-derived peptide secreted from hair follicle stem cells activates dermal papilla cells to promote hair growth

abstract: Hair follicle stem cells (HFSCs) and dermal papilla cells (DPCs) are crucial in the biogenesis and maintenance of hair follicles (HFs). In this study, a fragment derived from aminoacyl-tRNA synthetase-interacting multifunctional protein1 (AIMP1) was secreted from HFSCs to activate DPCs to maintain hair follicle homeostasis. A histological analysis revealed that AIMP1 levels in hair follicles decreased with hair loss. Hair regrowth in AIMP1-induced mice was faster than that in non-induced mice. Deletion mapping revealed 41 amino acids (TN41, aa 6-46) as the active region of AIMP1. The N-terminal peptide fragment of AIMP1 generated by MMP1 was secreted from Wnt-treated HFSCs to activate DPCs via FGFR2. TN41 activated Akt and ERK, increased {beta}-catenin, and enhanced DPCs activation. TN41 also promoted hair shaft elongation in cultured human hair follicles and improved the hair-inducing activity of cultured DPC spheroids. In summation, the AIMP1 fragment secreted from HFSCs appears to stimulate active hair regrowth through activating DPCs.

category: cell biology


title: Induction of tumor-initiating cells and glioma-initiating cells from fetal neural stem cells through p53 genome editing

abstract: The elucidation of the properties of malignant glioma and development of therapeutic methods require model cells with characteristics such as invasiveness, multinuclearity, and ability for mitosis. A previous study has shown that overexpression of active HRas (HRasL61) in neural stem/progenitor cells isolated from fetal telencephalic neuroepithelial cells with homozygous deletion of the p53 locus forms glioma-initiating cells (GICs). The orthotopically transplantation of 10 cells into the forebrain of immunodeficient mice, resulted in the development of glioblastoma multiforme-like malignant brain tumors. In this study, GICs were induced from wild-type fetal neural stem/progenitor cells. Through forced expression of CRISPR-associated protein 9 together with a guide RNA that recognizes the p53 gene, neural stem/progenitor cells containing p53 knockout cells were obtained. Furthermore, HRasL61 was forced-expressed and transplanted into the immunodeficient mice. These cells were tumor-initiating cells (TICs) with tumorigenicity in the brain. However, no invasiveness to the brain was observed. The p53 homozygous-deleted cells obtained through single-cell cloning from TICs were GICs, forming glioblastoma multiforme-like tumors. The induced GICs strongly expressed the GIC marker. These results indicate that GICs can be induced through genetic recombination using genome editing. This study suggests that the use of genome editing may lead to the elucidation of the oncogenic mechanisms of human cells that cannot delete genes.


title: AN IMAGING FLOW CYTOMETRY-BASED METHODOLOGY FOR THE ANALYSIS OF SINGLE EXTRACELLULAR VESICLES IN UNPROCESSED HUMAN PLASMA

abstract:
Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. To rule out selection bias or introduction of artefacts caused by EV isolation techniques, we present a clinically feasible, imaging flow cytometry (IFCM)-based methodology to phenotype and determine the concentration of EVs with a diameter [≤]400 nm in human platelet-poor plasma (PPP) without prior isolation of EVs.

Instrument calibration (both size and fluorescence) were performed with commercial polystyrene beads. Detergent treatment of EVs was performed to discriminate true vesicular events from artefacts. Using a combination of markers (CFSE & Tetraspanins, or CD9 & CD31) we found that >90% of double-positive fluorescent events represented single EVs.

Through this work, we provide a framework that will allow the application of IFCM for EV analysis in peripheral blood plasma in a plethora of experimental and potentially diagnostic settings. Additionally, this direct approach for EV analysis will enable researchers to explore new corners of EV as cellular messengers in healthy and pathological conditions.


title: Myopia alters the structural organization of the retinal astrocyte template, associated vasculature and ganglion layer thickness.

abstract:
PurposeTo describe the effect of myopic eye growth on the structure and distribution of astrocytes, vasculature and ganglion cell thickness, critical for inner retinal tissue homeostasis and survival.

MethodsAstrocyte and capillary distribution, retinal nerve fiber (RNFL) and ganglion cell layer (GCL) thicknesses were assessed using immunochemistry and spectral domain optical coherence tomography on eleven retinas of juvenile common marmosets (Callithrix Jacchus), six of which were induced with lens-induced myopia (refraction, Rx: -7.01{+/-}1.8D). Five untreated age-matched juvenile marmoset retinas were used as controls (Rx: -0.74{+/-}0.4D).

ResultsAs control marmoset eyes grew normally, there was an age-related increase in astrocyte numbers associated with RNFL thickening. Marmosets with induced myopia did not show this trend and, on the contrary, had reduced astrocyte numbers, increased positive GFAP immunopositive staining, thinner RNFL, lower peripheral capillary branching, and increased numbers of string vessels.

ConclusionThe myopic changes in retinal astrocytes, vasculature, and ganglion cell layer thickness suggest a reorganization of the astrocyte and vascular templates during myopia development and progression. Whether these adaptations are beneficial or harmful to the retina remains to be investigated.

Summary StatementThis article provides new information on how progressive myopia affects key elements of the retinal neurovascular unit.

category: developmental biology


title: Optimization of Whole Mount RNA multiplexed in situ Hybridization Chain Reaction with Immunohistochemistry, Clearing and Imaging to visualize octopus neurogenesis

abstract: Gene expression analysis has been instrumental to understand the function of key factors during embryonic development of many species. Marker analysis is also used as a tool to investigate organ functioning and disease progression. As these processes happen in three dimensions, the development of technologies that enable detection of gene expression in the whole organ or embryo is essential. Here, we describe an optimized protocol of whole mount multiplexed RNA in situ hybridization chain reaction version 3.0 (HCR v3.0) in combination with immunohistochemistry (IHC), followed by fructose-glycerol clearing and light sheet fluorescence microscopy (LSFM) imaging on whole-mount Octopus vulgaris embryos. We developed a code to automate probe design which can be applied for designing HCR v3.0 type probe pairs for fluorescent in situ mRNA visualization. As proof of concept, neuronal (Ov-elav) and glial (Ov-apolpp) markers were used for multiplexed HCR v3.0. Neural progenitor (Ov-ascl1) and precursor (Ov-neuroD) markers were combined with an immunostaining for phosphorylated-histone H3, a marker for mitosis. After comparing several tissue clearing methods, fructose-glycerol clearing was found optimal in preserving the fluorescent signal of HCR v3.0. The expression that was observed in whole-mount octopus embryos matched with the previous expression data gathered from paraffin-embedded transverse sections. Three-dimensional reconstruction revealed additional spatial organization that had not been discovered using two-dimensional methods.


title: Epidermal basal domains organization highlights skin robustness to environmental exposure

abstract:
Adult interfollicular epidermis (IFE) renewal is likely orchestrated by physiological demands of its complex tissue architecture comprising spatial and cellular heterogeneity. Mouse tail and back skin display two kinds of basal IFE spatial domains that regenerate at different rates. Here we elucidate the molecular and cellular states of basal IFE domains by marker expression and single cell transcriptomics in mouse and human skin. We uncover two paths of basal cell differentiation that reflect in part the IFE spatial domain organization. We unravel previously unrecognized similarities between mouse tail IFE basal domains defined as scales and interscales versus human rete ridges and inter-ridges, respectively. Second, our basal IFE transcriptomics and gene targeting in mice provide evidence supporting a physiological role of IFE domains: adaptation to differential UV exposure. We identify Sox6 as a novel UV-induced and interscale/inter-ridge basal IFE-domain transcription factor, important for IFE proliferation and survival. The spatial, cellular, and molecular organization of IFE basal domains underscores skin adaptation to environmental exposure and its unusual robustness in adult homeostasis.

Synopsis

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title: Nr2f1a maintains atrial nkx2.5 expression to repress pacemaker identity within venous atrial cardiomyocytes

abstract: Maintenance of cardiomyocyte identity is vital for normal heart development and function. However, our understanding of cardiomyocyte plasticity remains incomplete. Here, we show that sustained expression of the zebrafish transcription factor (TF) Nr2f1a prevents the progressive acquisition of ventricular cardiomyocyte (VC) and pacemaker cardiomyocyte (PC) identities within distinct regions of the atrium. Transcriptomic analysis of isolated atrial cardiomyocytes (ACs) from nr2f1a mutant zebrafish embryos showed increased VC marker gene expression and altered expression of core PC regulatory genes, including decreased expression of nkx2.5, a critical repressor of PC differentiation. At the arterial pole of the atrium in nr2f1a mutants, cardiomyocytes resolve to VC identity within the expanded atrioventricular canal. However, at the venous pole, there is a progressive wave of AC transdifferentiation into PCs across the atrium toward the arterial pole. Restoring Nkx2.5 is sufficient to repress PC identity in nr2f1a mutant atria and analysis of chromatin accessibility identified a Nr2f1a-dependent nkx2.5 enhancer expressed in the atrial myocardium directly adjacent to PCs, supporting that Nr2f1a limits PC differentiation within venous ACs via maintaining nkx2.5 expression. The Nr2f-dependent maintenance of AC identity within discrete atrial compartments may provide insights into the molecular etiology of concurrent structural congenital heart defects and associated arrhythmias.


title: Trunk neural crest migratory position and asymmetric division predict terminal differentiation

abstract: The generation of complex structures during embryogenesis requires the controlled migration and differentiation of cells from distant origins. How migration and differentiation are coordinated and impact each other to form functional structures is not fully understood. Neural crest cells migrate extensively giving rise to many cell types. In the trunk, neural crest migrate collectively forming chains comprised of cells with distinct migratory identities: one leader cell at the front of the group directs migration, while followers track the leader forming the body of the chain. Herein we analysed the relationship between trunk neural crest migratory identity and terminal differentiation. We found that trunk neural crest migration and fate allocation is coherent. Leader cells that initiate movement give rise to the most distal derivativities. Interestingly, the asymmetric division of leaders separates migratory identity and fate. The distal daughter cell retains the leader identity and clonally forms the Sympathetic Ganglia. The proximal sibling migrates as a follower and gives rise to Schwann cells. The sympathetic neuron transcription factor phox2bb is strongly expressed by leaders from early stages of migration, suggesting that specification and migration occur concomitantly and in coordination. Followers divide symmetrically and their fate correlates with their position in the chain.


title: EARLY AND MID-GESTATION ZIKA VIRUS (ZIKV) INFECTION IN THE OLIVE BABOON (PAPIO ANUBIS) LEADS TO FETAL CNS PATHOLOGY BY TERM GESTATION

abstract: Zika virus (ZIKV) infection in pregnancy can produce catastrophic teratogenic damage to the developing fetus including microcephaly and congenital Zika syndrome (CZS). We previously described fetal CNS pathology occurring by three weeks post-ZIKV infection in Olive baboons at mid-gestation, including neuroinflammation, loss of radial glia (RG), RG fibers, neuroprogenitor cells (NPCs) resulting in disrupted NPC migration. In the present study, we explored fetal brain pathologies at term gestation resulting from ZIKV infection during either first or second trimester in the Olive baboon. In all dams, viremia resolved after 7 days post infection (dpi). One first trimester infected dam aborted at 5 dpi. All dams developed IgM and IgG response to ZIKV with ZIKV IgG detected in fetal serum. Substantial placental pathology and inflammation was observed including disruption of syncytiotrophoblast layers, delayed villous maturation, partially or fully thrombosed vessels, calcium mineralization and fibrin deposits. In the uterus, ZIKV was detected in [3/4] first trimester but not in second trimester infected dams. While ZIKV was not detected in any fetal tissue at term, all fetuses exhibited varying degrees of neuropathology. Fetal brains from ZIKV infected dams exhibited a range of gross brain pathologies including irregularities of the major gyri and sulci of the cerebral cortex and cerebellar pathology. Frontal cortices of ZIKV fetuses showed a general disorganization of the six-layered cortex with degree of disorganization varying among the fetuses from the two groups. Frontal cortices from first but not second trimester infections exhibited increased microglia and astrocyte numbers (white matter) in both trimester infections. In the cerebellum, increased microglia were observed in both first and second trimester infected fetuses and decreased oligodendrocyte precursor cell populations in the cerebellar white matter in first trimester infections. In general, our observations are in accordance with those described in human ZIKV infected fetuses.

category: pathology


title: Metabolic Engineering of Oleaginous Yeast Rhodotorula toruloides for Overproduction of Triacetic Acid Lactone

abstract:
The plant-sourced polyketide triacetic acid lactone (TAL) has been recognized as a promising platform chemical for the biorefinery industry. However, its practical application was rather limited due to low natural abundance and inefficient cell factories for biosynthesis. Here we report the metabolic engineering of oleaginous yeast Rhodotorula toruloides for TAL overproduction. We first introduced a 2-pyrone synthase gene from Gerbera hybrida (GhPS) into R. toruloides and investigated the effects of different carbon sources on TAL production. We then systematically employed a variety of metabolic engineering strategies to increase the flux of acetyl-CoA by enhancing its biosynthetic pathways and disrupting its competing pathways. We found that overexpression of citrate lyase (ACL1) improved TAL production by 45% compared to the GhPS overexpressing strain, and additional overexpression of acetyl-CoA carboxylase (ACC1) further increased TAL production by 29%. Finally, we characterized the resulting strain I12-ACL1-ACC1 using fed-batch bioreactor fermentation in glucose or oilcane juice medium with acetate supplementation and achieved a titer of 28 g/L or 23 g/L TAL, respectively. This study demonstrates that R. toruloides is a promising host for production of TAL and other acetyl-CoA-derived polyketides from low-cost carbon sources.

Graphical abstract

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org.highwire.dtl.DTLVardef@32eb52org.highwire.dtl.DTLVardef@f786dborg.highwire.dtl.DTLVardef@b6d3f5org.highwire.dtl.DTLVardef@115aa67_HPS_FORMAT_FIGEXP  M_FIG C_FIG Triacetic acid lactone (TAL) is a promising platform chemical. Cao et al. overexpressed 2-pyrone synthase in oleaginous yeast Rhodotorula toruloides to produce TAL. They systematically evaluated various metabolic gene targets to increase acetyl-CoA and malonyl-CoA levels for TAL production and found that overexpression of both ACL1 and ACC1 led to 28 g/L or 23 g/L of TAL from glucose or oilcane juice with acetate supplementation, respectively, in fed-batch fermentation.

category: synthetic biology


title: LUBAC is required for RIG-I sensing of RNA viruses

abstract: The ability of cells to mount an interferon response to virus infections depends on intracellular nucleic acid sensing pattern recognition receptors (PRRs). RIG-I is an intracellular PRR that binds short double stranded viral RNAs to trigger MAVS-dependent signalling. The RIG-I/MAVS signalling complex requires the coordinated activity of multiple kinases and E3 ubiquitin ligases to activate the transcription factors that drive type I and type III interferon production from infected cells. The linear ubiquitin chain assembly complex (LUBAC) regulates the activity of multiple receptor signalling pathways in both ligase-dependent and -independent ways. Here, we show that the three proteins that constitute LUBAC have separate functions in regulating RIG-I signalling. Both HOIP, the E3 ligase capable of generating M1-ubiquitin chains, and LUBAC accessory protein HOIL-1 are required for viral RNA sensing by RIG-I. The third LUBAC component, SHARPIN, is not required for RIG-I signalling. These data cement the role of LUBAC as a positive regulator of RIG-I signalling and as an important component of antiviral innate immune responses.

category: immunology


title: Modeling predicts mechanisms altered by mutations of the SARS-CoV-2 delta and omicron variants

abstract: We apply our mechanistic, within-host, pre-immunity, respiratory tract infection model for unvaccinated, previously uninfected, and immune-compromised individuals. Starting from published cell infection and viral replication data for the SARS-CoV-2 alpha variant, we explore variability in outcomes of viral load and cell infection due to three plausible mechanisms altered by SARS-CoV-2 mutations of delta and omicron. We seek a mechanistic explanation of clinical test results: delta nasal infections express [~]3 orders-of-magnitude higher viral load than alpha, while omicron infections express an additional 1 to 2 orders-of-magnitude rise over delta. Model simulations reveal shortening of the eclipse phase (the time between cellular uptake of the virus and onset of infectious viral replication and shedding) alone can generate 3-5 orders-of-magnitude higher viral load within 2 days post initial infection. Higher viral replication rates by an infected cell can generate at most one order-of-magnitude rise in viral load, whereas higher cell infectability has minimal impact and lowers the viral load.


title: A high-throughput yeast display approach to profile pathogen proteomes for MHC-II binding

abstract: T cells play a critical role in the adaptive immune response, recognizing peptide antigens presented on the cell surface by Major Histocompatibility Complex (MHC) proteins. While assessing peptides for MHC binding is an important component of probing these interactions, traditional assays for testing peptides of interest for MHC binding are limited in throughput. Here we present a yeast display-based platform for assessing the binding of tens of thousands of user-defined peptides in a high throughput manner. We apply this approach to assess a tiled library covering the SARS-CoV-2 proteome and four dengue virus serotypes for binding to human class II MHCs, including HLA-DR401, -DR402, and -DR404. This approach identifies binders missed by computational prediction, highlighting the potential for systemic computational errors given even state-of-the-art training data, and underlines design considerations for epitope identification experiments. This platform serves as a framework for examining relationships between viral conservation and MHC binding, and can be used to identify potentially high-interest peptide binders from viral proteins. These results demonstrate the utility of this approach for determining high-confidence peptide-MHC binding.


title: Pre-clinical testing of two serologically distinct chimpanzee-origin adenovirus vectors expressing spike of SARS-CoV-2

abstract: Two serologically distinct chimpanzee-origin, replication-defective adenovirus (AdC) vectors expressing the spike (S) protein of an early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate were generated and tested for induction of antibodies in young and aged mice. Both vectors induced S protein-specific antibodies including neutralizing antibodies. Levels of antibodies increased after a boost. The effectiveness of the boost depended on vector dose and timing between the two immunizations. Using two heterologous AdC vectors was more effective than vaccinating with the same vector repeatedly. Antibodies partially crossreacted between different S protein variants. Cross-reactivity increased after booster immunization with vectors carrying the same S gene, expression of two different S proteins by the AdC vectors used for the prime and the boost did not selectively increase responses against the variants.


title: Gut microbiota transplantation drives the adoptive transfer of colonic genotype-phenotype characteristics between mice lacking catestatin and their wild type counterparts

abstract: The gut microbiota is in continuous interaction with the intestinal mucosa via metabolic, neuro- immunological, and neuroendocrine pathways. Disruption in levels of antimicrobial peptides produced by the enteroendocrine cells, such as catestatin, has been associated with changes in the gut microbiota and imbalance in intestinal homeostasis. However, whether the changes in the gut microbiota have a causational role in intestinal dyshomeostasis has remained elusive. To this end, we performed reciprocal fecal microbial transplantation in wild-type mice and mice with a knockout in the catestatin coding region of the chromogranin-A gene (CST-KO mice). Combined microbiota phylogenetic profiling, RNA sequencing, and transmission electron microscopy were employed. Fecal microbiota transplantation from mice deficient in catestatin (CST-KO) to microbiota-depleted wild-type mice induced transcriptional and physiological features characteristic of a distorted colon in the recipient animals, including impairment in tight junctions, as well as an increased collagen area fraction indicating colonic fibrosis. In contrast, fecal microbiota transplantation from wild-type mice to microbiota-depleted CST-KO mice reduced collagen fibrotic area, restored disrupted tight junction morphology, and altered fatty acid metabolism in recipient CST-KO mice. This study provides a comprehensive overview of the murine metabolic- and immune-related cellular pathways and processes that are co-mediated by the fecal microbiota transplantation and supports a prominent role for the gut microbiota in the colonic distortion associated with the lack of catestatin in mice. Overall, the data show that the gut microbiota may play a causal role in the development of features of intestinal inflammation and metabolic disorders, known to be associated with altered levels of catestatin and may, thus, provide a tractable target in the treatment and prevention of these disorders.

category: microbiology


title: Prevalence and mechanisms of evolutionary contingency in human influenza H3N2 neuraminidase

abstract: Neuraminidase (NA) of human influenza H3N2 virus has evolved rapidly and been accumulating mutations for more than half-century. However, biophysical constraints that govern the evolutionary trajectories of NA remain largely elusive. Here, we show that among 70 natural mutations that are present in the NA of a recent human H3N2 strain, >10% are deleterious for an ancestral strain. By mapping the permissive mutations using combinatorial mutagenesis and next-generation sequencing, an extensive epistatic network is revealed. Biophysical and structural analyses further demonstrate that certain epistatic interactions can be explained by non-additive stability effect, which in turn modulates membrane trafficking and enzymatic activity of NA. Additionally, our results suggest that other biophysical mechanisms also contribute to epistasis in NA evolution. Overall, these findings not only provide mechanistic insights into the evolution of human influenza NA and elucidate its sequence-structure-function relationship, but also have important implications for the development of next-generation influenza vaccines.


title: Methionine Metabolism Controls the B-cell EBV Epigenome and Viral Latency

abstract:
Epstein-Barr virus (EBV) subverts host epigenetic pathways to switch between viral latency programs, colonize the B-cell compartment and reactivate. Within memory B-cells, the reservoir for lifelong infection, EBV genomic DNA and histone methylation marks restrict gene expression. But, this epigenetic strategy also enables EBV-infected tumors, including Burkitt lymphomas to evade immune detection. Little is known about host cell metabolic pathways that support EBV epigenome landscapes. We therefore used amino acid restriction, metabolomic and CRISPR approaches to identify that an abundant methionine supply, and interconnecting methionine and folate cycles, maintain Burkitt EBV gene silencing. Methionine restriction, or methionine cycle perturbation, hypomethylated EBV genomes, de-repressed latent membrane protein and lytic gene expression. Methionine metabolism also shaped EBV latency gene regulation required for B-cell immortalization. Dietary methionine restriction altered murine Burkitt xenograft metabolomes and de-repressed EBV immunogens in vivo. These results highlight epigenetic/immunometabolism crosstalk supporting the EBV B-cell lifecycle and suggest therapeutic approaches.

HighlightsO_LIMethionine metabolism is critical for Epstein-Barr virus B-cell latency
C_LIO_LIExtensive cross-talk enables methionine metabolism to control the EBV epigenome
C_LIO_LIMethionine restriction also impairs EBV-driven human B-cell immortalization
C_LIO_LIDietary methionine restriction unmasks EBV antigens in Burkitt xenografts in vivo
C_LI


title: Omicron BA.1 and BA.2 are antigenically distinct SARS-CoV-2 variants

abstract: The emergence and rapid spread of SARS-CoV-2 variants may impact vaccine efficacy significantly1. The Omicron variant termed BA.2, which differs genetically substantially from BA.1, is currently replacing BA.1 in several countries, but its antigenic characteristics have not yet been assessed2,3. Here, we used antigenic cartography to quantify and visualize antigenic differences between SARS-CoV-2 variants using hamster sera obtained after primary infection. Whereas early variants are antigenically similar, clustering relatively close to each other in antigenic space, Omicron BA.1 and BA.2 have evolved as two distinct antigenic outliers. Our data show that BA.1 and BA.2 both escape (vaccine-induced) antibody responses as a result of different antigenic characteristics. Close monitoring of the antigenic changes of SARS-CoV-2 using antigenic cartography can be helpful in the selection of future vaccine strains.


title: Screening and Identification of Azo Dye Decolourizers from Mangrove Rhizospheric soil

abstract:
Removal of synthetic textile dye poses a challenge to the textile industry and a threat to environment flora and fauna. These dyes are marginally degradable and recalcitrant, hence alternatives to physical and chemical techniques like various bioremediation studies involving plants, plant roots, single or consortium of microbes have been used as environment friendly methods for the removal of textile dye. In the present study potent bacteria for dye decolourization were isolated from rhizospheric soil collected from Kamothe, Navi Mumbai, India. Of the 20 isolates obtained after enrichment, seven isolates were used for further screening of efficient decolourization ability in MBM media containing 10% glucose, 2.5 % trace metal solution and 0.1% of MO dye concentration. Physiological parameters to optimize the decolourization of dye at optimum pH, temperature and incubation time was studied for all the seven isolates. UV-vis and Fourier Transform Infrared spectroscopy were used to investigate dye decolorization. The seven isolates were characterized morphologically, biochemically, and molecular identification of these strains were performed by 16S rRNA sequence analysis. The isolates were identified as Bacillus paramycoides, Pseudomonas taiwanensis, Citrobacter murliniae, Acinetobacter pitti, Exiguobacterium acetylicum, Psychrobacter celer, and Aeromonas taiwanensis. Out of these Aeromonas taiwanensis has shown exceptional capacity by 100% decolorization of azo dye in minimum time.

1. Graphical Abstract

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C_LIO_LIEnrichment of soil sample in media containing azo dye for dye decolourization.
C_LIO_LIScreening and isolation of potential dye decolourizers.
C_LIO_LIOptimize the condition for dye decolourization.
C_LIO_LIFTIR analysis and application based studies.
C_LIO_LIIdentification of microbes using 16SrRNA.
C_LI


title: Bacterial metatranscriptomes in wastewater can differentiate virally infected human populations

abstract: Monitoring wastewater samples at building-level resolution screens large populations for SARS-CoV-2, prioritizing testing and isolation efforts. Here we perform untargeted metatranscriptomics on virally-enriched wastewater samples from 10 locations on the UC San Diego campus, demonstrating that resulting bacterial taxonomic and functional profiles discriminate SARS-CoV-2 status even without direct detection of viral transcripts. Our proof-of-principle reveals emergent threats through changes in the human microbiome, suggesting new approaches for untargeted wastewater-based epidemiology.


title: Identification of cytoplasmic chaperone networks relevant for respiratory syncytial virus replication

abstract: RNA viruses have limited coding capacity and must therefore successfully subvert cellular processes to facilitate their replication. A fundamental challenge faced by both viruses and their hosts is the ability to achieve the correct folding and assembly of their proteome while avoiding misfolding and aggregation. In cells, this process is facilitated by numerous chaperone systems together with a large number of co-chaperones. In this work, we set out to define the chaperones and co-chaperones involved in the replication of respiratory syncytial virus (RSV). Using an RNAi screen, we identify multiple members of cellular protein folding networks whose knockdown alters RSV replication. The reduced number of chaperones and co-chaperones identified in this work can facilitate the unmasking of specific chaperone subnetworks required for distinct steps of the RSV life cycle and identifies new potential targets for antiviral therapy. Indeed, we show that the pharmacological inhibition of one of the genes identified in the RNAi screen, valosin-containing protein (VCP/p97), can impede the replication of RSV by interfering with the infection cycle at multiple steps.


title: A central protein complex essential for Invasionin Toxoplasma gondii

abstract:
Apicomplexa are obligate intracellular parasites. While most species are restricted to specific hosts and cell types, Toxoplasma gondii can invade every nucleated cell derived from warm-blooded animals. This broad host range suggests that this parasite can recognize multiple host cell ligands or structures, leading to the activation of a central protein complex, which should be conserved in all apicomplexans. During invasion, the unique secretory organelles (micronemes and rhoptries) are sequentially released and several micronemal proteins have been suggested to be required for host cell recognition and invasion. However, to date only few micronemal proteins have been demonstrated to be essential for invasion.

Cysteine Repeat Modular Proteins (CRMPs) are a family of apicomplexan specific proteins. In Toxoplasma gondii, two CRMPs are present in the genome. The Kringle domain containing protein (CRMPA) and the GCC2 GCC3 domain containing protein (CRMPB). Here we demonstrate that both proteins form a complex that contains additional micronemal proteins. Disruption of this complex results in a block of rhoptry secretion and parasites being unable to invade the host cell. In conclusion, this complex is a central invasion complex conserved in all apicomplexans.


title: Plasma membrane protrusions mediate host cell-cell fusion induced by Burkholderia thailandensis

abstract: Cell-cell fusion is important for biological processes including fertilization, development, immunity, and microbial pathogenesis. Bacteria in the pseudomallei group of Burkholderia species, including B. thailandensis, spread between host cells by inducing cell-cell fusion. Previous work showed that B. thailandensis-induced cell-cell fusion requires intracellular bacterial motility and a bacterial protein secretion apparatus called the type VI secretion system-5 (T6SS-5), including the T6SS-5 protein VgrG5. However, the cellular level mechanism and T6SS-5 proteins important for bacteria-induced cell-cell fusion remained incompletely described. Using live cell imaging, we found bacteria used actin-based motility to push on the host cell plasma membrane to form plasma membrane protrusions that extended into neighboring cells. Then, membrane fusion occurred within these membrane protrusions, either proximal to the bacterium at the tip or elsewhere within a protrusion. Expression of VgrG5 by bacteria within membrane protrusions was required to promote cell-cell fusion. Furthermore, a second predicted T6SS-5 protein, TagD5, was also required for cell-cell fusion. In the absence of VgrG5 or TagD5, bacteria in plasma membrane protrusions were engulfed into neighboring cells. Our results suggest that the T6SS-5 effectors VgrG5 and TagD5 are secreted within membrane protrusions and act locally to promote membrane fusion.


title: Evolution of bacterial persistence to antibiotics during a 50,000-generation experiment in an antibiotic-free environment.

abstract: Failure of antibiotic therapies causes > 700,000 deaths yearly and involves both bacterial resistance and persistence. Persistence results in the relapse of infections by producing a tiny fraction of pathogen survivors that stay dormant during antibiotic exposure. From an evolutionary perspective, persistence is either a  bet-hedging strategy that helps to cope with stochastically changing environments or an unavoidable minimal rate of  cellular errors that lock the cells in a low activity state. Here, we analyzed the evolution of persistence over 50,000 bacterial generations in a stable environment by improving a published method that estimates the number of persister cells based on the growth of the reviving population. Our results challenged our understanding of the factors underlying persistence evolution. In one case, we observed a substantial decrease in persistence proportion, suggesting that the naturally observed persistence level is not an unavoidable minimal rate of  cellular errors. However, although there was no obvious environmental stochasticity, in 11 of the 12 investigated populations, the persistence level was maintained during 50,000 bacterial generations.

category: evolutionary biology


title: Evolution of bacterial persistence to antibiotics during a 50,000-generation experiment in an antibiotic-free environment


title: Accurate and simple FXN-GAA repeats (Friedreich ataxia loci) estimation by long read targeted sequencing.

abstract: Friedreich ataxia, an autosomal recessive disorder is caused by tandem GAA nucleotide repeats expansion in intron 1 of the FXN (frataxin gene). The GAA repeats above 66 in length are considered as pathogenic and commonly occurring repeats are 600-1200. Clinically the spectrum of the features is confined mainly to the neurological tissue, however, cardiomyopathy and diabetes mellitus has been observed in 60% and 30% of the subjects. The accurate detection of GAA repeats count is of utmost importance for clinical genetic correlation as no study has attempted an approach which is of high throughput nature and defines the sequence of GAA repeats. Largely, the method for detection of GAA repeats so far is either conventional PCR based screening and southern blot which is the gold standard method. We describe for the first time a method of long read sequencing wherein we utilized approach of long range targeted amplification of FXN-GAA repeats and sequencing on oxford MinION platform. We were able to achieve the successful amplification of GAA repeats ranging from 180-1200 at 250x coverage. The total throughput achievable for 96 samples can be less than 24 hours on one flow cell as per our protocol and is scalable and deployable at clinical day to sequencing.

category: genetics


title: Diagnostic Ion Data Analysis Reduction (DIDAR) allows rapid quality control analysis and filtering of multiplexed single cell proteomics data

abstract:
Recent advances in the sensitivity and speed of mass spectrometers utilized for proteomics and metabolomics workflows has led to a dramatic increase in data file size and density. For a field already challenged by data complexity due to a dependence on desktop PC architecture and the Windows operating systems, further compromises appear inevitable as data density scales. As one method to reduce data complexity, we present herein a light-weight python script that can rapidly filter and provide analysis metrics from tandem mass spectra based on the presence and number of diagnostic fragment ions determined by the end user. Diagnostic Ion Data Analysis Reduction (DIDAR) can be applied to any mass spectrometry dataset to create smaller output files containing only spectra likely to contain post-translational modifications or chemical labels of interest. In this study we describe the application DIDAR within the context of multiplexed single cell proteomics workflows. When applied in this manner using reporter fragment ions as diagnostic signatures, DIDAR can provide quality control metrics based on the presence of reporter ions derived from single human cells and simplified output files for search engine analysis. The simple output metric text files can be used to rapidly flag entire LCMS runs with technical issues and remove them from downstream analysis based on end user minimum requirements. Acquisition files that pass these criteria are further improved through the automatic removal of spectra where insufficient signal from single cells is observed. We describe the application of DIDAR to two recently described multiplexed single cell proteomics datasets.

Abstract Graphic

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category: biochemistry


title: Chlamydomonas mutant hpm91 lacking PGR5 is a scalable and valuable strain for algal hydrogen (H2) production

abstract: Clean and sustainable H2 production is essential toward a carbon-neutral world. H2 generation by Chlamydomonas reinhardtii is an attractive approach for solar-H2 from H2O. However, it is currently not scalable because of lacking ideal strains. Here, we explore hpm91, a previously reported PGR5-deletion mutant with remarkable H2 production, that possesses numerous valuable attributes towards large-scale application and in-depth study issues. We show that hpm91 is at least 100-fold scalable (upto 10 liter) with H2 collection sustained for averagely 26 days and 7287 ml H2/10L-HPBR. Also, hpm91 is robust and active over the period of sulfur-deprived H2 production, most likely due to decreased intracellular ROS relative to wild type. Moreover, quantitative proteomic analysis revealed its features in photosynthetic antenna, primary metabolic pathways and anti-ROS responses. Together with success of new high-H2-production strains derived from hpm91, we highlight that hpm91 is a potent strain toward basic and applied research of algal-H2 photoproduction.


title: Sequence-directed RNA remodeling within a topologically complex RNP substrate

abstract: DEAD-box ATPases are ubiquitous enzymes essential in all aspects of RNA biology. However, the limited in vitro catalytic activities described for these enzymes is at odds with their complex cellular roles, most notably in driving large-scale RNA remodeling steps during the assembly of ribonucleoproteins (RNPs). We describe cryo-EM structures of 60S ribosomal biogenesis intermediates that reveal how context-specific RNA unwinding by the DEAD-box ATPase Spb4 results in extensive, sequence-directed remodeling of rRNA secondary structure. Multiple cis and trans interactions stabilize a post-catalytic, high-energy intermediate that drives the organization of the root helix structure within rRNA domain IV. This mechanism explains how limited strand separation by DEAD-box ATPases is leveraged to provide non-equilibrium directionality and ensure efficient and accurate RNP assembly.


title: The inhibitory effect of EGCG-AgNP-lysozyme bioconjugates on biofilm formation

abstract: Antibiotic resistance of bacteria in the biofilm has become a major problem for the treatment of chronic infections. Nanotechnology offers a new approach to fighting against biofilm-associated infections. Here, we report on the synthesis of silver nanoparticles (AgNPs) with epigallocatechin gallate (EGCG) ligand, which is known to present antibacterial activity. We further describe the interaction of EGCG-AgNPs with lysozyme, an antibacterial enzyme abundant in fluids like serum, saliva and tears, and the formation of EGCG-AgNP-lysozyme bioconjugates. Our results demonstrate the formation of lysozyme protein corona as shown by UV-Vis, dynamic light scattering and SDS-PAGE analyses. Using circular dichroism spectroscopy we found that lysozyme preserves its secondary structure at low concentration of EGCG-AgNPs, whereas higher concentrations induce protein structural changes. Furthermore, we have shown in bacterial growth and biofilm formation assays that EGCG presents no antibacterial activity on planktonic Bacillus subtilis cells when bound to AgNPs. It was found that EGCG-AgNPs (>40 nM) inhibit B. subtilis in biofilm forming conditions and the inhibition effect is reduced by lysozyme corona formation on EGCG-AgNPs. Our results demonstrate that EGCG-AgNPs could be used as antibiofilm agents because they showed lower cytotoxicity and significant inhibitory effect on pellicle formation.


title: A Pan-Pneumovirus vaccine based on immunodominant epitopes of the fusion protein

abstract:
Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are two leading causes of severe respiratory infections in children, the elderly, and immunocompromised patients. The fusion (F) protein is the major target of neutralizing antibodies. Recent developments in stabilizing the pre-fusion conformation of the F proteins, and identifying immunodominant epitopes that elicit potent neutralizing antibodies have led to testing of numerous pre-fusion RSV F-based vaccines in clinical trials. We designed and tested the immunogenicity and protective efficacy of a chimeric fusion protein that contains immunodominant epitopes of RSV F and hMPV F (RHMS-1). RHMS-1 has several advantages over vaccination with pre-fusion RSV F or hMPV F, including a focus on recalling B cells to the most important protective epitopes and the ability to induce protection against two viruses with a single antigen. RHMS-1 was generated as a trimeric recombinant protein, and negative-stain EM analysis demonstrated the protein resembles the pre-fusion conformation. Probing of RHMS-1 antigenicity using a panel of RSV and hMPV F-specific monoclonal antibodies (mAbs) revealed the protein retains features of both viruses, including the pre-fusion site O epitope of RSV F. BALB/c mice immunized with RHMS-1 had serum binding and neutralizing antibodies to both viruses. RHMS-1 vaccinated mice challenged with RSV or hMPV had undetectable virus in lung homogenates for both viruses, in contrast to RSV F or hMPV F vaccinated mice, which had detectable virus for hMPV and RSV, respectively. Overall, this study demonstrates protection against two viruses with a single antigen and supports further testing of RHMS-1 in additional pre-clinical animal models.

SignificanceRespiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are two Pneumoviruses that cause substantial respiratory tract infections in young children, the elderly, and immunocompromised adults. Yet vaccines against either of them are still unavailable. Multiple promising vaccine designs based on the fusion proteins of RSV and hMPV have been tested, but none of them can induce cross-protective antibody responses despite their similar structures and closely related amino acid sequence identity. Here, we report a vaccine candidate, RHMS-1, which combines the immunodominant epitopes of the RSV and hMPV fusion proteins into a single antigen. RHMS-1 maintains the immunological features of both RSV F and hMPV F, which can be recognized by epitope-specific mAbs and human B cells pre-exposed to RSV or hMPV. Furthermore, this is the first immunogen that induced potent cross-neutralizing antibodies and protected mice from RSV and hMPV challenge. Our results suggest RHMS-1 is a promising Pan-Pneumovirus vaccine.


title: Multiple scenarios for sexual crosses in the fungal pathogen Zymoseptoria tritici on wheat residues: potential consequences for virulence gene transmission

abstract:
Little is known about the impact of host immunity on sexual reproduction in fungal pathogens. In particular, it is unclear whether crossing requires both sexual partners to infect living plant tissues. We addressed this issue in a three-year experiment investigating different scenarios of Zymoseptoria tritici crosses on wheat according to the virulence ( vir) or avirulence ( avr) of the parents against a qualitative resistance gene. Co-inoculations ( vir x vir,  avr x vir,  avr x avr) and single inoculations were performed on a cultivar carrying the resistance gene (Cellule) and a susceptible cultivar (Apache), in the greenhouse. We assessed the intensity of asexual multiplication by scoring disease severity, and the intensity of sexual reproduction by counting the ascospores discharged from wheat residues. As expected, disease severity was more intense on Cellule for  vir x vir co-inoculations than for  avr x vir co-inoculations, with no disease for  avr x avr. However, all types of co-inoculation yielded sexual offspring, whether or not the parental strains caused plant symptoms. Parenthood was confirmed by genotyping (SSR markers), and the occurrence of crosses between (co-)inoculated and exogenous strains (other strains from the experiment, or from far away) was determined. We found that symptomatic asexual infection was not required for a strain to participate in sexual reproduction, and that avirulent strains could be maintained asymptomatically "on" or "in" leaf tissues of plants carrying the corresponding resistant gene for long enough to reproduce sexually. In two of the three years, the intensity of sexual reproduction did not differ significantly between the three types of co-inoculation in Cellule, suggesting that crosses involving avirulent strains are not anecdotal. We discuss the possible mechanisms explaining the maintenance of avirulence in Z. tritici populations and supporting the potential efficacy of cultivar mixtures for limiting resistance gene breakdown.

HighlightsO_LIAvirulent Zymoseptoria tritici strains can reproduce sexually in wheat plants carrying the corresponding resistant gene.
C_LIO_LISymptomatic infection of plant tissues is not essential for a strain to reproduce sexually.
C_LIO_LIAvirulent strains can be maintained asymptomatically "on" or "in" leaf tissues of plants carrying the corresponding resistant gene for long enough to reproduce sexually.
C_LIO_LICrosses of virulent strains with virulent and avirulent strains in a plant host carrying the corresponding resistance gene can produce offspring with similar population sizes.
C_LIO_LISeveral possible scenarios for sexual crosses can explain the maintenance of avirulence in Zymoseptoria tritici populations evolving in a wheat canopy, particular in cultivar mixtures.
C_LI


title: Time-course of host cell transcription during the HTLV-1 transcriptional burst

abstract:
The human T-cell leukemia virus type 1 (HTLV-1) transactivator protein Tax has pleiotropic functions in the host cell affecting cell-cycle regulation, DNA damage response pathways and apoptosis. These actions of Tax have been implicated in the persistence and pathogenesis of HTLV-1-infected cells. It is now known that tax expression occurs in transcriptional bursts of the proviral plus-strand, but the effects of the burst on host transcription are not fully understood. We carried out RNA sequencing of two naturally-infected T-cell clones transduced with a Tax-responsive Timer protein, which undergoes a time-dependent shift in fluorescence emission, to study transcriptional changes during successive phases of the HTLV-1 plus-strand burst. We found that the transcriptional regulation of genes involved in the NF-{kappa}B pathway, cell-cycle regulation, DNA damage response and apoptosis inhibition were immediate effects accompanying the plus-strand burst, and are limited to the duration of the burst. The results distinguish between the immediate and delayed effects of HTLV-1 reactivation on host transcription, and between clone-specific effects and those observed in both clones. The major transcriptional changes in the infected host T-cells observed here, including NF-kB, are transient, suggesting that these pathways are not persistently activated at high levels in HTLV-1-infected cells. The two clones diverged strongly in their expression of genes regulating the cell cycle. Up-regulation of senescence markers was a delayed effect of the proviral plus-strand burst and the up-regulation of some pro-apoptotic genes outlasted the burst. We found that activation of the arylhydrocarbon receptor (AhR) pathway enhanced and prolonged the proviral burst, but did not increase the rate of reactivation. Our results also suggest that sustained plus-strand expression is detrimental to the survival of infected cells.

Author SummaryHuman T-cell leukemia virus type 1 (HTLV-1) causes a lifelong infection that results in disease in [~]10% of cases. The HTLV-1 transactivator protein Tax is involved in both the persistence of infected host cells, and the pathogenesis of HTLV-1 infection. tax is transcribed from the plus-strand of the provirus, and tax expression is not constitutive, but limited to transcriptional bursts. How these bursts affect host cell transcription is not completely understood. Here, we studied the temporal changes in host transcription during successive phases of the plus-strand burst in two naturally-infected T-cell clones. We found that the deregulation of genes involved in Tax-associated processes, including NF-{kappa}B activation, cell-cycle regulation, DNA damage response and suppression of apoptosis, coincided with the early phase of the plus-strand burst: these transcriptional effects appear to be limited to the duration of the proviral plus-strand expression. Regulation of cell-cycle genes diverged between the clones, demonstrating the heterogeneity of naturally-infected cells. We observed a pro-apoptotic response, which outlasted the burst and may indicate increased risk of apoptosis following the burst. Finally, we observed that AhR activity regulated the intensity and duration of the burst, but not the dynamics of reactivation.


title: Plasmodium GPI-Anchored Micronemal Antigen is essential for parasite transmission through the mosquito host

abstract:
The complex life cycle of Plasmodium parasites, the eukaryotic pathogens that cause malaria, features three distinct invasive forms tailored specifically to the equally distinct host environment they must navigate and invade for progression of the life cycle. One conserved feature of all these invasive forms is the presence of micronemes, apically oriented secretory organelles involved in egress, motility, adhesion and invasion. Micronemes are tailored to their specific host environment and feature stage specific contents. Here we investigate the role of GPI-anchored micronemal antigen (GAMA), which shows a micronemal localization in all zoite forms of the rodent infecting species Plasmodium berghei. While GAMA is dispensable during asexual blood stages, GAMA knock out parasites are severely defective for invasion of the mosquito midgut, resulting in reduced numbers of oocysts. Once formed, oocysts develop normally, however sporozoites are unable to egress and these sporozoites exhibit defective motility. Epitope-tagging of GAMA revealed tight temporal expression late during sporogony and showed that GAMA is shed during sporozoite gliding motility in a similar manner to circumsporozoite protein. Complementation of P. berghei knock out parasites with full length P. falciparum GAMA partially restored infectivity to mosquitoes, indicating a conservation of function across Plasmodium species. A suite of parasites with GAMA expressed under the promoters of the known ookinete-to-sporozoite stage-specific genes: CTRP, CAP380 and TRAP, further confirmed the involvement of GAMA in midgut infection, motility and infection of the mammalian host and revealed a lethal consequence to overexpression of GAMA during oocyst development. Combined, the research suggest that GAMA plays independent roles in sporozoite motility, egress and invasion, possibly implicating GAMA as a regulator of microneme function.

AUTHOR SUMMARYMalaria remains a major source of morbidity and mortality across the globe. Completion of a complex life cycle between vertebrates and mosquitoes is required for the maintenance of parasite populations and the persistence of malaria disease and death. Three invasive forms across the complex lifecycle of the parasite must successfully egress and invade specific cell types within the vertebrate and mosquito hosts to maintain parasite populations and consequently disease and suffering. A conserved feature of all invasive forms are the micronemes, apically oriented secretory organelles which contain proteins required for motility, egress and invasion. Few proteins are expressed in the micronemes of all three invasive forms. One such protein is GPI-anchored micronemal antigen (GAMA). Here we reveal that GAMA is required for the invasion of the mosquito midgut, egress of sporozoites from oocysts and invasion of the vertebrate host. Our finding indicate that while GAMA is essential for sporozoite motility, the defects in oocyst egress and hepatocyte invasion occur independently of the motility defect, implicating the requirement of GAMA in all three processes.


title: The ankyrin repeat protein RARP-1 is a periplasmic factor that supports Rickettsia parkeri growth and host cell invasion

abstract:
Rickettsia spp. are obligate intracellular bacterial pathogens that have evolved a variety of strategies to exploit their host cell niche. However, the bacterial factors that contribute to this intracellular lifestyle are poorly understood. Here, we show that the conserved ankyrin repeat protein RARP-1 supports Rickettsia parkeri infection. Specifically, RARP-1 promotes efficient host cell entry and growth within the host cytoplasm, but it is not necessary for cell-to-cell spread or evasion of host autophagy. We further demonstrate that RARP-1 is not secreted into the host cytoplasm by R. parkeri. Instead, RARP-1 resides in the periplasm, and we identify several binding partners that are predicted to work in concert with RARP-1 during infection. Altogether, our data reveal that RARP-1 plays a critical role in the rickettsial life cycle.

ImportanceRickettsia spp. are obligate intracellular bacterial pathogens that pose a growing threat to human health. Nevertheless, their strict reliance on a host cell niche has hindered investigation of the molecular mechanisms driving rickettsial infection. This study yields much needed insight into the Rickettsia ankyrin repeat protein RARP-1, which is conserved across the genus but has not yet been functionally characterized. Earlier work had suggested that RARP-1 is secreted into the host cytoplasm. However, the results from this work demonstrate that R. parkeri RARP-1 resides in the periplasm and is important both for invasion of host cells and for growth in the host cell cytoplasm. These results reveal RARP-1 as a novel regulator of the rickettsial life cycle.


title: Targetome analysis of malaria sporozoite transcription factor AP2-Sp reveals its role as a master regulator

abstract: Malaria transmission to humans begins with sporozoite infection of the liver. The elucidation of gene regulation during the sporozoite stage will promote the investigation of mechanisms of liver infection by this parasite and contribute to the development of strategies for preventing malaria transmission. AP2-Sp is a transcription factor (TF) essential for the formation of sporozoites or sporogony, which takes place in oocysts in the midgut of infected mosquitoes. To understand the role of this TF in the transcriptional regulatory system of this stage, we performed ChIP-seq analyses using whole mosquito midguts containing late oocysts as starting material and explored its genome-wide target genes. We identified 697 target genes, comprising those involved in distinct processes parasites experience during this stage, from sporogony to development into the liver-stage, and representing the majority of genes highly expressed in the sporozoite stage. These results suggest that AP2-Sp determines basal patterns of gene expression by targeting a broad range of genes directly. The ChIP-seq analyses also showed that AP2-Sp maintains its own expression by a transcriptional auto-activation mechanism (positive feedback loop) and induces all TFs reported to be transcribed at this stage, including AP2-Sp2, AP2-Sp3, and SLARP. The results showed that AP2-Sp exists at the top of the transcriptional cascade of this stage and triggers the formation of this stage as a master regulator.


title: Dynamic genetic adaptation of Bacteroides thetaiotaomicron murine gut colonization

abstract:
To understand how a bacterium ultimately succeeds or fails in adapting to a new environment, it is essential to assess the temporal dynamics of its fitness over the course of colonization. The mammalian gut, into which exogenous microorganisms are regularly introduced, represents a biologically and clinically relevant system to explore microbial adaptational processes. In this study, we introduce a human-derived commensal organism, Bacteroides thetaiotaomicron, into the guts of germ-free mice to 1) determine whether the genetic requirements for colonization shift over time and, if so, 2) characterize the biological functions required for microbial survival at different points of colonization. The results of a high-throughput functional genetics assay (BarSeq), transcriptomics, and metabolomics converge on several conclusions. First, adaptation to the host gut occurs in distinct stages. We observed drastic changes in gene usage during the first week, shifting from high expression of amino acid biosynthesis to polysaccharide utilization genes. These changes were sustained thereafter, except for the continued upregulation of a single polysaccharide utilization locus responsible for the degradation of raffinose-family oligosaccharides rich in the standard chow diet fed to our mice. Spontaneous mutations in wildtype Bt also evolve around this locus, highlighting the importance of efficient carbohydrate metabolism in long-term persistence within a monoassociated gut. To improve microbiome-based therapies, it will be important to appreciate and meet the distinct needs of the organism during each stage of colonization.

ImportanceMicrobes regularly disperse across and adapt to new environments and ecological niches. A clinically significant microbial niche home to trillions of microbes is the mammalian gut. Temporal processes of microbial adaptation over the course of gut colonization are poorly understood on a genetic, transcriptional, and metabolite level. In this study, we leverage a three-pronged approach to characterize gut colonization as a dynamic process with shifting genetic determinants of microbial fitness. This study sheds light on host colonization by Bacteroides thetaiotaomicron, an organism that is prevalent and dominant across healthy human microbiomes, and not only identifies key pathways involved in colonization, but determines the timing of when these pathways are most vital to colonization success. By demonstrating that the key determinants of colonization success in the gut change over time, the results of this study highlight the importance of considering ecological dynamics in developing more effective microbiome-based therapies.


title: ΔNp63 drives dysplastic alveolar remodeling and restricts epithelial plasticity upon severe lung injury

abstract: Unlike many mammalian vital organs, the lung exhibits a robust, multifaceted regenerative response to severe injuries such as influenza infection, which primarily targets epithelial cells in the airways and alveoli. Quiescent lung-resident epithelial progenitors proliferate, migrate, and differentiate following lung injury, participating in two distinct reparative pathways: functionally beneficial regeneration and dysplastic tissue remodeling. Intrapulmonary airway-resident basal-like p63+ progenitors are one such progenitor cell type that migrates from the airways to form ectopic bronchiolar tissue in the alveoli, generating honeycomb-like cysts that fail to resolve after injury. Though this phenomenon is now well described, the cell-autonomous signals that drive dysplastic alveolar remodeling remain uncertain, a question made especially salient by observations that p63+ progenitors also expand dramatically upon diffuse alveolar damage in humans resulting from a variety of insults including SARS-CoV-2-induced ARDS. Here we show that the master basal cell transcription factor {Delta}Np63 is required for the immense migratory capacity of intrapulmonary p63+ progenitors and consequently for the dysplastic repair pathway manifest by these cells. We further demonstrate that {Delta}Np63 restricts the fate plasticity of intrapulmonary p63+ progenitors by regulating their epigenetic landscape, and that loss of {Delta}Np63 alters the deposition of active and repressive histone modifications at key differentiation gene loci, allowing {Delta}Np63KO progenitors to proceed towards airway or alveolar differentiation depending on their surrounding environment. These insights into the regulatory mechanisms of dysplastic repair and intrapulmonary p63+ progenitor fate choice highlight potential therapeutic targets to promote more effective alveolar regeneration following severe lung injuries.

category: molecular biology


title: Saliva metabolome alterations after acute stress

abstract:
Major stress has systemic effects on the body that can have adverse consequences for physical and mental health. However, the molecular basis of these damaging effects remains incompletely understood. Here we use a longitudinal approach to characterise the acute systemic impact of major psychological stress in a pig model. We perform untargeted metabolomics on non-invasively-obtained saliva samples from pigs before and 24-hours after transfer to the novel physical and social environment of a slaughterhouse. The main molecular changes occurring include decreases in amino acids, B-vitamins, and amino acid-derived metabolites synthesized in B-vitamin-dependent reactions, as well as yet-unidentified metabolite features. Decreased levels of several of the identified metabolites are implicated in the pathology of human psychological disorders and neurodegenerative disease, suggesting a possible neuroprotective function. Our results provide a fingerprint of the acute effect of psychological stress on the metabolome and suggest candidate biomarkers with potential roles in stress-related disorders.

One Sentence SummaryIdentification of metabolites decreased under acute psychological stress may lead to future discoveries for the prevention and treatment of stress-related disorders.


title: Three-dimensional cilia structures from animals' closest unicellular relatives, the Choanoflagellates

abstract: In most eukaryotic organisms, cilia perform a variety of life-sustaining roles related to environmental sensing and motility. Cryo-electron microscopy has provided considerable insight into the morphology and function of ciliary structures, but studies have been limited to less than a dozen of the millions of known eukaryotic species. Ultrastructural information is particularly lacking for unicellular organisms in the opisthokont clade, leaving a sizeable gap in our understanding of cilia evolution between unicellular species and multicellular metazoans (animals). Choanoflagellates are important aquatic heterotrophs, uniquely positioned within the opisthokonts as the metazoans closest living unicellular relatives. We performed cryo-focused ion beam milling and cryo-electron tomography on cilia from the choanoflagellate species Salpingoeca rosetta. We show that the axonemal dyneins, radial spokes, and central pair complex in S. rosetta more closely resemble metazoan structures than those of unicellular organisms from other suprakingdoms. In addition, we describe unique features of S. rosetta cilia, including microtubule holes, microtubule inner proteins, and the ciliary vane: a fine, net-like extension that has been notoriously difficult to visualize using other methods. Furthermore, we report barb-like structures of unknown function on the extracellular surface of the ciliary membrane. Together, our findings provide new insights into choanoflagellate biology and cilia evolution between unicellular and multicellular opisthokonts.


title: Chloride ion evokes taste sensation by binding to the extracellular ligand-binding domain of sweet/umami taste receptors

abstract:
The taste sensation of salt is multifaceted: table salt (NaCl) at low concentrations is perceived as a preferable taste through the salty taste receptor, while that at higher concentrations is perceived as an aversive taste through distinct pathways. In addition, Cl- is also thought to participate in taste sensation through a currently unknown mechanism. Here we describe a Cl- -ion binding and the response of taste receptor type 1 (T1r), a receptor family composing sweet/umami receptors. The T1r2a/T1r3 heterodimer from medaka fish, an amino-acid taste receptor and the sole T1r member whose structure has been solved, exhibited a specific Cl--binding in the vicinity of the amino-acid-binding site in the ligand-binding domain (LBD) of T1r3. Notably, the Cl--binding site is likely conserved among T1r3 from other species, including humans. The Cl--binding at the site is considered to organize the structures of the amino-acid binding site and heterodimer interface. Indeed, the Cl--binding was found to induce a conformational change of T1r2a/T1r3LBD at sub-mM to low-mM concentrations in a similar manner to canonical taste substances. In order to address the physiological significance of the Cl- action on T1r, single fiber responses from mouse taste nerves connected to T1r-expressing taste cells were investigated. As a result, a Cl- application increased impulse frequencies, which were inhibited by a lingual application of a T1r-specific blocker. These results suggest that the Cl- evokes taste sensations by binding to T1r, underlying a reported "sweet" taste sensation by table salt at a low concentration.

Significance statementSalt taste sensation is essential for regulating salt intake and maintaining body fluid volume and concentration. Therefore, in concentrations close to that of body fluid, the sodium ion is perceived as an appealing taste through a specific taste transduction pathway. In this study, we found that a low concentration of chloride ion, a sodium ion counter anion in table salt, is perceived via the sweet taste transduction pathway mediated by the sweet taste receptor, one of the attractive taste qualities in animals. The chloride perception system discovered in this study could be another attractive pathway for promoting salt intake in an appropriate concentration range.


title: Epstein-Barr virus infection promotes Th1-mediated disease in a humanized immune mouse model of multiple sclerosis

abstract: Infection with the human-tropic Epstein-Barr virus (EBV) is a strong risk factor for multiple sclerosis (MS), though the underlying mechanisms remain unclear. To investigate EBV infection directly, we induced experimental autoimmune encephalomyelitis (EAE) in immunocompromised mice humanized with peripheral blood mononuclear cells (PBMCs) from individuals with or without a history of EBV infection and/or diagnosis of relapsing MS. HuPBMC EAE mice generated from EBV seronegative healthy donors (HD) were less susceptible to developing severe clinical symptoms than EBV seropositive cohorts. Donor EBV seropositivity and RRMS diagnosis led to a significant incremental increase in the human Th1:Treg CD4+ T cell ratio in the brain and spinal cord, as well as increased human cytotoxic CD8+ T cell and murine macrophage infiltration and demyelination. The data indicate that a history of EBV infection, further compounded by a diagnosis of RRMS, promotes Th1-mediated disease in a novel humanized mouse model of MS.


title: Population Dynamics and Resource Availability Drive Seasonal Shifts in the Consumptive and Competitive Impacts of Introduced House Mice (Mus musculus) on an Island Ecosystem

abstract:
BackgroundHouse mice (Mus musculus) are widespread and invasive on many islands where they can have both direct and indirect ecological impacts on native ecological communities. Given their opportunistic, omnivorous nature the consumptive and competitive impacts of house mice on islands have the potential to vary over time in concert with resource availability and mouse population dynamics.

MethodsWe examined the ecological niche of invasive house mice on Southeast Farallon Island, California, USA using a combination of mouse trapping, food resource surveys, and stable isotope analysis to better understand their trophic interactions with native flora and fauna.

ResultsWe found that plants were the important resource for house mice during the spring months when vegetation is abundant and mouse populations are low. However, plants decline in dietary importance throughout the summer and fall as mouse populations increase, and seabird and insect resources become relatively more available and consumed by house mice. Mouse abundance peaks and other resource availability are low in the fall months when the isotopic niches of house mice and salamanders overlap significantly indicating the potential for competition, most likely for insect prey.

DiscussionThese results indicate how seasonal shifts in both the mouse abundance and resource availability are key factors that mediate the consumptive and competitive impacts of introduced house mice on this island ecosystem.

category: ecology


title: Phenotypic and transcriptional response of Daphnia pulicaria to the combined effects of temperature and predation

abstract: Daphnia, an ecologically important zooplankton species in lakes, shows both genetic adaptation and phenotypic plasticity in response to temperature and fish predation, but little is known about the molecular basis of these responses and their potential interactions. We performed a factorial experiment exposing laboratory-propagated Daphnia pulicaria clones from two lakes in the Sierra Nevada mountains of California to normal or high temperature (15{degrees}C or 25{degrees}C) in the presence or absence of fish kairomones, then measured changes in life history and gene expression. Exposure to kairomones increased upper thermal tolerance limits for physiological activity in both clones. Cloned individuals matured at a younger age in response to higher temperature and kairomones, while size at maturity, fecundity and population intrinsic growth were only affected by temperature. At the molecular level, both clones expressed more genes differently in response to temperature than predation, but specific genes involved in metabolic, cellular, and genetic processes responded differently between the two clones. Although gene expression differed more between clones from different lakes than experimental treatments, similar phenotypic responses to predation risk and warming arose from these clone-specific patterns. Our results suggest that phenotypic plasticity responses to temperature and kairomones interact synergistically, with exposure to fish predators increasing the tolerance of Daphnia pulicaria to stressful temperatures, and that similar phenotypic responses to temperature and predator cues can be produced by divergent patterns of gene regulation.


title: The Eucalyptus grandis chloroplast proteome: leaf development and seasonal variations

abstract: Chloroplast metabolism is very sensitive to environmental fluctuations and is intimately related to plant leaf development. Characterization of the chloroplast proteome dynamics may contribute to enlarge the understanding on plant adaptation to different climate scenarios and leaf development processes. Herein, we carried out a discovery-driven proteome analysis of the Eucalyptus grandis chloroplast proteome during leaf maturation and throughout different seasons of the year. The chloroplast proteome from young leaves differed the most from all assessed samples. Most up-regulated proteins identified in mature and young leaves were those related to catabolic-redox signaling and biogenesis processes, respectively. Seasonal dynamics revealed unique proteome features in the autumn and spring periods. The most abundant chloroplast protein in humid (wet) seasons (spring and summer) was a small subunit of RuBisCO, while in the dry periods (fall and winter) the proteins that showed the most pronounced accumulation were associated with photo-oxidative damage, Calvin cycle, shikimate pathway, and detoxification. Our investigation of the chloroplast proteome dynamics during leaf development revealed significant alterations in relation to the maturation event. Our findings also suggest that transient seasons induced the most pronounced chloroplast proteome changes over the year. This study contributes to a more comprehensive understanding on the subcellular mechanisms that lead to plant leaf adaptation and ultimately to Eucalyptus grandis productivity. Mass spectrometric data are available via ProteomeXchange under identifier PXD029004.

category: plant biology


title: Brain structure, phenotypic and genetic correlates of reading abilities

abstract: Reading is an evolutionary new development that recruits and tunes brain circuitry connecting visual- and language-processing regions. We investigated the structural correlates of reading and whether genetics influence brain-reading associations. First, we identified left hemisphere cortical surface area (CSA) and cortical thickness (CT) correlates of reading in the large ABCD dataset (N=9,013) of 9-to-10-year-olds. Next, the heritability of cognitive and brain measures of interest was examined through complementary approaches. Last, shared genetic effects between reading, reading-related cognitive traits and reading-associated brain measures were examined by computing genetic correlations and polygenic score analyses, and through mediation analyses. Our results support that morphometric brain measures are related to reading abilities, and that the total left CSA in general, and left superior temporal gyrus CSA in particular, contribute to reading partially through genetic factors.

category: neuroscience


title: Mapping of promoter usage QTL using RNA-seq data reveals their contributions to complex traits

abstract: Genomic variations are associated with gene expression levels, which are called expression quantitative trait loci (eQTL). Most eQTL may affect the total gene expression levels by regulating transcriptional activities of a specific promoter. However, the direct exploration of genomic loci associated with promoter activities using RNA-seq data has been challenging because eQTL analyses treat the total expression levels estimated by summing those of all isoforms transcribed from distinct promoters. Here we propose a computational framework for identifying genomic loci associated with promoter activities, called promoter usage quantitative trait loci (puQTL), using conventional RNA-seq data. By leveraging public RNA-seq datasets from the lymphoblastoid cell lines of 438 individuals from the GEUVADIS project, we obtained promoter activity estimates and mapped 2,592 puQTL at the 10% FDR level. The results of puQTL mapping enabled us to interpret the manner in which genomic variations regulate gene expression. We found that 310 puQTL genes (16.1%) were not detected by eQTL analysis, suggesting that our pipeline can identify novel variant-gene associations. Furthermore, we identified genomic loci associated with the activity of "hidden" promoters, which the standard eQTL studies have ignored. We found that most puQTL signals were concordant with at least one genome-wide association study (GWAS) signal, enabling novel interpretations of the molecular mechanisms of complex traits. Our results emphasize the importance of the re-analysis of public RNA-seq datasets to obtain novel insights into gene regulation by genomic variations and their contributions to complex traits.


title: Oxidation-reduction and photophysical properties of isomeric forms of Safranin

abstract: Safranine O is widely used in the bioenergetics community as an indicator dye to determine membrane potentials and as an electron transfer mediator in potentiometric titrations. Here we show that two different commercial preparations of Safranine O contain less than sixty percent by weight of the title compound, with the rest primarily consisting of two closely related safranine isomers. All three major isomer components were isolated using reverse phase HPLC and their structures determined using mass spectrometry and two-dimensional NMR. These Safranines have two-electron midpoint potentials ranging from -272 to -315 mV vs. SHE. We have also investigated the absorption and fluorescence spectra of the compounds and found that they display distinct spectral and photophysical properties. While this mixture may aid in Safranine Os utility as a mediator compound, membrane potential measurements must take this range of dye potentials into account.


title: Age influences audiovisual speech processing in multi-talker scenarios - Evidence from cortical oscillations

abstract: Age-related differences in the processing of audiovisual speech in a multi-talker environment were investigated analyzing event-related spectral perturbations (ERSPs), focusing on theta, alpha, and beta oscillations that are assumed to reflect conflict processing, multisensory integration, and attentional mechanisms, respectively. Eighteen older and 21 younger healthy adults completed a two-alternative forced-choice word discrimination task, responding to audiovisual speech stimuli. In a cocktail-party scenario with two competing talkers (located at-15{degrees} and 15{degrees} azimuth), target words (/yes/ or /no/) appeared at a pre-defined (attended) position, distractor words at the other position. In two audiovisual conditions, acoustic speech was combined either with congruent or uninformative visual speech. While a behavioral benefit for congruent audiovisual speech occurred for both age groups, differences between audiovisual conditions in the theta and beta band were only present for older adults. A stronger increase in theta perturbations for stimuli containing uninformative visual speech could be associated with early conflict processing, while a stronger suppression in beta perturbations for congruent audiovisual speech could be associated to audiovisual integration. Compared to the younger group, the older group showed generally stronger beta perturbations. No condition differences in the alpha band were found. Overall, the findings suggest age-related differences in audiovisual speech integration in a multi-talker environment. While the behavioral benefit of congruent audiovisual speech was unaffected by age, older adults had a stronger need for cognitive control when processing conflicting audiovisual speech input. Furthermore, mechanisms of audiovisual integration are differently activated depending on the informational content of the visual information.


title: Myelin lipids as nervous system energy reserves

abstract: Neuronal functions and impulse propagation depend on the continuous supply of glucose1,2. Surprisingly, the mammalian brain has no obvious energy stores, except for astroglial glycogen granules3. Oligodendrocytes make myelin for rapid axonal impulse conduction4 and also support axons metabolically with lactate5-7. Here, we show that myelin itself, a lipid-rich membrane compartment, becomes a local energy reserve when glucose is lacking. In the mouse optic nerve, a model white matter tract, oligodendrocytes survive glucose deprivation far better than astrocytes, by utilizing myelin lipids which requires oxygen and fatty acid beta-oxidation. Importantly, fatty acid oxidation also contributes to axonal ATP and basic conductivity. This metabolic support by fatty acids is an oligodendrocyte function, involving mitochondria and myelin-associated peroxisomes, as shown with mice lacking Mfp2. To study reduced glucose availability in vivo without physically starving mice, we deleted the Slc2a1 gene from mature oligodendrocytes. This caused a significant decline of the glucose transporter GLUT1 from the myelin compartment leading to myelin sheath thinning. We suggest a model in which myelin turnover under low glucose conditions can transiently buffer axonal energy metabolism. This model may explain the gradual loss of myelin in a range of neurodegenerative diseases8 with underlying hypometabolism9.


title: Increased vulnerability of Purkinje cells in the posterior cerebellum of SCA1 mice is associated with molecular and cellular alterations related to disease pathology

abstract:
Spinocerebellar Ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by abnormal expansion of the polyglutamine (polyQ) repeats in the ATAXIN1 (ATXN1) protein. Patients with SCA1 suffer from progressive motor and cognitive impairments, without any disease modifying treatments available. Although mutant ATXN1 is expressed throughout the brain, the cerebellum undergoes the most severe degeneration. It remains unclear why the cerebellum is particularly vulnerable in SCA1.

A majority of previous studies using mouse models of SCA1 have investigated pathological changes in the cerebellar cortex by examining a singular region in the cerebellar cortex, the vermal primary fissure (lobules V and VI). Gene expression changes have mostly been examined using the bulk cerebellum. However, recent studies in patients indicate that SCA1 pathogenesis is not uniform across cerebellum, and in fact demonstrate that the posterior vermis and hemispheres exhibit worse pathology. Pathological and molecular alterations in the posterior cerebellum and hemispheres remain mostly unknown in mouse models of SCA1. This study addresses these questions using Atxn1154Q/2Q line, a knock-in SCA1 mouse model in which mutant ATXN1 is widely expressed. We found more pronounced pathology of Purkinje cells and reactive gliosis in the posterior vermis of Atxn1154Q/2Q mice at mid -disease stages, similar to what has been described in SCA1 patients.

Comparison of the gene expression changes demonstrates that several cerebellar cell types, including Purkinje cells, and Bergmann glia, are more affected in the posterior vermis, and hemispheres. Pathway analysis identified discrete, region specific SCA1 pathogenesis pathways as well as commonly enriched biological pathways.


title: Evaluation of Three Forms of Rhizoctonia solani Mediated Pathogenicity to Sugar Beet Cultivars in Greenhouse Studies

abstract: Rhizoctonia solani causes damping-off, as well as crown and root rot of sugar beet (Beta vulgaris L). This pathogen overwinters as sclerotia or melanized mycelia. Traditionally, the resistance of cultivars to R. solani is evaluated by scoring disease reactions of the crowns and roots of older seedlings, instead of evaluating at seed germination. Most studies that have evaluated cultivar resistance to R. solani have used colonized whole barley grains as artificial inocula. Colonized grains are prone to contamination with other pathogens and are often lost to rodents/birds when applied in the field. Considering those limitations, a study was undertaken (1) to develop in vitro methods to generate natural sclerotia of R. solani on a large scale, (2) to compare pathogenic potentials of R. solani sclerotia, mycelia, and colonized barley grains for optimization of damping-off assays, and (3) to evaluate resistance of selected commercial cultivars to R. solani. Of the six-culture media, amended clarified V8 (ACV8) was the most suitable culture medium to grow and produce sclerotia on a large scale and 10% PDA was the least suitable. Three testing sizes of sclerotia were found to be equally effective in causing plant losses. Sclerotia inocula were comparable with mycelial discs and colonized barley grains in causing pre-emergence damping off under aseptic in vitro conditions. Sclerotia also were equally or more effective than mycelia plug or barley grain inocula in reducing seedling emergence, inducing damping off, and increased root rot under greenhouse in vivo conditions. To conclude, sclerotia can be prepared on a feasible scale and used as natural inocula to screen response to R. solani on sugar beet.


title: Low-cost and open-source super-resolution fluorescence microscope with autofocus for teaching and research

abstract: Super-resolution fluorescence microscopy revolutionized optical microscopy by breaking the theoretical resolution limit and thus providing an unprecedented view of biological structure in exquisite detail. Additionally, the relative ease of use and implementation for some super-resolution microscopes provided hope that this technology would be widely available and accessible. Despite initial excitement, super-resolution microscopy is still largely limited to facilities that can afford expensive commercial instruments or with personnel experienced in optical engineering that can construct an instrument. Recently, the open scientific hardware movement has attempted to make scientific instrumentation more widely available and accessible in a similar way to the open software movement. The widespread availability and affordability of prototyping devices, such as three-dimensional printers and interfaces (e.g., Arduino) have all contributed to unlocking the potential of the open hardware movement, turning expensive "black box" hardware into open-source, affordable, and robust instrumentation. Many others have recognized the potential of describing low-cost implementations of super-resolution microscope designs. However, we have found these microscope designs lacking in the documentation and details required to meet the requirements of open-source hardware. In this paper, we attempt to provide the documentation, details, and instructions necessary for the construction of an open-source and low-cost super-resolution microscope at a targeted cost of <$15,000.

category: scientific communication and education


title: SUCCESSFUL IN VITRO EMBRYO PRODUCTION WITH OOCYTES ASPIRATED FROM LIVE WHITE-TAILED DEER (Odocoileus virginianus texanus) DONNORS UNDER CAPTIVITY IN NORTHEAST MEXICO

abstract: Assisted reproductive technologies (ARTs), such as artificial insemination, semen sorting and freezing, embryo production in vivo and in vitro, are all methods in which animal reproduction management of several species has become considerably more efficient. These ARTs have been applied both in domestic and wild ruminants. In this study, in vitro embryo production was attempted with oocytes collected surgically during the mating season, from live white-tailed deer (WTD) hinds, maintained under captivity, in Northeast Mexico. The study was conducted in two WTD farms nearby Cd. Victoria, Tamps., Mexico, and another WTD farm located in Guerrero Coahuila, Mexico. The laboratory work was carried at the Centro de Desarrollo de la Capacidad Productiva y Mejoramiento Genetico de la Ganaderia, property of the Union Ganadera Regional de Tamaulipas (Centro-UGRT). The deer hinds are kept in captivity year-around, with feeding and health management provided accordingly to their requirements (50-60 kg females). Fresh green forage and clean fresh water is supplied daily. Oocytes were collected on each farm, while the hinds were maintained under general anesthesia and by means of a mid-ventral laparotomy, ovaries were exposed and follicles greater than 1-2 mm were aspirated with a 20 G needle, connected by a two-way plastic and latex hose to a vacum machine (WTA, Brazil); the oocytes were collected into a 50 ml. centrifuge tube containing wash media with heparine (Vitrogen, Brazil). Each oocyte collection lasted approximately 20 minutes, after which and while still on the farm, the oocytes were filtered with 50 {micro} mesh filters and rinsed several times with wash media, then placed on search Petri plates. Oocytes were then counted and classified (total, viable and non-viable oocytes) and placed into cryovials with maturation media (3 ml. MIV, Vitrogen, Brazil) and placed into a portable incubator with 5 % CO2 gas mix (LabiMixWTA, Brazil), after all hinds were done, oocytes were transported to the in vitro fertilization laboratory (IVF Lab) at the Centro-UGRT. Once at the IVF Lab, the oocytes were placed on a larger incubator (Eve, WTA, Brazil) and kept there for 18-20 hours, after which, the MIV media was changed for fertilization media (FIV, Vitrogen, Brazil) and fertilization was initiated by adding 10,000-12,000 live sperm per cryovial, and incubated for an additional period of 24 hours; after which, FIV media was replaced by the same media and at this point, cleavage rate was estimated by counting the oocytes that initiated cell division. At 72 hours after cell division started, fertilization rate was estimated; and 7 days after, the blastocysts were counted and classified. The whole process of oocyte maturation and embryo production in vitro, was conducted based on a beef cattle embryo production system and adapted to a deer embryo production system using media for small ruminants (Vitrogen, Brazil). Data collected per hind included total number of oocytes, viable and non-viable oocytes, cleavage rate (ratio of viable oocytes that initiated cell division over viable oocytes), fertilization rate (ratio of embryos that initiated cleavage over those that continued development to the blastocyst stage) and blastocyst rate (embryos reaching the blastocyst stage over cleaved embryos); averages were calculted for each parameter. The main results from this study on a per hind basis for total viable and non-viable oocytes were 9.8, 6.3 and 4.5, respectively; cleavage and blastocyst rates were 39.5 and 36.8, respectively and 2.3 blastocysts. In conclusion, oocyte collection from live WTD hinds and in vitro embryo production was succesfully done under farming conditions in Northeast Mexico.

category: zoology