title: Genetic and epigenetic determinants of reactivation of Mecp2 and the inactive X chromosome in neural stem cells.

abstract: Rett Syndrome is a neurodevelopmental disorder in girls that is caused by heterozygous inactivation of the chromatin remodeler gene MECP2. Rett Syndrome may therefore be treated by reactivation of the wild type copy of MECP2 from the inactive X chromosome. Most studies that model Mecp2 reactivation have used mouse fibroblasts rather than neural cells, which would be critical for phenotypic reversal, and rely on fluorescent reporters that lack adequate sensitivity. Here, we present a mouse model system for monitoring Mecp2 reactivation that is more sensitive and versatile than any bioluminescent and fluorescent system currently available. The model consists of neural stem cells derived from female mice with a dual reporter system where MECP2 is fused to NanoLuciferase and TdTomato on the inactive X chromosome. We show by bioluminescence and fluorescence that Mecp2 is synergistically reactivated by 5-Aza treatment and Xist knockdown. As expected, other genes on the inactive X chromosome are also reactivated, the majority of which overlaps with genes reactivated early during reprogramming of mouse embryonic fibroblasts to iPSCs. Genetic and epigenetic features such as CpG density, SINE elements, distance to escapees and CTCF binding are consistent indicators of reactivation, whereas different higher order chromatin areas are either particularly prone or resistant to reactivation. Our MeCP2 reactivation monitoring system thereby suggests that genetic and epigenetic features on the inactive X chromosome affect reactivation of its genes, irrespective of cell type or procedure of reactivation.


title: Larvae and adults exhibit contrasting patterns of immune gene expression and infection resistance in wild flour beetle populations

abstract: In nature, hosts face shifting patterns of parasite exposure and life history trade-offs as they develop from birth to old age. As a result, the net fitness benefit of immunological investment can change dramatically from one life stage to the next. Previous work has revealed a puzzling diversity of relative immune investment patterns among juvenile and adult stages, and it is not clear whether lessons learned from one particular population or species can be generalized to wild populations, after accounting for local adaptation and other variance-generating processes. In this study, we quantify larval and adult immune gene expression and resistance to bacterial infection in two flour beetle species (Tribolium castaneum and T. confusum) from two lab-adapted and five wild-derived populations. Our results provide a clear signal of higher infection-induced immunological investment and resistance in adults relative to larvae, despite variation among species in immune gene regulation. Better characterization of stage-specific investment in infection resistance in natural populations can inform our understanding of life history evolution and improve predictions of disease dynamics in the wild.

category: evolutionary biology


title: The evolutionary history of a gammaretrovirus currently colonizing the mule deer genome is marked by extensive recombination

abstract: All vertebrate genomes have been colonized by retroviruses along their evolutionary trajectory. While endogenous retroviruses (ERVs) can contribute important physiological functions to contemporary hosts, such benefits are attributed to long-term co-evolution of ERV and host because germline infections are rare and expansion is slow, because the host effectively silences them. The genomes of several outbred species including mule deer (Odocoileus hemionus) are currently being colonized by ERVs, which provides an opportunity to study ERV dynamics at a time when few are fixed. Because we have locus-specific data on the distribution of cervid endogenous retrovirus (CrERV) in populations of mule deer, in this study we determine the molecular evolutionary processes acting on CrERV at each locus in the context of phylogenetic origin, genome location, and population prevalence. A mule deer genome was de novo assembled from short and long insert mate pair reads and CrERV sequence generated at each locus. CrERV composition and diversity have recently measurably increased by horizontal acquisition of a new retrovirus lineage. This new lineage has further expanded CrERV burden and CrERV genomic diversity by activating and recombining with existing CrERV. Resulting inter-lineage recombinants endogenized and subsequently retrotransposed. CrERV loci are significantly closer to genes than expected if integration were random and gene proximity might explain the recent expansion by retrotransposition of one recombinant CrERV lineage. Thus, in mule deer, retroviral colonization is a dynamic period in the molecular evolution of CrERV that also provides a burst of genomic diversity to the host population.

category: genomics


title: Recombination marks the evolutionary dynamics of a recently endogenized retrovirus


title: Visualization of SARS-CoV-2 Infection Scenes by 'Zero-Shot' Enhancements of Electron Microscopy Images

abstract: Electron microscopy (EM) recordings of infected tissues serve to diagnose a disease, and they can contribute to our understanding of infection processes. Consequently, a large number of EM images of the interaction of SARS-CoV-2 viruses with cells have been made available by numerous labs. However, due to EM recording techniques at high resolution, images of infection scenes are very noisy and they appear two dimensional ( flat). Current research consequently aims (A) at methods that can remove noise, and (B) at techniques that allow for recovering a 3D impression of the virus or its parts. Here we discuss a novel method which can recover a spatial impression of a whole infection scene at high resolution. In contrast to previous approaches which aim at the reconstruction of single spike proteins or a single virus, the here used method can be applied to a single noisy EM image of an infection scene. As one example image, we show a high resolution image of SARS-CoV-2 viruses in Vero cell cultures (Fig. 1). The method we use is based on probabilistic machine learning algorithms which can operate in a  zero-shot setting, i.e., in a setting when just one noisy image (and no large and clean image corpus) is available. The probabilistic method we apply can estimate non-noisy images by inferring first order statistics (pixel means) across image patches using a previously learned probabilistic image representation. Estimating higher order statistics and appropriately chosen probabilistic models then allow for the generation of images that enhance details and give a spatial impression of a full nanoscopic scene.

category: bioinformatics


title: Towards a systematic characterization of protein complex function: a natural language processing and machine-learning framework

abstract: It is a general assumption of molecular biology that the ensemble of expressed molecules, their activities and interactions determine biological processes, cellular states and phenotypes. Quantitative abundance of transcripts, proteins and metabolites are now routinely measured with considerable depth via an array of "OMICS" technologies, and recently a number of methods have also been introduced for the parallel analysis of the abundance, subunit composition and cell state specific changes of protein complexes. In comparison to the measurement of the molecular entities in a cell, the determination of their function remains experimentally challenging and labor-intensive. This holds particularly true for determining the function of protein complexes, which constitute the core functional assemblies of the cell. Therefore, the tremendous progress in multi-layer molecular profiling has been slow to translate into increased functional understanding of biological processes, cellular states and phenotypes. In this study we describe PCfun, a computational framework for the systematic annotation of protein complex function using Gene Ontology (GO) terms. This work is built upon the use of word embedding-- natural language text embedded into continuous vector space that preserves semantic relationships-- generated from the machine reading of 1 million open access PubMed Central articles. PCfun leverages the embedding for rapid annotation of protein complex function by integrating two approaches: (1) an unsupervised approach that obtains the nearest neighbor (NN) GO term word vectors for a protein complex query vector, and (2) a supervised approach using Random Forest (RF) models trained specifically for recovering the GO terms of protein complex queries described in the CORUM protein complex database. PCfun consolidates both approaches by performing the statistical test for the enrichment of the top NN GO terms within the child terms of the predicted GO terms by RF models. Thus, PCfun amalgamates information learned from the gold-standard protein-complex database, CORUM, with the unbiased predictions obtained directly from the word embedding, thereby enabling PCfun to identify the potential functions of putative protein complexes. The documentation and examples of the PCfun package are available at https://github.com/sharmavaruns/PCfun. We anticipate that PCfun will serve as a useful tool and novel paradigm for the large-scale characterization of protein complex function.


title: Elementary growth modes/vectors and minimal autocatalytic sets for kinetic/constraint-based models of cellular growth

abstract:
Elementary vectors are fundamental objects in polyhedral geometry. In metabolic pathway analysis, elementary vectors range from elementary flux modes (of the flux cone) and elementary flux vectors (of a flux polyhedron) via elementary conversion modes (of the conversion cone) to minimal cut sets (of a dual polyhedron) in computational strain design.

To better understand cellular phenotypes with optimal (or suboptimal) growth rate, we introduce and analyze classes of elementary vectors for models of cellular growth. Growth modes (GMs) only depend on stoichiometry, but not on growth rate or concentrations; they are elements of the growth cone. Elementary growth modes (EGMs) are conformally nondecomposable GMs; unlike elementary flux modes, they are not support-minimal, in general. Most importantly, every GM can be written as a conformal sum of EGMs. Growth vectors (GVs) and elementary growth vectors (EGVs) also depend on growth rate, concentrations, and linear constraints; they are elements of a growth polyhedron. Again, every GV can be written as a conformal sum of EGVs. To relate the new concepts to other branches of theory, we define autocatalytic GMs and the corresponding (minimal) autocatalytic sets of reactions.

As a case study, we consider whole cell models (simple kinetic models of self-fabrication). First, we use EGMs to derive an upper bound for growth rate that only depends on enzyme kinetics. Next, we study growth rate maximization (via control parameters for ribosome kinetics). In particular, we analyze growth states (GSs) and elementary growth states (EGSs) as introduced in [de Groot et al, 2020]. Unlike EGMs, EGSs depend on (metabolite) concentrations and growth rate. Most importantly, (i) we show that EGSs are support-minimal, (ii) we give a simple proof for the fact that maximum growth rate is attained at an EGS, and (iii) we show that, at every optimal EGS, the ribosome capacity constraint is active. Finally, we determine the dependence of EGSs on growth rate, and we study the relation between EGSs and minimal autocatalytic sets, EGMs, and elementary flux modes. Along the way, we point out (and resolve) mathematical issues in [de Groot et al, 2020].

category: systems biology


title: Postembryonic development of the two-spotted field cricket (Gryllus bimaculatus): a staging system

abstract: The two-spotted field cricket Gryllus bimaculatus has emerged as a central model for studies on insect development, regeneration, and physiology. G. bimaculatus has the most sophisticated functional genetic toolkit of any hemimetabolous insect, making it a foremost model to understand the evolutionary developmental biology and comparative physiology of insects. However, the morphology and stages of postembryonic development have never been comprehensively reported. Here, 8 morphologically defined stages are described. Size, coloration, and the morphology of wing buds, hind tibial spines, and the ovipositor are the best landmarks for staging. The stages correspond to the 8-12 moult-based instars present in the literature. The staging system aims to standardise studies on the postembryonic development of G. bimaculatus and serve as a point of reference for delineating interspecific postembryonic homologies within Orthoptera.

category: zoology


title: Domestication effects on immune response: comparison of cell-mediated immune competence in wild and domesticated Bengalese finch

abstract: Domesticated Bengalese finch (Lonchura striata var. domestica) lack natural selection pressures and consequently have more complex songs and altered behavioural and psychological traits when compared to their wild ancestor, the white-rumpud munia (Lonchura striata). Clarifying the sexual traits and life history trade-offs in Bengal finches will be help to improve our understanding of the evolution of complex songs and domesticated traits. Here, we have assessed the immune competence of the Bengalese finch and the white-rumped munia using phytohemagglutinin (PHA) tests to create an index of cell-mediated immune responses. We found that the Bengalese finch had a significantly larger immunocompetence than the white-rumped munia, indicating that they devote more resources to both immunity and reproduction. Thus, there is no trade-off but a positive relationship between immunocompetence and reproductive traits, which may be related to the release from natural selection pressures. These results will be useful in understanding the mechanisms by which domestication-induced changes due to a lack of natural selection pressure affect behavioural and physiological changes.


title: Can we harness digital technologies and physiology to hasten genetic gain in U.S. maize breeding?

abstract: Genetic gain in breeding programs depends on the predictive skill of genotype-to-phenotype algorithms and precision of phenotyping, both integrated with well-defined breeding objectives for a target population of environments (TPE). The integration of physiology and genomics could improve predictive skill by capturing additive and non-additive interaction effects of genotype (G), environment (E), and management (M). Precision phenotyping at managed stress environments (MSEs) can elicit physiological expression of processes that differentiate germplasm for performance in target environments, thus enabling algorithm training. Gap analysis methodology enables design of GxM technologies for target environments by assessing the difference between current and attainable yields within physiological limits. Harnessing digital technologies such as crop growth model-whole genome prediction (CGM-WGP) and gap analysis, and MSEs, can hasten genetic gain by improving predictive skill and definition of breeding goals in the U.S. maize production TPE. A half-diallel maize experiment resulting from crossing 9 elite maize inbreds was conducted at 17 locations in the TPE and 6 locations at MSEs between 2017 and 2019. Analyses over 35 families represented by 2367 hybrids demonstrated that CGM-WGP offered a predictive advantage (y) compared to WGP that increased with occurrence of drought as measured by decreasing whole-season evapotranspiration (ET; log(y) = 0.80({+/-}0.6) - 0.006({+/-}0.001) x ET; r2 = 0.59; df = 21). Predictions of unobserved physiological traits using the CGM, akin to digital phenotyping, were stable. This understanding of germplasm response to ET enables predictive design of opportunities to close productivity gaps. We conclude that enabling physiology through digital methods can hasten genetic gain by improving predictive skill and defining breeding objectives bounded by physiological realities.

category: plant biology


title: Comparative genomics analysis of Chryseobacterium sp. KMC2 reveals metabolic pathways involved in keratinous utilization and natural product biosynthesis

abstract:
Several efforts have been made to valorize keratinous materials, an abundant and renewable resource. Despite these attempts to valorize products generated from keratin hydrolysate, either via chemical or microbial conversion, they generally remain with an overall low value. In this study, a promising keratinolytic strain from the genus Chryseobacterium (Chryseobacterium sp. KMC2) was investigated using comparative genomic tools against publicly available reference genomes to reveal the metabolic potential for biosynthesis of valuable secondary metabolites. Genome and metabolic features of four species were compared, shows different gene numbers but similar functional categories. We successfully mined eleven different secondary metabolite gene clusters of interest from the four genomes, including five common ones shared across all genomes. Among the common metabolites, we identified gene clusters involved in biosynthesis of flexirubin-type pigment, microviridin, and siderophore, all showing remarkable conservation across the four genomes. Unique secondary metabolite gene clusters were also discovered, for example, ladderane from Chryseobacterium sp. KMC2. Additionally, this study provides a more comprehensive understanding of the potential metabolic pathways of keratin utilization in Chryseobacterium sp. KMC2, with the involvement of amino acid metabolism, TCA cycle, glycolysis/gluconeogenesis, propanoate metabolism, and sulfate reduction. This work uncovers the biosynthesis of secondary metabolite gene clusters from four keratinolytic Chryseobacterium spp. and shades lights on the keratinolytic potential of Chryseobacterium sp. KMC2 from a genome-mining perspective, providing alternatives to valorize keratinous materials into high-value natural products.

ImportanceKeratin is an abundant and renewable resource from slaughterhouses or the poultry industry. Low-value products such as animal feed and fertilizer were generated from these feedstocks based on conventional processing like chemical conversion. In fact, microorganisms possess the potential to synthesize valuable natural products. In this work, we explored the metabolic potential of Chryseobacterium sp. KMC2, which was isolated with efficient keratinolytic capacity from a previous study. Comparative genomics analysis displayed similar functional categories against three publicly available reference genomes of keratin-degrading Chryseobacterium spp.. Eleven different secondary metabolite gene clusters of interest were mined among four genomes, including five common and unique ones. Furthermore, we provide a more comprehensive understanding of metabolic pathways on keratin utilization in Chryseobacterium sp. KMC2, with the involvement of amino acid assimilation and sulfate reduction. These findings contribute to expanding the application of Chryseobacterium sp. KMC2 on the valorization of keratinous materials.

category: microbiology


title: Bacterial Cytological Profiling Identifies Rhodanine-containing PAINS Analogs as Specific Inhibitors of E. coli Thymidylate Kinase in Vivo

abstract:
In this study, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules inhibiting the growth of E. coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite their structural similarity to related PAINS. A genetic analysis of resistant mutants revealed that thymidylate kinase (an enzyme essential for DNA synthesis) is an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by assays of in vitro activity as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the lowest IC50 in vitro and MIC in vivo displayed the greatest specificity for the inhibition of DNA replication in E. coli, despite containing a rhodamine moiety. While its generally thought that PAINS cannot be developed as antibiotics, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics, and it showcases novel inhibitors of E. coli thymidylate kinase.

ImportanceWe demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotics in vivo mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and non-specific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While in vitro inhibition assays are susceptible to false positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.


title: Expression patterns of Plasmodium falciparum clonally variant genes at the onset of a blood infection in non-immune humans

abstract:
Clonally variant genes (CVGs) play fundamental roles in the adaptation of Plasmodium falciparum parasites to the fluctuating conditions of the human host. However, their expression patterns under the natural conditions of the blood circulation have been characterized in detail only for a few specific gene families. Here we provide a detailed characterization of the complete P. falciparum transcriptome across the full intraerythrocytic development cycle (IDC) at the onset of a blood infection in non-immune human volunteers. We found that the vast majority of transcriptional differences between parasites obtained from the volunteers and the parental parasite line maintained in culture occur in CVGs. Specifically, we observed a major increase in the transcript levels of most members of the pfmc-2tm and gbp families and of specific genes of other families, in addition to previously reported changes in var and clag3 genes expression. Large transcriptional differences correlate with changes in the distribution of heterochromatin, confirming their epigenetic nature. The analysis of parasites collected at different time points along the infection indicates that when parasites pass through transmission stages, the epigenetic memory at CVG loci is lost, resulting in a reset of their expression state and reestablishment of new epigenetic patterns.

ImportanceThe ability of malaria parasites to adapt to changes in the human blood environment, where they produce long term infection associated with clinical symptoms, is fundamental for their survival. Clonally variant genes, regulated at the epigenetic level, play a major role in this adaptive process, as changes in the expression of these genes result in antigenic and functional alterations that enable immune evasion and provide phenotypic plasticity. However, the way these genes are expressed under the natural conditions of the human circulation or how their expression is affected by passage through transmission stages is not well understood. Here we provide a comprehensive characterization of the expression patterns of these genes at the onset of human blood infections, which reveals major differences with in vitro cultured parasites and also distinctive alterations between different families of clonally variant genes. We also show that epigenetic patterns are erased and reestablished during transmission stages.


title: Cervical Squamous Intraepithelial Lesions are Associated with Changes in the Vaginal Microbiota of Mexican Women

abstract:
Cervical cancer is an important health concern worldwide and is one of the leading causes of deaths in Mexican women. Previous studies have shown changes in the female genital tract microbe community related to Human Papillomavirus (HPV) infection and cervical cancer, yet this link remains unexplored in many human populations. This study evaluated the vaginal bacterial community among Mexican women with pre-cancerous Squamous Intraepithelial Lesions (SIL). We sequenced the V3 region of the 16S rRNA gene (Illumina Miseq) in cervical samples from 300 Mexican women, including 157 patients with SIL, most of which were HPV positive, and 143 healthy women without HPV infection or SIL. Beta-diversity analysis showed that 14.6% of the variance in vaginal bacterial community structure is related to the presence of SIL. Presence of SIL was also associated with a higher species richness (Chao 1). MaAsLiN analysis yielded independent associations between SIL/HPV status and an increase in the relative abundance Brachybacterium conglomeratum, as well as a decrease in Sphingobium yanoikuyae and Lactobacillus spp. We also identified independent associations between HPV-16, the most common HPV subtype linked to SIL, and Brachybacterium conglomeratum. Our work indicates that the presence of SIL and HPV infection is associated with important changes in the vaginal microbiome, some of which may be specific to this human population.

IMPORTANCEHPV plays a critical role in cervical carcinogenesis but is not sufficient for cervical cancer development, indicating involvement of other factors. Vaginal microbiota is an important factor in controlling infections caused by HPV and depending on its composition it can modulate the microenvironment in vaginal mucosa against viral infection. Ethnic and sociodemographic factors influence differences in vaginal microbiome composition, which underlies the dysbiotic patterns linked to HPV infection and cervical cancer across different women populations. Here, we provide evidence for associations between vaginal microbiota patterns and HPV infection, linked to ethnic and sociodemographic factor. To our knowledge, this is the first report of Brevibacterium aureum and Brachybacterium conglomeratum species linked to HPV infection or SIL.


title: Miltefosine enhances infectivity of miltefosine-resistant Leishmania infantum by attenuating innate immune recognition

abstract:
Miltefosine (MIL) is currently the only oral drug available to treat visceral leishmaniasis but its use as first-line monotherapy has been compromised by an increasing treatment failure. Despite the scarce number of resistant clinical isolates, MIL-resistance by mutations in a single aminophospholipid transporter gene can easily be selected in a laboratory environment. These mutations result in a reduced survival in the mammalian host, which can partially be restored by exposure to MIL, suggesting a kind of drug-dependency. To enable a combined study of the infection dynamics and underlying immunological events for differential in vivo survival, firefly luciferase (PpyRE9) / red fluorescent protein (DsRed) double-reporter strains were generated of MIL-resistant (MIL-R) and syngeneic MIL-sensitive (MIL-S) Leishmania infantum. Results in C57Bl/6 and BALB/c mice show that MIL-R parasites induce an increased innate immune response that is characterized by enhanced influx and infection of neutrophils, monocytes and dendritic cells in the liver and elevated serum IFN-{gamma} levels, finally resulting in a less efficient establishment in liver macrophages. The elevated IFN-{gamma} levels were shown to originate from an increased response of hepatic NK and NKT cells to the MIL-R parasites. In addition, we demonstrated that MIL could increase the in vivo fitness of MIL-R parasites by lowering NK and NKT cell activation, leading to a reduced IFN-{gamma} production. These data provide an immunological basis for the MIL-R-associated attenuated phenotype and for the peculiar drug-dependency that may constitute one of the mechanisms of treatment failure.

ImportanceRecently, our laboratory demonstrated an in vivo fitness loss of experimentally selected MIL-R parasites in both the sand fly vector and vertebrate host. These findings could explain the scarce number of MIL-R clinical isolates. Surprisingly, MIL-R parasites developed a MIL-dependency which could partially rescue their fitness loss and which may constitute a mechanism of treatment failure. This research aimed to better understand the immunological basis of the attenuated phenotype and the effect of MIL on infectivity traits. Together, this study provides new insights into the complex interplay between the parasite, drug and host and discloses an immune-related mechanism of treatment failure.


title: Apical contacts stemming from incomplete delamination guide progenitor cell allocation through a dragging mechanism

abstract:
The developmental strategies used by progenitor cells to endure a safe journey from their induction place towards the site of terminal differentiation are still poorly understood. Here we uncovered a progenitor cell allocation mechanism that stems from an incomplete process of epithelial delamination that allows progenitors to coordinate their movement with adjacent extra-embryonic tissues. Progenitors of the zebrafish laterality organ originate from the surface epithelial enveloping layer by an apical constriction process of cell delamination. During this process, progenitors retain long-term apical contacts that enable the epithelial layer to pull a subset of progenitors along their way towards the vegetal pole. The remaining delaminated progenitors follow apically-attached progenitors movement by a co-attraction mechanism, avoiding sequestration by the adjacent endoderm, ensuring their fate and collective allocation at the differentiation site. Thus, we reveal that incomplete delamination serves as a cellular platform for coordinated tissue movements during development.

Impact StatementIncomplete delamination serves as a cellular platform for coordinated tissue movements during development, guiding newly formed progenitor cell groups to the differentiation site.

category: developmental biology


title: The Functional Significance of Bacterial Predators

abstract: Predation structures food webs, influences energy flow, and alters rates and pathways of nutrient cycling through ecosystems, effects that are well documented for macroscopic predators. In the microbial world, predatory bacteria are common, yet little is known about their rates of growth and roles in energy flows through microbial food webs, in part because these are difficult to quantify. Here, we show that growth and carbon uptake were higher in predatory bacteria compared to non-predatory bacteria, a finding across 15 sites, synthesizing 82 experiments and over 100,000 taxon-specific measurements of element flow into newly synthesized bacterial DNA. Obligate predatory bacteria grew 36% faster and assimilated carbon at rates 211% higher than non-predatory bacteria. These differences were less pronounced for facultative predators (6% higher growth rates, 17% higher carbon assimilation rates), though high growth and carbon assimilation rates were observed for some facultative predators, such as members of the genera Lysobacter and Cytophaga, both capable of gliding motility and wolfpack hunting behavior. Added carbon substrates disproportionately stimulated growth of obligate predators, with responses 63% higher than non-predators for the Bdellovibrionales and 81% higher for the Vampirovibrionales, whereas responses of facultative predators to substrate addition were no different from non-predators. This finding supports ecological theory that higher productivity increases predator control of lower trophic levels. These findings also indicate that the functional significance of bacterial predators increases with energy flow, and that predatory bacteria influence element flow through microbial food webs.

category: ecology


title: Genetic variation in haemoglobin regulates breathing in high-altitude deer mice

abstract: Physiological systems often have emergent properties but the effects of genetic variation on physiology are often unknown, which presents a major challenge to understanding the mechanisms of phenotypic evolution. We investigated the in vivo effects on respiratory physiology of genetic variants in haemoglobin (Hb) that contribute to hypoxia adaptation in high-altitude deer mice (Peromyscus maniculatus). We created F2 inter-population hybrids of highland and lowland deer mice to test the phenotypic effects of - and {beta}-globin variants on a mixed genetic background. High-altitude genotypes were associated with breathing phenotypes that enhance O2 uptake in hypoxia, including a deeper more effective breathing pattern and an augmented hypoxic ventilatory response. These effects could not be explained by erythrocyte Hb-O2 affinity or globin gene expression in the brainstem. Therefore, adaptive variation in haemoglobin can have unexpected effects on physiology that are distinct from the canonical function of this protein in circulatory O2 transport.


title: ExOrthist: a tool to infer exon orthologies at any evolutionary distance

abstract: Several bioinformatic tools have been developed for genome-wide identification of orthologous and paralogous genes among species. However, no existing tool allows the detection of orthologous/paralogous exons. Here, we present ExOrthist, a fully reproducible Nextflow-based software enabling to (i) infer exon homologs and orthogroups, (ii) visualize evolution of exon-intron structures, and (iii) assess conservation of alternative splicing patterns. ExOrthist not only evaluates exon sequence conservation but also considers the surrounding exon-intron context to derive genome-wide multi-species exon homologies at any evolutionary distance. We demonstrate its use in various evolutionary scenarios, from whole genome duplication to convergence of alternative splicing networks.


title: HDAC6 promote HCCLM3 cell migration and invasion by regulating TGF-β1pathway

abstract: Histone deacetylase 6 (HDAC6) have been demonstrated to play critical roles in the progression of tumor migration and invasion in recent years.However, little research has been done on hepatocellular carcinoma (HCC). This study investigated the role of HDAC6 in the migration and invasion of HCCLM3 cells and its potential mechanism. Western blot and immunohistochemistry were used to detect the expression of HDAC6 in normal hepatic cell line LO2 and HCC cell line HCCLM3. Transforming growth factor 1 (TGF-{beta}1) was proved as a direct target of HDAC6 by Western blot and qRT-PCR.The changes of migration and invasion ability of HCCLM3 cells were confirmed by wound healing and Transwell assays.Morphological changes of HCCLM3 cells were observed by microscope.Our study proved that drownregulated HDAC6 supress HCCLM3 cells metastasis and invasion. Meanwhile, our study verified that HDAC6 regulate EMT by targeting TGF-{beta}1.In conclusion, HDAC6 inhibits the EMT of HCCLM3 cells and further inhibits the migration and invasion of HCCLM3 cells by downregulating the expression of TGF-{beta}1.

category: biochemistry


title: Dual mechanisms of opioid-induced respiratory depression in the inspiratory rhythm generating network

abstract: The analgesic utility of opioid-based drugs is limited by the life-threatening risk of respiratory depression. Opioid-induced respiratory depression (OIRD), mediated by the -opioid receptor (MOR), is characterized by a pronounced decrease in the frequency and regularity of the inspiratory rhythm, which originates from the medullary preBotzinger Complex (preB[o]tC). To unravel the cellular- and network-level consequences of MOR activation in the preBotC, MOR-expressing neurons were optogenetically identified and manipulated in transgenic mice in vitro and in vivo. Based on these results, a model of OIRD was developed in silico. We conclude that hyperpolarization of MOR-expressing preBotC neurons alone does not phenocopy OIRD. Instead, the effects of MOR activation are twofold: 1) pre-inspiratory spiking is reduced and 2) excitatory synaptic transmission is suppressed, thereby disrupting network-driven rhythmogenesis. These dual mechanisms of opioid action act together to make the normally robust inspiratory-rhythm-generating network particularly prone to collapse when challenged with exogenous opioids.

category: neuroscience


title: Elucidation of fibril structure responsible for swimming in Spiroplasma using electron microscopy

abstract:
Spiroplasma, known pathogens and commensals of arthropods and plants, are helical-shaped bacteria lacking the peptidoglycan layer. They swim by alternating between left- and right-handed cell helicity, which is driven by an internal structure called the ribbon. This system is unrelated to flagellar motility that is widespread in bacteria. The ribbon comprises the bacterial actin homolog MreB and fibril, the protein specific to Spiroplasma. Here, we isolated the ribbon and its core, the fibril filament, and using electron microscopy, found that the helicity of the ribbon and the cell is linked to the helicity of the fibril. Single particle analysis using the negative-staining method revealed that the three-dimensional structures of the fibril filament comprise a repeated ring structure twisting along the filament axis. The handedness of these structures were verified by the structures reconstituted through tomogram from quick-freeze deep-etch replica electron microscopy. Based on these observations, we propose a scheme for the helicity-switching mechanism in which the twists caused by the conformational changes in the fibril filament are accumulated, transmitted to the ribbon, and then propel the cells by rotating the cell body like a screw.

Significance StatementSpiroplasma are commensal and pathogenic bacteria widespread globally in animals and plants. A few species are also recognized as male-killing bacteria of insects. Their special swimming mechanism is caused by helicity switching, which could be the simplest swimming mechanism, because helicity shift can be caused by contraction and extension of paired filaments. This mechanism has attracted research attention for many years because of the possible application in the field of nano actuators; however, the details of this mechanism remain to be clarified. Here, we propose a model for the swimming mechanism based on the structure of the core of the Spiroplasma ribbon.


title: Elucidation of fibril helix structure responsible for swimming in Spiroplasma using electron microscopy


title: Sequence-function relationships in phage-encoded bacterial cell wall lytic enzymes and their implications for phage-derived products design

abstract:
Phage (endo)lysins are thought to be a viable alternative to usual antibiotic chemotherapy to fight resistant bacterial infections. However, a landscape view of lysins structure and properties regarding their function, with an applied focus, is somewhat lacking. Current literature suggests that specific features typical of lysins from phages infecting Gram-negative bacteria (G-) (higher net charge, amphipathic helices) are responsible for an improved interaction with G- envelope. Such antimicrobial peptide (AMP)-like elements are also of interest for antimicrobial molecules design. Thus, this study aims to provide an updated view on the primary structural landscape of phage lysins to clarify the evolutionary importance of several sequence-predicted properties, particularly for the interaction with the G- surface. A database of 2,182 lysin sequences was compiled, containing relevant information such as domain architectures, data on the phages host bacteria and sequence-predicted physicochemical properties. Based on such classifiers, an investigation on the differential appearance of certain features was conducted. Such analyses revealed different lysin architectural variants that are preferably found in phages infecting certain bacterial hosts. Particularly, some physicochemical properties (higher net charge, hydrophobicity, hydrophobic moment and aliphatic index) were associated to G- phage lysins, appearing specifically at their C-terminal end. Evidences on the remarkable genetic specialization of lysins regarding the features of the bacterial hosts have been provided, specifically supporting the nowadays common hypothesis that lysins from G- usually contain AMP-like regions.

IMPORTANCEPhage-encoded lytic enzymes, also called lysins, are one of the most promising alternatives to common antibiotics. The lysins potential as novel antimicrobials to tackle antibiotic-resistant bacteria not only arises from features such as a lower chance to provoke resistance, but also from their versatility as synthetic biology parts. Functional modules derived from lysins are currently being used for the design of novel antimicrobials with desired properties. This study provides a view of the lysins diversity landscape by examining a set of phage lysin genes. This way, we have uncovered the fundamental differences between the lysins from phages that infect bacteria with different superficial architectures, and, thus, also the reach of their specialization regarding cell wall structures. These results provide clarity and evidences to sustain some of the common hypothesis in current literature, as well as make available an updated and characterized database of lysins sequences for further developments.


title: Identification of mutations associated with Macozinone-resistant in Mycobacterium Tuberculosis

abstract:
Macozinone is identified as a drug candidate and is currently under clinical development for the treatment of tuberculosis, but the mutations conferring resistance to Macozinone remain inadequately characterized. Herein, we investigated the Macozinone -resistance -associated mutations through selecting resistant isolates in vitro. Macozinone-resistant isolates were obtained through induction in vitro. The level of Macozinone -resistant strains was confirmed by MABA test. PCR sequencing analysis was carried out on dprE1 gene. Whole Genome Sequencing was performed to identify mutations associated with Macozinone -resistance. The totals of isolates obtained at Macozinone concentrations of 6.4ng/ml, 25.6ng/ml, 50ng/ml and 100ng/ml were 49, 20, 20 and 4 respectively. Among the 49 strains obtained by 6.4ng/ml Macozinone only one strain had C387S mutation in dprE1. C387S is only occurred in high-level resistant isolates (MIC > 500ng/ml). Meanwhile high-level resistance to Macozinone can occur in strains induced at 6.4ng/ml and the frequency of occurrence is low (1/49, 2.04%). The MIC90 of other strains except the strains carrying C387S mutation is at the same level (11.5ng/ml > MIC90 > 2ng/ml). The G61A or G248A mutations in dprE1 was discovered for the first time. Other gene mutations (rv0678, rrs, mbtF, rv2956, et al) were found in low-level resistant strains.

ConclusionsHigh-level resistant isolates can be produced at low concentration of Macozinone. C387S mutation in dprE1 is directly related to high-level resistance. There may be new mechanisms involved in Macozinone-resistance independent of dprE1 mutations.

category: molecular biology


title: Agency rescues competition for credit assignment among predictive cues from adverse learning conditions

abstract: A fundamental assumption of learning theories is that the credit assigned to predictive cues is not simply determined by their probability of reinforcement, but by their ability to compete with other cues present during learning. This assumption has guided behavioral and neural science research for decades, and tremendous empirical and theoretical advances have been made identifying the mechanisms of cue competition. However, when learning conditions are not optimal (e.g., when training is massed), credit assignment is no longer competitive. This is a catastrophic failure of the learning system that exposes the individuals vulnerability to form spurious associations in the real world. Here, we uncover that cue competition can be rescued when conditions are suboptimal provided that the individual has agency over the learning experience. Our findings reveal a new connection between agency over learning and credit assignment to cues, and open new avenues of investigation into the underlying mechanisms.

category: animal behavior and cognition


title: Genome-wide survey of odorant-binding proteins in the dwarf honey bee Apis florea

abstract: Odorant binding proteins (OBPs) in insects bind to volatile chemical cue and help in their binding to odorant receptors. The odor coding hypothesis states that OBPs may bind with specificity to certain volatiles and aid the insect in various behaviours. Honeybees are eusocial insects with complex behaviour that requires olfactory inputs. Here, we have identified and annotated odorant binding proteins from the genome of the dwarf honey bee, Apis florea using an exhaustive homology-based bioinformatic pipeline and analyzed the evolutionary relationships between the OBP subfamilies. Our study suggests that Minus-C subfamily may have diverged from the Classic subfamily of odorant binding proteins in insects.


title: An integrated multi-wavelength SCATTIRSTORM microscope combining TIRFM and IRM modalities for imaging cellulases and other processive enzymes

abstract: We describe a multimodal SCATTIRSTORM microscope for visualizing processive enzymes moving on immobilized substrates. The instrument combines Interference Reflection Microscopy (IRM) with multi-wavelength Total Internal Reflectance Fluorescence Microscopy (TIRFM). The microscope can localize quantum dots with a precision of 2.8 nm at 100 frames/s, and was used to image the dynamics of the cellulase, Cel7a interacting surface-immobilized cellulose. The instrument, which was built with off-the-shelf components and controlled by custom software, is suitable for tracking other degradative enzymes such as collagenases, as well as motor proteins moving along immobilized tracks.

category: biophysics


title: Odor Blocking Of Stress

abstract: Scents have been employed for millennia to allay fear and stress, but whether they do so is poorly understood. In response to fear and stress, hypothalamic corticotropin releasing hormone neurons (CRHNs) induce increases in blood stress hormones. Here, we find that certain structurally and perceptually dissimilar odorants can block mouse stress hormone responses to three potent stressors: physical restraint, predator odor, and male-male social confrontation. Both odorants activate GABAergic inhibitory neurons presynaptic to CRHNs in the hypothalamic ventromedial nucleus (VMH). Stimulation of those neurons inhibits restraint-induced activation of CRHNs and stress hormone increase, mimicking a blocking odorant. Conversely, their silencing prevents odorant blocking of both responses. Notably, we also observed odor blocking of stressor activation in neurons presynaptic to CRHNs in the bed nucleus of the stria terminalis (BNST). Together, these findings indicate that selected odorants can indeed block stress responses, and that odor blocking can occur via two routes: a direct route in which blocking odor signals directly inhibit CRHNs and an indirect route in which they inhibit stressor activation of neurons presynaptic to CRHNs and prevent them from transmitting stress signals to CRHNs.


title: Brain Morphometry and Diminished Physical Growth in Bangladeshi Children Growing up in Extreme Poverty: a Longitudinal Study

abstract: Diminished physical growth is a common marker of malnutrition and it affects approximately 200 million children worldwide. Despite its importance and prevalence, it is not clear whether diminished growth affects brain development and neurocognitive outcomes. Further, diminished growth is more common in areas of extreme poverty, raising the possibility that it may serve as a mechanism for previously shown links between poverty and brain development. To address these questions, 79 children growing up in an extremely poor, urban area of Bangladesh underwent MRI at 6 years. Structural brain images were submitted to Mindboggle software, a Docker-compliant and highly reproducible tool for tissue segmentation and regional estimations of volume, surface area, cortical thickness, travel depth, and mean curvature. Diminished growth predicted brain morphometry and mediated the link between poverty and brain morphometry most consistently for white matter and subcortical volumes. Meanwhile, brain volume in left pallidum and right ventral diencephalon mediated the relationship between diminished growth and full-scale IQ. These findings offer malnutrition as one possible mechanism by which poverty affects brain development and neurocognitive outcomes in areas of extreme poverty.


title: Local Accumulation of Axonal Mitochondria in the Optic Nerve Glial Lamina Precedes Myelination

abstract: Mitochondria are essential for neurons and must be optimally distributed along their axon to fulfil local functions. A high density of mitochondria has been observed in retinal ganglion cell (RGC) axons of an unmyelinated region of the optic nerve, called the glial lamina (GL) in mouse (lamina cribrosa in human). In glaucoma, the worlds leading cause of irreversible blindness, the GL is the epicenter of RGC degeneration and is connected to mitochondrial dysfunction. It is generally accepted that the local accumulation of mitochondria in the GL is established due to the higher energy requirement of unmyelinated axons. Here we revisit the connection between mitochondrial positioning and myelin in RGC axons. We show that the high density of mitochondria in the GL is restricted to larger axons and is established before myelination. Thus, contrary to a longstanding belief in the field, the myelination pattern is not responsible for the establishment of the local accumulation of mitochondria in GL axons. Our findings open new research avenues likely critical to understanding the pathophysiology of glaucoma.


title: Natural selections on both amino acid sequences and expression levels are determinants of ohnolog retention

abstract: The mechanism of ohnolog retention is a subject of concern in evolutionary biology. Natural selections on coding sequences and gene dosages have been proposed to be determinants of ohnolog retention. However, the relationship between the two models is not widely accepted, and the role of regulatory sequences on ohnolog retention has long been neglected. In this study, based on a model of complex traits genetic architecture, we compared the natural selections strength on corresponding sequences between ohnologs and non-ohnologs by comparing complex traits heritability enrichments. We showed that complex traits regulatory sequences heritability enrichments (p = 1.1 x 10-5 in 5 kb flanking regions) and expression-mediated heritability enrichments (p = 2.1 x 10-5) of ohnologs were significantly higher than non-ohnologs. Then, we deduced that regulatory sequences of ohnologs were under substantial natural selection, which was also a determent of ohnolog retention. Meanwhile, we showed that in coding sequences, the complex traits heritability enrichments of ohnologs were significantly higher than of non-ohnologs (p = 9.9 x 10-5), supporting the ohnolog retention model of natural selection on coding sequences. We also showed that complex traits causal gene expression effect sizes of ohnologs were significantly larger than of non-ohnologs (p = 8.8 x 10-6), supporting the ohnolog retention model of natural selection on gene dosages. In conclusion, we provide the first unified framework to show that both amino acid sequences and expression levels of ohnologs are under substantial selection, which may end the long-standing debate on ohnolog retention models.


title: The Diameter Factor of Aligned Electrospinning Membranes Facilitates Cutaneous Wound Healing by Promoting Epithelialization

abstract:
Topographical properties (such as pattern and diameter) of biomaterials play important roles in influencing cell activities and manipulating the related immune response during the skin wound healing. We prepared aligned electrospinning membranes with different fiber diameters including 319{+/-}100 nm (A300), 588{+/-}132 nm (A600) and 1048{+/-}130 nm (A1000). The A300 membranes significantly improved cell proliferation and spreading, and facilitated skin wound healing (epithelization and vascularization) with the regeneration of hair follicles. The transcriptome revealed the underlying molecular mechanism that A300 could promote macrophages transformation to reparative phenotypes with highly expressed IL10 and TGF{beta}, subsequently promoting epidermal cell migration via secreting matrix metalloproteinases 12 (MMP12). All the results indicate the A300 to be a potential candidate for guided skin regeneration applications.

Graphic abstract

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category: bioengineering


title: Antiviral Resistance against Viral Mutation: Praxis and Policy for SARS CoV-2

abstract: AO_SCPLOWBSTRACTC_SCPLOWNew tools developed by Moderna, BioNTech/Pfizer, and Oxford/Astrazeneca, among others, provide universal solutions to previously problematic aspects of drug or vaccine delivery, uptake and toxicity, portending new tools across the medical sciences. A novel method is presented based on estimating protein backbone free energy via geometry to predict effective antiviral targets, antigens and vaccine cargos that are resistant to viral mutation. This method, partly described in earlier work of the author, is reviewed and reformulated here in light of the recent proliferation of structural data on the SARS-CoV-2 spike glycoprotein and its latest mutations in the variants of concern and several further variants of interest including all international lineages. Particular attention to structures computed with Cryo Electron Microscopy allows the novel approach of probing the pH dependence of free energy in order to infer function. Key findings include: collections of recurring mutagenic residues occur across strains, presumably through cooperative convergent evolution; the preponderance of mutagenic residues do not participate in backbone hydrogen bonds; metastability of the spike glycoprotein limits the change of free energy from before to after mutation and thereby constrains selective pressure; and there are mRNA or virus-vector cargos which target low free energy peptides proximal to conserved high free energy peptides providing specific recipes for vaccines with greater specificity than the current full-spike approach. These results serve to limit peptides in the spike glycoprotein with high mutagenic potential and thereby provide a priori constraints on viral and attendant vaccine evolution. Scientific and regulatory challenges to nucleic acid therapeutic and vaccine development and deployment are finally discussed.


title: Transposable elements as sensors of SARS-CoV-2 infection

abstract:
Transposable element (TE) transcription is induced in response to viral infections. TE induction triggers a robust and durable interferon (IFN) response, providing a host defense mechanism. Still, the connection between SARS-CoV-2 IFN response and TEs remains largely unknown. Here, we analyzed TE expression changes in response to SARS-CoV-2 infection in different human cellular models. We find that compared to other viruses, which cause global upregulation of TEs, SARS-CoV-2 infection results in a significantly milder TE response in both primary lung epithelial cells and in iPSC-derived lung alveolar type 2 cells. TE activation precedes, and correlates with, the induction of IFN-related genes, suggesting that the limited activation of TEs following SARS-CoV-2 infection may be the reason for the weak IFN response. Diminished TE activation was not observed in lung cancer cell lines with very high viral load. Moreover, we identify two variables which explain most of the observed diverseness in immune responses: basal expression levels of TEs in the pre-infected cells, and the viral load. Finally, analyzing the SARS-CoV-2 interactome, as well as the epigenetic landscape around the TEs that are activated following infection, we identify SARS-CoV-2 interacting proteins, which may regulate chromatin structure and TE transcription in response to a high viral load. This work provides a functional explanation for SARS-CoV-2s success in its fight against the host immune system, and suggests that TEs could be used as sensors and serve as potential drug targets for COVID-19.

Key points O_LIUnlike other viruses, SARS-CoV-2 invokes a weak and inefficient transposable element (TE) response
C_LIO_LITE induction precedes and predicts IFN response
C_LIO_LIBasal TE expression and viral load explain immune responses
C_LIO_LIDistinct chromatin and enhancer binding factors occupancy on TEs induced by SARS-CoV-2
C_LI


title: Impaired activation of Transposable Elements in SARS-CoV-2 infection


title: Vascular and renal telomere shortening in mice exposed to chronic intermittent hypoxia

abstract: Obstructive sleep apnea (OSA) is a chronic condition characterized by chronic intermittent hypoxia (IH) and is associated with cardiovascular (CVD) and chronic kidney diseases (CKD). There is increased biomarkers of aging, such as telomere shortening, in patients with OSA. We assessed telomere lengths in aortic and renal tissues from mice exposed to 8 weeks of IH using a PCR protocol, and demonstrate significant telomere shortening in both tissues. This data indicates that IH, a hallmark of OSA, can accelerate vascular and renal aging that may contribute to OSA-induced CVD and CKD

category: physiology


title: Regulation of plasmodesmata at specific cell-cell interfaces

abstract: Precise exchange of information and resources among cells is essential for multicellular organisms. Intercellular communication among diverse cell types requires differential mechanisms to achieve the specific regulation. Despite the significance of intercellular communication, it is largely unknown how the communication between different cells is regulated. Here, we report that two members of plasmodesmata-located proteins modulate plasmodesmata at two distinct cell-cell interfaces.


title: Defining kinetic roles of transcriptional activators in the early Drosophila embryo

abstract: Most animal transcription factors are categorized as activators or repressors without specifying their mechanisms of action. Defining their specific roles is critical for deciphering the logic of transcriptional regulation and predicting the function of regulatory sequences. Here, we define the kinetic roles of three activating transcription factors in the Drosophila embryo--Zelda, Bicoid and Stat92E--by introducing their binding sites into the even skipped stripe 2 enhancer and measuring transcriptional output with live imaging. We find that these transcription factors act on different subsets of kinetic parameters, and these subsets can change over the course of nuclear cycle (NC) 14. These transcription factors all increase the fraction of active nuclei. Zelda dramatically shortens the time interval between the start of NC 14 and initial activation, and Stat92E increases the duration of active transcription intervals throughout NC 14. Zelda also decreases the time intervals between instances of active transcription early in NC 14, while Stat92E does so later. Different transcription factors therefore play distinct kinetic roles in activating transcription; this has consequences for understanding both regulatory DNA sequences as well as the biochemical function of transcription factors.


title: Tissue-resident, extravascular CD64-Ly6C- population forms a critical barrier for inflammation in the synovium

abstract: Monocytes are one of the most abundant immune cells infiltrating the inflamed organs. However, the majority of studies on monocytes focus on circulating cells, rather than those in the tissue. Here, we identify and characterize an intravascular (i.v.) and extravascular (e.v.) synovial population (Syn Ly6C- cells) which lack cell surface markers of classical monocytes (Ly6C and CD62) or tissue macrophages (CD64 and Tim4), are transcriptionally distinct and conserved in RA patients. e.v. Syn Ly6C- cells are independent of NR4A1 and CCR2, long-lived and embryonically derived while the i.v. Syn Ly6C- cells are dependent on NR4A1, short lived and derived from circulating NCM. e.v. Syn Ly6C- cells undergo increased proliferation and reverse diapedesis dependent on LFA1 in response to arthrogenic stimuli and are required for the development of RA-like disease. These findings uncover a new facet of mononuclear cell biology and are imperative to understanding tissue-resident myeloid cell function in RA.

category: immunology


title: Aerobic growth physiology of Saccharomyces cerevisiae on sucrose is strain-dependent

abstract:
Present knowledge on the quantitative aerobic physiology of the yeast Saccharomyces cerevisiae during growth on sucrose as sole carbon and energy source is limited to either adapted cells or to the model laboratory strain CEN.PK113-7D. To broaden our understanding of this matter and open novel opportunities for sucrose-based biotechnological processes, we characterized three strains, with distinct backgrounds, during aerobic batch bioreactor cultivations. Our results reveal that sucrose metabolism in S. cerevisiae is a strain-specific trait. Each strain displayed a distinct extracellular hexose concentration and invertase activity profiles. Especially, the inferior maximum specific growth rate (0.21 h-1) of the CEN.PK113-7D strain, with respect to that of strains UFMG-CM-Y259 (0.37 h-1) and JP1 (0.32 h-1), could be associated to its low invertase activity (0.04 to 0.09 U mgDM-1). Moreover, comparative experiments with glucose or fructose alone, or in combination, suggest mixed mechanisms of sucrose utilization by the industrial strain JP1, and points out the remarkable ability of the wild isolate UFMG-CM-259 to grow faster on sucrose than on glucose in a well-controlled cultivation system. This work hints to a series of metabolic traits that can be exploited to increase sucrose catabolic rates and bioprocess efficiency.

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title: Proanthocyanidin-enriched cranberry extract induces resilient bacterial community dynamics in a gnotobiotic mouse model

abstract: Cranberry consumption has numerous health benefits, with experimental reports showing its anti-inflammatory and anti-tumor properties. Importantly, microbiome research has demonstrated that the gastrointestinal bacterial community modulates host immunity, raising the question whether the cranberry-derived effect may be related to its ability to modulate the microbiome. Only a few studies have investigated the effect of cranberry products on the microbiome to date. Especially because cranberry is rich in dietary fibers, we do not know the extent of microbiome modulation that is caused solely by polyphenols, particularly proanthocyanidins (PACs). Since previous work has only focused on the long-term effects of cranberry extracts, in this study we investigated the effect of a water-soluble, polyphenol-rich cranberry juice extract (CJE) on the short-term dynamics of human-derived bacterial community in a gnotobiotic mouse model. CJE characterization revealed a high enrichment in PACs (57% PACs), the highest ever utilized in a microbiome study. In a 37-day experiment with a 10-day CJE intervention and 14-day recovery time, we profiled the microbiota via 16 rDNA sequencing and applied diverse time-series analytics methods to identify individual bacterial responses. We show that daily administration of CJE induces distinct dynamical patterns in bacterial abundances during and after treatment before recovering resiliently to pre-treatment levels. Specifically, we observed an increase of the immunomodulatory mucin degrading Akkermansia muciniphila after treatment, suggesting intestinal mucus accumulation due to CJE. Interestingly, this expansion coincided with an increase in the abundance of butyrate-producing Clostridia, a group of microbes known to promote numerous adaptive and innate anti-inflammatory phenotypes.


title: Metagenomic sequencing reveals a lack of virus exchange between native and invasive freshwater fish across the Murray-Darling Basin, Australia

abstract:
Biological invasions are among the biggest threats to freshwater biodiversity. This is increasingly relevant in the Murray-Darling Basin, Australia, particularly since the introduction of the common carp (Cyprinus carpio). This invasive species now occupies up to 90% of fish biomass, with hugely detrimental impacts on native fauna and flora. To address the ongoing impacts of carp, cyprinid herpesvirus 3 (CyHV-3) has been proposed as a potentially effective biological control. Crucially, however, it is unknown whether CyHV-3 and other cyprinid herpesviruses already exist in the Murray-Darling. Further, little is known about those viruses that naturally occur in wild freshwater fauna, and the frequency with which these viruses jump species boundaries. To document the evolution and diversity of freshwater fish viromes and better understand the ecological context to the proposed introduction of CyHV-3, we performed a meta-transcriptomic viral survey of invasive and native fish across the Murray-Darling Basin, covering over 2,200 km of the river system. Across a total of 36 RNA libraries representing 10 species, we failed to detect CyHV-3 nor any closely related viruses. Rather, meta-transcriptomic analysis identified 18 vertebrate-associated viruses that could be assigned to the Arenaviridae, Astroviridae, Bornaviridae, Caliciviridae, Coronaviridae, Chuviridae, Flaviviridae, Hantaviridae, Hepeviridae, Paramyxoviridae, Picornaviridae, Poxviridae, Reoviridae and Rhabdoviridae families, and a further 27 that were deemed to be associated with non-vertebrate hosts. Notably, we revealed a marked lack of viruses that are shared among invasive and native fishes sampled here, suggesting that there is little virus transmission from common carp to native fish species. Overall, this study provides the first data on the viruses naturally circulating in a major river system and supports the notion that fish harbour a large diversity of viruses with often deep evolutionary histories.

Author SummaryThe ongoing invasion of the common carp in the Murray-Darling Basin, Australia, has wreaked havoc on native freshwater ecosystems. This has stimulated research into the possible biological control of invasive carp through the deliberate release of the virus cyprinid herpesvirus 3 (CyHV-3). However, little is known on the diversity of viruses that naturally circulate in wild freshwater fauna, whether these viruses are transmitted between invasive and native species, nor if CyHV-3 or other cyprinid herpesviruses are already present in the basin. To address these fundamental questions we employed meta-transcriptomic next-generation sequencing to characterise the total assemblage of viruses (i.e. the viromes) in three invasive and seven native fish species cohabiting at 10 sites across 2,200 km of the river system. From this analysis we identified 18 vertebrate-associated viruses across 14 viral families, yet a marked lack of virus transmission between invasive and native species. Importantly, no CyHV-3 was detected. This study shows that freshwater fish harbour a high diversity and abundance of viruses, that viruses have likely been associated with fish for millennia, and that there is likely little direct virus transmission between introduced and native species.


title: Comparison of the pathogenicity and virus shedding of SARS CoV-2 VOC 202012/01 and D614G variant in hamster model

abstract:
The emergence of SARS-CoV-2 variants has posed a serious challenge to public health system and vaccination programs across the globe. We have studied the pathogenicity and virus shedding pattern of the SARS-CoV-2 VOC 202012/01 and compared with D614G variant in Syrian hamsters. VOC 202012/01 could produce disease in hamsters characterized by body weight loss and respiratory tract tropism but mild lung pathology. Further, we also documented that neutralizing antibodies developed against VOC 202012/01 could equally neutralize D614G variant. Higher load of VOC 202012/01 in the nasal wash specimens was observed during the first week of infection outcompeting the D614G variant. The findings suggest increased fitness of VOC 202012/01 to the upper respiratory tract which could lead to higher transmission. Further investigations are needed to understand the transmissibility of new variants.

One-Sentence SummarySARS-CoV-2 VOC 202012/01 infected hamsters demonstrated high viral RNA shedding through the nasal secretions and significant body weight loss with mild lung pathology compared to the D614G variant.


title: Structural and molecular characterization of the Rpv3 locus towards the development of KASP markers for downy mildew resistance in grapevine (Vitis spp.)

abstract: Plasmopara viticola is the oomycete that causes downy mildew in grapevine. Varying levels of resistance to P. viticola across grape cultivars allowed quantitative trait loci to be identified. The Rpv3 locus is located at chromosome 18, in a region enriched in TIR-NBS-LRR genes, and the phenotype associated is a high hypersensitive response. In this work, we aimed to identify candidate genes associated with resistance to downy mildew on the Rpv3 locus and to evaluate their transcriptional profiles in a susceptible and a resistant grapevine cultivar after challenging with P. viticola. Candidate genes were identified by representational differential analysis and also by functional enrichment tests. Many predicted genes associated with resistance to diseases were found at the Rpv3 locus. In total, seventeen genes were evaluated by RT-qPCR. Differences in the steady-state expression of these genes were observed between the two cultivars. Four genes were found to be expressed only in Villard Blanc, suggesting their association to the hypersensitivity reaction. Concerning marker assisted-selection for downy mildew resistance, we show the efficient use of a haplotype of SSR markers. Furthermore, based on Rpv3-located SNPs between grapevine cultivars contrasting in downy mildew resistance, we developed and tested forty-one new markers for assisted selection. After genotypic and phenotypic evaluations on segregant populations, two markers, Rpv3_15 and Rpv3_33, were considered efficient for downy mildew resistance identification. This study constitutes an in-depth genomic characterization of the Rpv3 locus, confirms its involvement in resistance against P. viticola infection and presents promising biotechnological tools for the selection of young resistant individuals.


title: Endoribonuclease activity of XRN-2 is critical for RNA metabolism and survival of Caenorhabditis elegans

abstract: microRNAs (miRNAs) are known to regulate a vast majority of the eukaryotic genes by post-transcriptional means, and multiple nucleases play critical roles in the biogenesis and turnover of these regulators. A number of studies have indicated that turnover is important for determining the abundance of miRNAs, and thus, in turn govern their functionality. Recent research in Caenorhabditis elegans has revealed an ATP-independent endoribonuclease activity of the  miRNase-XRN-2. Here, we report the characterization of this new enzymatic activity of the fundamentally important XRN-2, and show that it is critical for miRNA turnover and survival of quiescent dauer worms. The dual enzymatic activity of XRN-2 capacitates the mechanism of miRNA turnover to be dynamic, which might confer adaptive advantage to the organism. In continuously growing worms, this new enzymatic activity does not act on miRNAs, but it is important for the generation of mature ribosomal RNAs, which in turn is critical for translation, and thus indispensable for the survival of worms.


title: High-resolution, genome-wide mapping of positive supercoiling in chromosomes

abstract: Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently-oriented genes, consistent with the "twin-domain" model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.


title: Versatile Cell-Based Assay for Measuring Base Excision Repair of DNA Alkylation Damage

abstract: DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair.


title: Versatile Cell-Based Assay for Measuring DNA Alkylation Damage and its repair


title: The CCL2 Chemokine Promotes Early Seeding of the Latent HIV Reservoir

abstract:
HIV infects long-lived CD4 memory T cells establishing a latent viral reservoir that necessitates lifelong anti-retroviral therapy (ART). How this reservoir is formed so swiftly remains unknown. We now show the innate inflammatory response to HIV infection results in CCL2 chemokine release, which can drive recruitment of cells expressing the CCR2 receptor including a subset of central memory CD4 T cells. Supporting a role for the CCL2/CCR2 axis in rapid reservoir formation, we find 1) treatment of humanized mice with anti-CCL2 antibodies during HIV infection decreases reservoir seeding and 2) CCR2/5+ cells from the blood of HIV-infected individuals on long term ART contain significantly more provirus than CCR2/5-negative memory or naive cells. Together, these studies support a model where the hosts innate inflammatory CCL2 response to HIV infection recruits CCR2/5+ central memory CD4 T cells to zones of virus-associated inflammation likely contributing to rapid formation of the latent HIV reservoir.

GRAPHICAL ABSTRACTWhy is the latent HIV reservoir established so early following infection? An innate immune response occurs during acute infection that establishes a "zone of inflammation" (step 1). The CCL2 chemokine is produced in part through IFI16 sensing of HIV DNA in abortively infected cells. CCL2 promotes rapid recruitment of CCR2/5+ memory CD4 T cells (step 2). Many of these cells become productively infected (step 3) and a fraction become latently infected (step 4). Thus, HIV hijacks the host inflammatory response to rapidly establish the latent reservoir. In support of this model, we find HIV reservoir reduction in humanized mice treated with anti-CCL2 antibodies during early infection. Further, we find that CCR2/5+ CD4 T cells harbor a substantial fraction of detectable proviruses in the blood of HIV-infected individuals on long-term suppressive ART.

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category: cell biology


title: Apolar mode of gastrulation leads to the formation of polarized larva in a marine hydroid, Dynamena pumila

abstract:
BackgroundIn almost all metazoans examined to this respect, the axial patterning system based on canonical Wnt (cWnt) signaling operates throughout the course of development. In most metazoans, gastrulation is polar, and embryos develop morphological landmarks of axial polarity, such as blastopore under control/regulation from Wnt signaling. However, in many cnidarian species, gastrulation is morphologically apolar. The question remains whether [c]Wnt signaling providing the establishment of a body axis controls morphogenetic processes involved in apolar gastrulation.

ResultsIn this study, we focused on the embryonic development of Dynamena pumila, a cnidarian species with apolar gastrulation. We thoroughly described cell behavior, proliferation, and ultrastructure and examined axial patterning in the embryos of this species. We revealed that the first signs of morphological polarity appear only after the end of gastrulation, while molecular prepatterning of the embryo does exist during gastrulation. We have shown experimentally that in D. pumila, the morphological axis is highly robust against perturbations in cWnt activity.

ConclusionOur results suggest that morphogenetic processes are uncoupled from molecular axial patterning during gastrulation in D. pumila. Investigation of D. pumila might significantly expand our understanding of the ways in which morphological polarization and axial molecular patterning are linked in Metazoa.


title: Wolbachia in the spittlebug Prosapia ignipectus: Variable infection frequencies, but no apparent effect on host reproductive isolation

abstract: Animals serve as hosts for complex communities of microorganisms, including endosymbionts that live inside their cells. Wolbachia bacteria are perhaps the most common endosymbionts, manipulating host reproduction to propagate. Many Wolbachia cause intense cytoplasmic incompatibility (CI) that promotes their spread to high and relatively stable frequencies. Wolbachia that cause weak or no CI tend to persist at intermediate, often variable, frequencies. Wolbachia could also contribute to host reproductive isolation (RI), although current support for such contributions is limited to a few systems. To test for Wolbachia frequency variation and effects on host RI, we sampled several local Prosapia ignipectus (Fitch)(Hemiptera: Cercopidae) spittlebug populations in the northeastern USA over two years, including closely juxtaposed Maine populations with different monomorphic color forms, "black" and "lined". We discovered a group-B Wolbachia (wPig) infecting P. ignipectus that diverged from group-A Wolbachia--like model wMel and wRi strains in Drosophila--6 to 46 MYA. Populations of the sister species Prosapia bicincta (Say) from Hawaii and Florida are uninfected, suggesting that P. ignipectus acquired wPig after their initial divergence. wPig frequencies were generally high and variable among sites and between years. While phenotyping wPig effects on host reproduction is not currently feasible, the wPig genome contains three divergent sets of CI loci, consistent with high wPig frequencies. Finally, Maine monomorphic black and monomorphic lined populations of P. ignipectus share both wPig and mtDNA haplotypes, implying no apparent effect of wPig on the maintenance of this morphological contact zone. We hypothesize P. ignipectus acquired wPig horizontally as observed for many Drosophila species, and that significant CI and variable transmission produce high but variable wPig frequencies.

category: evolutionary biology


title: Low-input ATAC&mRNA-Seq: a simple and robust method for simultaneous dual-omics profiling with low cell number

abstract: Deciphering epigenetic regulation of gene expression requires measuring the epigenome and transcriptome jointly. Single-cell multi-omics technologies have been developed for concurrent profiling of chromatin accessibility and gene expression. However, multi-omics profiling of low-input bulk samples remains challenging. Therefore, we developed low-input ATAC&mRNA-seq, a simple and robust method for studying the role of chromatin structure in gene regulation in a single experiment with thousands of cells, to maximize insights from limited input material by obtaining ATAC-seq and mRNA-seq data simultaneously from the same cells with data quality comparable to conventional mono-omics assays. Integrative data analysis revealed similar strong association between promoter accessibility and gene expression using the data of low-input ATAC&mRNA-seq as using single-assay data, underscoring the accuracy and reliability of our dual-omics assay to generate both data types simultaneously with just thousands of cells. We envision our method to be widely applied in many biological disciplines with limited materials.

category: genomics


title: A single-cell and spatial atlas of autopsy tissues reveals pathology and cellular targets of SARS-CoV-2

abstract: The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.


title: Decoupling the bridge helix of Cas12a results in a reduced trimming activity and impaired conformational transitions

abstract: The widespread and versatile prokaryotic CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats and associated Cas proteins) constitute powerful weapons against foreign nucleic acids. Recently, the single-effector nuclease Cas12a that belongs to the type V CRISPR-Cas system was added to the Cas enzymes repertoire employed for gene editing purposes. Cas12a is a bilobal enzyme composed of the REC and Nuc lobe connected by a central structural element, the so-called bridge helix (BH). We generated BH mutants and integrated biochemical and single-molecule FRET (smFRET) studies to elucidate the role of the BH for the enzymatic activity and conformational flexibility of Francisella novicida Cas12a. We demonstrate that the BH impacts the trimming activity of Cas12a resulting in Cas12a variants with improved cleavage accuracy. Single-molecule FRET measurements reveal the hitherto unknown open and closed state of apo Cas12a. BH mutants preferentially adopt the open state. Transition to the closed state of the Cas12a-crRNA complex is inefficient in BH mutants but the semi-closed state of the ternary complex can be adopted even if the BH is deleted in its entirety. Taken together, these insights reveal that the BH is a structural element that influences the catalytic activity and impacts conformational transitions of FnCas12a.


title: Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA

abstract: The complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. In Escherichia coli, replication termination, the final stage of the duplication process, is confined to the  replication fork trap region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomic ter sites. Termination typically occurs away from Tus-ter complexes, but they become part of the fork fusion process when a delay to one replisome allows the second to travel more than halfway around the chromosome. In this instance, replisome progression is blocked at the non-permissive interface of Tus-ter and termination occurs when a converging replisome meets the non-permissive interface. To investigate the consequences of replication fork fusion at Tus-ter complexes, we established a plasmid-based replication system where we could mimic the termination process at Tus-ter in vitro. We developed a termination mapping assay to measure leading strand replication fork progression and demonstrate that the DNA template is under-replicated by 15-24 bases when replication forks fuse at Tus-ter complexes. This gap could not be closed by the inclusion of lagging strand processing enzymes as well as several helicases that promote DNA replication. Our results indicate that accurate fork fusion at Tus-ter barriers requires further enzymatic processing, highlighting large gaps that still exist in our understanding of the final stages of chromosome duplication and the evolutionary advantage of having a replication fork trap.


title: Polyvalent Guide RNAs for CRISPR Antivirals

abstract: CRISPR biotechnologies, where CRISPR effectors recognize and degrade specific nucleic acid targets that are complementary to their guide RNA (gRNA) cofactors, have been primarily used as a tool for precision gene editing1 but possess an emerging potential for novel antiviral diagnostics, prophylactics, and therapeutics.2-5 In gene editing applications, significant efforts are made to limit the natural tolerance of CRISPR effectors for nucleic acids with imperfect complementarity to their gRNAs in order to prevent degradation and mutation at unintended or "off-target" sites; here we exploit those tolerances to engineer gRNAs that are optimized to promote activity at multiple viral target sites, simultaneously, given that multiplexed targeting is a critical tactic for improving viral detection sensitivity,3 expanding recognition of clinical strain variants,6 and suppressing viral mutagenic escape from CRISPR antivirals.7 We demonstrate in vitro and in higher plants that single "polyvalent" gRNAs (pgRNAs) in complex with CRISPR effectors Cas9 or Cas13 can effectively degrade pairs of viral targets with significant sequence divergence (up to 40% nucleotide differences) that are prevalent in viral genomes. We find that CRISPR antivirals using pgRNAs can robustly suppress the propagation of plant RNA viruses, in vivo, better than those with a "monovalent" gRNA counterpart. These results represent a powerful new approach to gRNA design for antiviral applications that can be readily incorporated into current viral detection and therapeutic strategies, and highlight the need for specific approaches and tools that can address the differential requirements of precision gene editing vs. CRISPR antiviral applications in order to mature these promising biotechnologies.


title: Chronos: a CRISPR cell population dynamics model

abstract: CRISPR loss of function screens are a powerful tool to interrogate cancer biology but are known to exhibit a number of biases and artifacts that can confound the results, such as DNA cutting toxicity, incomplete phenotype penetrance and screen quality bias. Computational methods that more faithfully model the CRISPR biological experiment could more effectively extract the biology of interest than typical current methods. Here we introduce Chronos, an algorithm for inferring gene knockout fitness effects based on an explicit model of the dynamics of cell proliferation after CRISPR gene knockout. Chronos is able to exploit longitudinal CRISPR data for improved inference. Additionally, it accounts for multiple sources of bias and can effectively share information across screens when jointly analyzing large datasets such as Project Achilles and Score. We show that Chronos outperforms competing methods across a range of performance metrics in multiple types of experiments.

category: bioinformatics


title: ARYL HYDROCARBON RECEPTOR BLOCKS AGING-INDUCED SENESCENCE IN THE LIVER AND FIBROBLAST CELLS

abstract: Aging induces progressive organ degeneration and worsening of tissue homeostasis leading to multiple pathologies. Yet, little is known about the mechanisms and molecular intermediates involved. Here, we report that aged aryl hydrocarbon receptor-null mice (AhR-/-) had exacerbated senescence and larger numbers of liver progenitor cells. Senescence-associated markers {beta}-galactosidase (SA-{beta}-Gal), p16Ink4a and p21Cip1 and genes of the senescence-associated secretory phenotype (SASP) TNF and IL1 were overexpressed in aged AhR-/- livers. AhR binding to the promoter of those genes, as shown by chromatin immunoprecipitation, likely had a repressive effect maintaining their physiological levels in AhR+/+ livers. Furthermore, factors secreted by senescent cells MCP-2, MMP12 and FGF were also produced at higher levels in aged AhR-null livers. Supporting the linkage between senescence and stemness, liver progenitor cells were more abundant in AhR-/- mice, which could probably contribute to their increased hepatocarcinoma burden. These roles of AhR are not liver-specific since adult and embryonic AhR-null fibroblasts acquired cellular senescence upon culturing with overexpression of SA-{beta}-Gal, p16Ink4a and p21Cip1. Notably, depletion of senescent cells with the senolytic agent navitoclax restored basal expression of senescent markers in AhR-/- fibroblasts. Oppositely, senescence promoter palbociclib induced an AhR-null like phenotype in AhR+/+ fibroblasts. Moreover, doxycycline-induced senescence reduced AhR levels while depletion of p16Ink4a-expressing senescent cells restored basal AhR levels in mouse lungs. Thus, AhR is needed to restrict age-induced senescence, and such activity seems to correlate with a more differentiated phenotype and with increased resistance to liver tumorigenesis.

category: cancer biology


title: The chemotherapeutic CX-5461 primarily targets TOP2B and exhibits selective activity in high-risk neuroblastoma.

abstract: Survival in high-risk pediatric neuroblastoma has remained around 50% for the last 20 years, with immunotherapies and targeted therapies having had minimal impact. Here, we identify the small molecule CX-5461 as selectively cytotoxic to high-risk neuroblastoma and synergistic with low picomolar concentrations of topoisomerase I inhibitors improving survival in vivo in orthotopic patient-derived xenograft neuroblastoma mouse models. CX-5461 recently progressed through phase I clinical trial as a first-in-human inhibitor of RNA-POL I. However, we also use a comprehensive panel of in vitro and in vivo assays to demonstrate that CX-5461 has been mischaracterized and that its primary target at pharmacologically relevant concentrations, is in fact topoisomerase II beta (TOP2B), not RNA-POL I. These findings are important because existing clinically approved chemotherapeutics have well-documented off-target interactions with TOP2B, which have previously been shown to cause both therapy-induced leukemia and cardiotoxicity--often-fatal adverse events, which can emerge several years after treatment. Thus, while we show that combination therapies involving CX-5461 have promising anti-tumor activity in vivo in neuroblastoma, our identification of TOP2B as the primary target of CX-5461 indicates unexpected safety concerns that should be examined in ongoing phase II clinical trials in adult patients before pursuing clinical studies in children.


title: Systems biology analysis identifies TCF7L1 as a key regulator of metastasis in Ewing sarcoma

abstract:
Identification of cancer stemness genes is crucial to understanding the underlying biology of therapy resistance, relapse, and metastasis. Ewing sarcoma (EwS) is the second most common bone tumor in children and adolescents. It is a highly aggressive cancer associated with a dismal survival rate (<30%) for patients with metastatic disease at diagnosis ([~]25% of cases). Hence, deciphering the underlying mechanisms of metastasis is imperative. EwS tumors are characterized by a remarkably  silent genome with a single driver mutation generating an oncogenic fusion transcription factor (EWSR1-ETS). Thus, EwS constitutes an ideal model to study how perturbation of a transcriptional network by a dominant oncogene can mediate metastasis, even though canonical metastasis-associated genes are not mutated.

Here, through the implementation of an integrative systems biology approach, we identified transcription factor 7 like 1 (TCF7L1, alias TCF3) as a prognostically-relevant and EWSR1-ETS suppressed determinant of metastasis in EwS. We demonstrated that conditional TCF7L1 re-expression significantly reduces EwS single-cell migration, invasion and anchorage-independent growth in 3D assays in vitro, and tumorigenesis in vivo mediated by its DNA binding domain. In primary EwS tumors as well as in functional orthotopic in vivo models, low TCF7L1 expression was associated with pro-metastatic gene signatures and a much higher migratory and metastatic capacity of EwS cells, which correlated with poor outcome of EwS patients.

Collectively, our findings establish TCF7L1 as a major regulator of metastasis in EwS, which may be utilized as a prognostic biomarker and open inroads to future therapeutic intervention.


title: MtrA regulation of essential peptidoglycan cleavage in Mycobacterium tuberculosis  during infection

abstract: The success of Mycobacterium tuberculosis (Mtb) is largely due to its ability to withstand multiple stresses encountered in the host. Here, we present a data-driven model that captures the dynamic interplay of environmental cues and genome-encoded regulatory programs in Mtb. The model captures the genome-wide distribution of cis-acting gene regulatory elements and the conditional influences of transcription factors at those elements to elicit environment-specific responses. Analysis of transcriptional responses that may be essential for Mtb to survive acidic stress within the maturing macrophage, identified regulatory control by the MtrAB two-component signal system. Using genome-wide transcriptomics as well as imaging studies, we have characterized the MtrAB circuit by tunable CRISPRi knockdown in both Mtb and the non-pathogenic organism, M. smegmatis (Msm). These experiments validated the essentiality of MtrA in Mtb, but not Msm. We identified that MtrA regulates multiple enzymes that cleave cell wall peptidoglycan and is required for efficient cell division. Moreover, our results suggest that peptidoglycan cleavage, regulated by MtrA, is necessary for Mtb to survive intracellular stress. Further, we present MtrA as an attractive drug target, as even weak repression of mtrA results in loss of Mtb viability and completely clears the bacteria with low-dose isoniazid or rifampicin treatment.

category: systems biology