title: Distributions of extinction times from fossil ages and tree topologies: the example of some mid-Permian synapsid extinctions

abstract:
Given a phylogenetic tree that includes only extinct, or a mix of extinct and extant taxa, where at least some fossil data are available, we present a method to compute the distribution of the extinction time of a given set of taxa under the Fossilized-Birth-Death model.

Our approach differs from the previous ones in that it takes into account (i) the possibility that the taxa or the clade considered may diversify before going extinct and (ii) the whole phylogenetic tree to estimate extinction times, whilst previous methods do not consider the diversification process and deal with each branch independently. Because of this, our method can estimate extinction times of lineages represented by a single fossil, provided that they belong to a clade that includes other fossil occurrences.

We assess and compare our new approach with a standard previous one using simulated data. Results show that our method provides more accurate confidence intervals.

This new approach is applied to the study of the extinction time of three Permo-Carboniferous synapsid taxa (Ophiacodontidae, Edaphosauridae, and Sphenacodontidae) that are thought to have disappeared toward the end of the Cisuralian (early Permian), or possibly shortly thereafter. The timing of extinctions of these three taxa and of their component lineages supports the idea that the biological crisis in the late Kungurian/early Roadian consisted of a progressive decline in biodiversity throughout the Kungurian.


title: Distributions of extinction times from fossil ages and tree topologies: the example of mid-Permian synapsid extinctions

category: evolutionary biology


title: Mitogenomics and mitochondrial gene phylogeny decipher the evolution of Saccharomycotina yeasts

abstract: Saccharomycotina yeasts contain diverse clades within the kingdom of Fungi and are important to human everyday life. This work investigates the evolutionary relationships among these yeasts from a mitochondrial (mt) genomic perspective. A comparative study of 141 yeast mt genomes representing all major phylogenetic lineages of Saccharomycotina was performed, including genome size and content variability, intron and intergenic regions diversity, genetic code alterations and syntenic variation. Findings from this study suggest that mt genome size diversity is the result of a ceaseless random process mainly based on genetic recombination and intron mobility. Gene order analysis revealed conserved syntenic units and many occurring rearrangements, which can be correlated with major evolutionary events as shown by the phylogenetic analysis of the concatenated mt protein matrix. For the first time, molecular dating indicated a slower mt genome divergence rate in the early stages of yeast evolution, in contrast with a faster rate in the late evolutionary stages, compared to their nuclear time divergence. Genetic code reassignments of mt genomes are a perpetual process happening in many different parallel evolutionary steps throughout Saccharomycotina evolution. Overall, this work shows that phylogenetic studies that employ the mt genome of yeasts highlight major evolutionary events.


title: Vertebrate whole genome duplications shaped the current 3D genome architecture

abstract: Topologically associated domains (TADs) are interaction sub-networks of 3D genomes. TAD boundaries frequently coincide with genome breaks while their deletion is under negative selection, suggesting that TADs act as modules facilitating genome rearrangements and metazoan evolution. However, the role of TADs in the evolution of gene regulation and essentiality is not well understood. Here, we show that TADs play a role organising ancestral functions and evolutionary novelty. We discovered that genes co-localise by evolutionary age in the human and mouse genomes, resulting in TADs that have different proportions of younger and older genes. A major transition in the TAD age co-localisation patterns is observed between the genes born as a result of the vertebrate whole genome duplications (WGDs) or before, and those born afterwards. We also found that primate- and rodentspecific genes are more frequently essential when they are located in  aged TADs and connected to genes that have not duplicated since the WGD. Our data suggests that evolutionary success of recent genes may increase when located in functionally relevant TADs with established regulatory networks.

category: genomics


title: Endogenous retroviruses co-opted into divergently transcribed regulatory elements shape the regulatory landscape of embryonic stem cells

abstract: Transposable elements are an abundant source of transcription factor binding sites and favorable genomic integration may lead to their recruitment by the host genome for gene regulatory functions. However, it is unclear how frequent co-option of transposable elements as regulatory elements is, to which regulatory programs they contribute and how they compare to regulatory elements devoid of transposable elements. Here, we report a transcription initiation-centric, in-depth characterization of the transposon-derived regulatory landscape of mouse embryonic stem cells. We demonstrate that a substantial number of transposable elements, in particular endogenous retroviral elements, carry open chromatin regions that are divergently transcribed into unstable RNAs in a cell-type specific manner, and that these elements contribute to a sizable proportion of active enhancers and gene promoters. We further show that transposon subfamilies contribute differently and distinctly to the pluripotency regulatory program through their repertoires of transcription factor binding sites, shedding light on the formation of regulatory programs and the origins of regulatory elements.


title: Endogenous retroviruses co-opted as divergently transcribed regulatory elements shape the regulatory landscape of embryonic stem cells


title: Recirculation of H2, CO2, and ethylene improves carbon fixation and carboxylate yields in anaerobic fermentation

abstract: Anaerobic fermentation with mixed cultures has gained momentum as a bioprocess for its promise to produce platform carboxylates from low-value biomass feedstocks. Anaerobic fermenters are net carbon emitters and their carboxylate yields are limited by electron donor availability. In a new approach to tackle these two disadvantages, we operated two bioreactors fed with acetate and lactate as a model feedstock while recirculating H2/CO2 to stimulate concomitant autotrophic activity. After 42 days of operation, hydrogenotrophic methanogenesis was predominant and ethylene ([≥]1.3 kPa) was added to one of the reactors, inhibiting methanogenesis completely and recovering net carbon fixation (0.20 g CO2 L-1 d-1). When methanogenesis was inhibited, exogenous H2 accounted for 17% of the consumed electron donors. Lactate-to-butyrate selectivity was 101% (88% in the control without ethylene) and lactate-to-caproate selectivity was 17% (2.3% in the control). Community analysis revealed that ethylene caused Methanobacterium to be washed out, giving room to acetogenic bacteria. In contrast to 2-bromoethanosulfonate, ethylene is a scalable methanogenesis inhibition strategy that did not collaterally block i-butyrate formation. By favoring the bacterial share of the community to become mixotrophic, the concept offers a way to simultaneously increase selectivity to medium-chain carboxylates and to develop a carbon-fixing chain elongation process.

category: bioengineering


title: Daily transient coating of the intestine leads to weight loss and improved glucose tolerance

abstract: The worsening obesity epidemic has driven a parallel increase in incidence of type-2 diabetes (T2D) found in 13% of the adult US population. Roux-en-Y gastric bypass surgery (RYGB) is the golden standard surgical treatment for obesity-related T2D. However, over 70% of T2D patient do not meet the NIH criteria for surgery, and many that do, are reluctant to consider such an invasive intervention. Mechanisms behind the antidiabetic effect of RYGB are not clearly understood, but isolation of proximal bowel from nutrient exposure plays a critical role. In this study, we tested the impact of daily administration of an oral gut coating agent, LuCI (Luminal Coating of the Intestine), in controlling weight and insulin resistance. LuCI is not systemically absorbed and provides a transient coating of the proximal intestines and excludes the coated bowel from nutrient contact, thus mimicking the effects of RYGB. We demonstrate that LuCI results in weight loss and improved insulin sensitivity in a rat DIO model. LuCI also appears to replicate hormonal changes seen after RYGB, by increasing post-prandial GLP-1 and GIP levels. LuCI has the potential to replicate the metabolic benefit of surgery and be a novel therapeutic strategy for obesity associated T2D.


title: Highly potent photoinactivation of bacteria using a water soluble, cell permeable, DNA-binding photosensitizer

abstract: Antimicrobial photodynamic therapy (APDT) employs a photosensitizer, light, and molecular oxygen to treat infectious diseases via oxidative damage, with a low likelihood for the development of resistance. For optimal APDT efficacy, photosensitizers with cationic charges that can permeate bacteria cells and bind intracellular targets are desired to not limit oxidative damage to the outer bacterial structure. Here we report the application of brominated DAPI (Br-DAPI), a water-soluble, DNA-binding photosensitizer for eradication of both gram-negative and gram-positive bacteria (as demonstrated on N99 E. coli and B. subtilis, respectively). We observe intracellular uptake of Br-DAPI, ROS-mediated bacterial cell death via 1- and 2-photon excitation, and selective photocytotoxicity of bacteria over mammalian cells. Photocytotoxicity of both N99 E. coli and B. subtilis occurred at sub-micromolar concentrations (IC50 = 0.2 M - 0.4 M) and low light doses (5-minute irradiation times, 4.5 J cm-2 dose) making it superior to commonly employed APDT phenothiazinium photosensitizers such as methylene blue. Given its high potency and 2-photon excitability, Br-DAPI is a promising novel photosensitizer for in vivo APDT applications.

category: biophysics


title: Stx2 Induces Differential Gene Expression by Activating Several Pathways and Disturbs Circadian Rhythm Genes in the Proximal Tubule.

abstract:
(1) BackgroundShiga toxin-producing Escherichia coli (STEC) causes proximal tubular defects in the kidney. However, factors altered by Shiga toxin (Stx) within the proximal tubules are yet to be shown.

(2) MethodsWe determined Stx receptor Gb3 in murine and human kidneys and confirmed the receptor expression in the proximal tubules. Stx2-injected mouse kidney tissues and Stx2-treated human primary renal proximal tubular epithelial cell (RPTEC) were collected, and microarray analysis was performed.

(3) ResultsWe compared murine kidney and RPTEC arrays and selected common 58 genes that are differentially expressed vs. control (0 h, no toxin-treated). We found that the most highly expressed gene was GDF15, which may be involved in Stx2-induced weight loss. Genes associated with previously reported Stx2 activities such as src kinase Yes phosphorylation pathway activation, unfolded protein response (UPR) and ribotoxic stress response (RSR) showed differential expressions. Moreover, circadian clock genes were differentially expressed suggesting Stx2-induced renal circadian rhythm disturbance. Circadian rhythm-regulated proximal tubular Na+-glucose transporter SGLT1(SLC5A1) was down-regulated, indicating proximal tubular functional deterioration, and mice developed glucosuria confirming proximal tubular dysfunction.

(4) ConclusionsStx2 alters gene expression in murine and human proximal tubules through known activities and newly investigated circadian rhythm disturbance resulting in proximal tubular dysfunctions.

category: pathology


title: Antimicrobial Agent Triclosan Inhibits Acetylcholinesterase Activity in Vitro in a Dose-Dependent Manner

abstract: The antimicrobial agent, Triclosan, is widely used in many consumer products. It has been designated as a "contaminant of emerging concern (CEC)" because its exposure is known to cause adverse ecological and human health effects. Triclosan is not labelled as GRAS/GRAE (generally recognized as safe and effective), but its use is still prevailing. In vivo studies have revealed that exposure to triclosan results in a decreased acetylcholinesterase (AChE) activity. However mechanistic insights into AChE inhibition by triclosan are missing. Using in vitro AChE activity assay with purified AChE, we show that triclosan acts as a direct inhibitor of AChE and inhibits AChE activity in a dose-dependent manner. Given the function of AChE, any alteration in its activity can be neurotoxic. Our results provide important mechanistic insights into triclosan induced neurotoxicity with AChE as a target.

category: pharmacology and toxicology


title: A new method to identify global targets of RNA-binding proteins in plants

abstract:
BackgroundRNA-binding proteins (RBPs) play crucial roles in various aspects of post-transcriptional gene expression; their functions can vary between tissues, cell types, developmental stages, and environmental conditions. Identifying RBP target RNAs and investigating whether they are differentially bound by RBPs in different cell types, stages, or conditions could shed light on RBP functions. Although several strategies have been designed to identify RBP targets, they involve complicated biochemical steps and require large quantities of material, and only a few studies using these techniques have been performed in plants. The TRIBE (targets of RNA binding proteins identified by editing) method was recently developed to identify RBP targets using a RBP coupled to the catalytic domain of a Drosophila RNA editing enzyme and expressing this fusion protein in vivo. The resulting novel editing events can be identified by sequencing. This technique uses little material and does not require complex biochemical steps, however it is not yet adapted for use in plants.

ResultsWe successfully applied an optimized genome-wide TRIBE method in plants. We selected the splicing regulator polypyrimidine tract-binding protein (PTB) as a model protein for testing the TRIBE system in the moss Physcomitrium patens. We demonstrated that 13.81% of protein-coding gene transcripts in P. patens are targets of PTB. Most potential PTB binding sites are located in coding sequences and 3 untranslated regions, suggesting that PTB performs multiple functions besides pre-mRNA splicing in this moss. In addition, TRIBE showed reproducible results compared to other methods.

ConclusionsWe have developed an alternative method based on the TRIBE system to identify RBP targets in plants globally, and we provide guidance here for its application in plants.

category: plant biology


title: Provenance Attestation of Human Cells Using Physical Unclonable Functions

abstract: We introduce a novel methodology, namely CRISPR-Engineered Attestation of Mammalian Cells using Physical Unclonable Functions (CREAM-PUFs), which can serve as the cornerstone for formally verifying transactions in human cell line distribution networks. A PUF is a physical entity which provides a measurable output that can be used as a unique and irreproducible identifier for the artifact wherein it is embedded. Popularized by the electronics industry, silicon PUFs leverage the inherent physical variations of semiconductor manufacturing to establish intrinsic security primitives for attesting integrated circuits. Owing to the stochastic nature of these variations and the multitude of steps involved, photo-lithographically manufactured silicon PUFs are impossible to reproduce (thus unclonable). Inspired by the success of silicon PUFs, we sought to exploit a combination of sequence-restricted barcodes and the inherent stochasticity of CRISPR-induced non-homologous end joining DNA error repair to create the first generation of genetic physical unclonable functions in three distinct human cells (HEK293, HCT116, and HeLa). We demonstrate that these CREAM-PUFs are robust (i.e., they repeatedly produce the same output), unique (i.e., they do not coincide with any other identically produced PUF), and unclonable (i.e., they are virtually impossible to replicate). Accordingly, CREAM-PUFs can serve as a foundational principle for establishing provenance attestation protocols for protecting intellectual property and confirming authenticity of engineered cell lines.

category: synthetic biology


title: The acquisition of additional feedback loops may optimize and speed up the response of quorum sensing

abstract: 1.Bacterial quorum sensing (QS) is a cell-to-cell communication system in which specific signals are activated to coordinate, for example, pathogenic behaviors and help bacteria collectively respond to perturbations. QS in Gram-negative bacteria is typically regulated by a N-acyl-homoserine lactone (AHL) molecules-mediated system, homologous of Vibrio fischeri LuxI-R. In many cases, bacteria possess more than one QS system, based on different types of molecules, that interact through a complex regulatory network. Presumably, these configurations have emerged over time from simpler ones through the acquisition of novel players (e.g. transcription factors) that have been successfully integrated into the native regulatory systems. However, the advantages provided by these alternative/additional configurations on QS-related phenotypes is poorly predictable only based on their underlying network structure. Here, we have adopted a modelling approach to infer the possible improvements conferred by the acquisition of additional control over bacterial regulation of QS. We use the Burkholderia genus as a case study because some of these strains, besides the LuxIR-like system (named CepIR), possess an integrated regulatory module named CciIR that interferes with the CepIR system through the implementation of several positive and negative control loops. Being associated to a genomic island (cci island), this additional module is prone to being horizontally transferred, giving rise to a potentially patchy genomic distribution and, in turn, to a complete (CepIR and CciIR systems together) vs. core (CepIR only) organization of QS regulation in this group of microorganisms. By using both deterministic and stochastic modelling we show that, upon their activation, the two regulatory schemes may lead to different phenotypes and to distinct responses to the extracellular concentration of signalling molecules. In particular, our simulations show that the presence of the additional regulatory module may confer specific improvements, including a faster response time and optimized control of QS regulation. Interestingly, some of these features may be particularly advantageous during host invasion, thus highlighting once more the importance of QS in the establishment and maintenance of bacterial infections.

category: systems biology


title: A combination of RBD and NTD neutralizing antibodies limits the generation of SARS-CoV-2 spike neutralization-escape mutants

abstract:
Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150 and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent sera were reduced by 4-16-fold against rVSV-SARS2 bearing Y145D, K150E or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs modestly enhanced their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC.

ImportanceThe US FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (mAb) therapies for the treatment of mild to moderate COVID-19. These mAb therapeutics are solely targeting the receptor binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor binding and N-terminal domains may have higher efficacy and is beneficial to combat the emergence of virus variants.

category: microbiology


title: Target-Dependent Coordinated Biogenesis Ensure Cascaded Expression of miRNAs in Activated Macrophage

abstract:
MicroRNAs (miRNAs) repress protein expression by binding to the 3 UTR of the target mRNAs. By exploring the effect of target mRNA on biogenesis of cognate miRNAs, we have noted miRNA with higher number of binding sites (primary miRNA) coordinates the biogenesis and activity of another miRNA (secondary miRNA) having binding sites on the 3 UTR of a common target mRNA. From the quantitative data obtained from macrophage cells, we detected miR-146a-5p as a "primary" miRNA that coordinates biogenesis of "secondary" miR-125b, miR-21 or miR-142-3p to target new sets of mRNAs to balance the immune response in activated macrophage cells. Interestingly, target dependent coordinated biogenesis of miRNAs, happening on the rough endoplasmic reticulum attached membrane, ensures a cumulative mode of action of primary and secondary miRNAs on the secondary target mRNAs where a cascaded effect of primary miRNA on its secondary targets has been detected. Extensive computational analysis for the presence of coordinated biogenesis pairs of miRNAs in mammalian cells has also allowed us to construct a coordinate biogenesis repository to determine context specific coordinated biogenesis relationships exists for specific pairs of miRNAs in mammalian cells.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/448041v1_ufig1.gif" ALT="Figure 1">
View larger version (16K):
org.highwire.dtl.DTLVardef@318871org.highwire.dtl.DTLVardef@1f4d3a3org.highwire.dtl.DTLVardef@72adceorg.highwire.dtl.DTLVardef@f0d79e_HPS_FORMAT_FIGEXP  M_FIG C_FIG O_LImiRNA with higher number of binding sites (primary miRNA) can coordinates the biogenesis and activity of another miRNA (secondary miRNA)
C_LIO_LITarget dependent coordinated biogenesis of miRNAs ensures a cumulative mode of action of primary and secondary miRNAs on the secondary target mRNAs on rER attached polysomes
C_LIO_LImiR-146a-5p acts as a "primary" miRNA to coordinate biogenesis of "secondary" miR-125b, miR-21 or miR-142-3p in activated macrophage cells
C_LIO_LICoordinate biogenesis balance the immune response in activated macrophage cells by ensuring propagation of primary miRNAs effect to diverse target mRNAs through secondary miRNAs.
C_LI

category: molecular biology


title: The emergence of a birth-dependent mutation rate in asexuals: causes and consequences

abstract: AO_SCPLOWBSTRACTC_SCPLOWIn unicellular organisms such as bacteria and in most viruses, mutations mainly occur during reproduction. Thus, genotypes with a high birth rate should have a higher mutation rate. However, standard models of asexual adaptation such as the  replicator-mutator equation often neglect this generation-time effect. In this study, we investigate the emergence of a positive dependence between the birth rate and the mutation rate in models of asexual adaptation and the consequences of this dependence. We show that it emerges naturally at the population scale, based on a large population limit of a stochastic time-continuous individual-based model with elementary assumptions. We derive a reaction-diffusion framework that describes the evolutionary trajectories and steady states in the presence of this dependence. When this model is coupled with a phenotype to fitness landscape with two optima, one for birth, the other one for survival, a new trade-off arises in the population. Compared to the standard approach with a constant mutation rate, the symmetry between birth and survival is broken. Our analytical results and numerical simulations show that the trajectories of mean phenotype, mean fitness and the stationary phenotype distribution are in sharp contrast with those displayed for the standard model. The reason for this is that the usual weak selection limit does not hold in a complex landscape with several optima associated with different values of the birth rate. Here, we obtain trajectories of adaptation where the mean phenotype of the population is initially attracted by the birth optimum, but eventually converges to the survival optimum, following a hook-shaped curve which illustrates the antagonistic effects of mutation on adaptation.


title: Development of the myzozoan aquatic parasite Perkinsus marinus as a versatile experimental genetic model organism

abstract: The phylum Perkinsozoa is an aquatic parasite lineage that has devastating effects on commercial and natural mollusc populations, and also comprises parasites of algae, fish and amphibians. They are related to, and share much of their biology with, dinoflagellates and apicomplexans and thus offer excellent genetic models for both parasitological and evolutionary studies. Genetic transformation has been previously achieved for select Perkinsus spp. but with few tools for transgene expression and only limited selection efficacy. We thus sought to expand the power of experimental genetic tools for Perkinsus marinus -- the principal perkinsozoan model to date. We constructed a modular plasmid assembly system that enables expression of multiple genes simultaneously. We developed an efficient selection system for three drugs, puromycin, bleomycin and blasticidin, that achieves transformed cell populations in as little as three weeks. We developed and quantified eleven new promoters of variable expression strength. Furthermore, we identified that genomic integration of transgenes is predominantly via non-homologous recombination and often involves transgene fragmentation including deletion of some introduced elements. To counter these dynamic processes, we show that bi-cistronic transcripts using the viral 2A peptides can couple selection systems to the maintenance of the expression of a transgene of interest. Collectively, these new tools and insights provide new capacity to efficiently genetically modify and study Perkinsus as an aquatic parasite and evolutionary model.

category: genetics


title: Skeletal muscle mitochondrial respiration in a model of age-related osteoarthritis is impaired after dietary rapamycin

abstract: A decline in skeletal muscle mitochondrial function is associated with the loss of skeletal muscle size and function during knee osteoarthritis (OA). We have recently reported that the 12-weeks of dietary rapamycin (Rap, 14ppm), with or without metformin (Met, 1000ppm), increased plasma glucose and OA severity in male Dunkin Hartley (DH) guinea pigs, a model of naturally occurring, age-related OA. The purpose of the current study was to determine if increased OA severity after dietary Rap and Rap+Met was accompanied by impaired skeletal muscle mitochondrial function. Mitochondrial respiration and hydrogen peroxide (H2O2) emissions were evaluated in permeabilized muscle fibers via high-resolution respirometry and fluorometry using either a saturating bolus or titration of ADP. Rap and Rap+Met decreased complex I (CI)-linked respiration and increased ADP sensitivity, consistent with previous findings in patients with end-stage OA. Rap also tended to decrease mitochondrial H2O2 emissions, however, this was no longer apparent after normalizing to respiration. The decrease in CI-linked respiration was accompanied with lower CI protein abundance. This is the first inquiry into how lifespan extending treatments Rap and Rap+Met can influence skeletal muscle mitochondria in a model of age-related OA. Collectively, our data suggest that Rap with or without Met inhibits CI-linked capacity and increases ADP sensitivity in DH guinea pigs that have greater OA severity.

category: physiology


title: The nucleolar DExD/H protein Hel66 is involved in ribosome biogenesis in Trypanosoma brucei

abstract: The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process.


title: A panel of KSHV mutants in the polycistronic kaposin locus for precise analysis of individual protein products

abstract:
Kaposis sarcoma-associated herpesvirus (KSHV) is the cause of several human cancers including the endothelial cell (EC) malignancy, Kaposis sarcoma. Unique KSHV genes absent from other human herpesvirus genomes, the "K-genes", are important for KSHV replication and pathogenesis. Among these, the kaposin transcript is highly expressed in all phases of infection, but its complex polycistronic nature has hindered functional analysis to date. At least three proteins are produced from the kaposin transcript: Kaposin A (KapA), B (KapB), and C (KapC). To determine the relative contributions of kaposin proteins during KSHV infection, we created a collection of mutant viruses unable to produce kaposin proteins individually or in combination. Kaposin-deficient latent iSLK cell lines displayed reduced viral genome copy number and often exhibited small LANA nuclear bodies; despite this, all were capable of progeny virion production. Primary infection with {Delta}KapB virus revealed decreased LANA expression and viral genome copy number, yet providing KapB protein in trans failed to complement these defects, suggesting a requirement for the kaposin locus in cis. Our previous work showed that KapB was sufficient to recapitulate the elevated proinflammatory cytokine transcripts associated with KS via the disassembly of RNA granules called processing bodies (PBs). We now show that KapB is necessary for PB disassembly during latent KSHV infection. These findings demonstrate that our panel of kaposin-deficient viruses enables precise analysis of the respective contributions of individual kaposin proteins to KSHV replication. Moreover, our mutagenesis approach serves as a guide for the functional analysis of other complex multicistronic viral loci.

ImportanceKaposis sarcoma-associated herpesvirus (KSHV) expresses high levels of the kaposin transcript during both latent and lytic phases of replication. Due to its repetitive, GC-rich nature and polycistronic coding capacity, until now no reagents existed to permit a methodical analysis of the role of individual kaposin proteins in KSHV replication. We report the creation of a panel of recombinant viruses and matched producer cell lines that delete kaposin proteins individually or in combination. We demonstrate the utility of this panel by confirming the requirement of one kaposin translation product to a key KSHV latency phenotype. This study describes a new panel of molecular tools for the KSHV field to enable precise analysis of the roles of individual kaposin proteins during KSHV infection.


title: TopFlash transgenic quail reveals dynamic TCF/beta-catenin signaling during avian embryonic development

abstract: The Wnt/{beta}-catenin signaling pathway is highly conserved throughout evolution and it plays crucial roles in several developmental and pathological processes. Wnt ligands can act at a considerable distance from their sources and it is therefore necessary to examine not only the Wnt-producing but also the Wnt-receiving cells and tissues to fully appreciate the many functions of this pathway. To monitor Wnt activity, multiple tools have been designed which consist of multimerized Wnt signaling response elements (TCF/LEF binding sites) driving the expression of fluorescent reporter proteins (e.g. GFP, RFP) or of LacZ. The high stability of those reporters leads to a considerable accumulation in cells activating the pathway, thereby making them easily detectable. However, this makes them unsuitable to follow temporal changes of the pathways activity during dynamic biological events. Even though fluorescent transcriptional reporters can be destabilized to shorten their half-lives, this dramatically reduces signal intensities, particularly when applied in vivo. To alleviate these issues, we developed two transgenic quail lines in which high copy number (12x or 16x) of the TCF/LEF binding sites drive the expression of destabilized GFP variants. Translational enhancer sequences derived from viral mRNAs were used to increase signal intensity and specificity. This resulted in transgenic lines efficient for the characterisation of TCF/{beta}-catenin transcriptional dynamic activities during embryogenesis, including using in vivo imaging. Our analyses demonstrate the use of this transcriptional reporter to unveil novel aspects of Wnt signaling, thus opening new routes of investigation into the role of this pathway during amniote embryonic development.

category: developmental biology


title: Disruption of folate metabolism causes poor alignment and spacing of mouse conceptuses for multiple generations

abstract: Abnormal uptake or metabolism of folate increases risk of human pregnancy complications, though the mechanism is unclear. Here, we explore how defective folate metabolism influences early development by analysing mice with an Mtrrgt hypomorphic mutation. MTRR is necessary for methyl group utilisation from the folate cycle, and the Mtrrgt allele disrupts this process. We show that the spectrum of phenotypes previously observed in Mtrrgt/gt conceptuses at embryonic day (E) 10.5 is apparent from E8.5 including developmental delay, congenital malformations, and placental phenotypes (e.g., eccentric chorioallantoic attachment). Notably, we report misalignment of some Mtrrgt conceptuses within their implantation sites from E6.5. The degree of skewed growth occurs across a continuum, with eccentric chorioallantoic attachment now re-characterised as a severe form of conceptus misalignment. Additionally, some Mtrrgt/gt conceptuses display twinning. Therefore, we implicate folate metabolism in blastocyst orientation and spacing at implantation. Embryo development is influenced by skewed growth since developmental delay and heart malformations (but not neural tube defects) associate with severe misalignment of Mtrrgt/gt conceptuses. Patterning of trophoblast lineage markers is largely unaffected in skewed Mtrrgt/gt conceptuses at E8.5 indicating trophoblast differentiation was normal when misaligned. Typically, the uterus guides conceptus orientation. Accordingly, we manipulate the maternal Mtrr genotype and assess conceptus alignment. Mtrr+/gt, and Mtrrgt/gt mothers, plus Mtrr+/+ mothers, exhibit misaligned conceptuses at E6.5. While progesterone and/or BMP2 signalling required for decidualisation might be disrupted, normal gross decidual morphology, patterning, and blood perfusion is evident regardless of conceptus alignment, arguing against a uterine defect. Given the important finding that Mtrr+/+ mothers also display conceptus misalignment, a grandparental effect is explored. Multigenerational phenotype inheritance is characteristic of the Mtrrgt model, though the mechanism remains unclear. Genetic pedigree analysis reveals that severe skewing associates with the Mtrr genotype of either maternal grandparent. Moreover, misalignment is independent of the uterus and instead is attributed to an embryonic mechanism based on blastocyst transfer experiments. Overall, our data indicates that abnormal folate metabolism influences conceptus orientation over multiple generations with implications for subsequent development. Our study casts light on the complex role of folate metabolism during development beyond a direct maternal effect.


title: Complete loss of mitochondrial complex I genes in mistletoes (Viscaceae) and evidence of polyadenylated mitochondrial transcripts shown by whole transcriptome sequencing

abstract: The profound absence of mitochondrial complex I (NADH-ubiquinone oxidoreductase) genes from the mitogenome of Viscum spp. and the rapid rates of molecular evolution characterizing most of their remaining mitochondrial genes raise questions regarding the possible transfer of the entire nad gene set to the nucleus, as well as for the functionality of the remaining highly divergent genes. Using whole transcriptome sequencing in three species of Viscaceae: V. album, V. crassulae, and Phoradendron leucarpum we were able to confirm transcription of all previously identified genes. However, we did not detect any nad gene transcripts, thus, providing further evidence of the complete loss of complex I in Viscaceae. The results from transcriptome sequencing also revealed that levels and patterns of RNA editing were not different from those found in autotrophic plant species. Hence, RNA editing is not a means of restoring conserved domains or folding sites of the proteins coded for by the divergent mitochondrial genes. Since we were able to recover mitochondrial genes transcripts following a sequencing protocol targeted towards polyadenylated mRNA molecules, it is suggested that mitochondrial genes undergo post-transcriptional polyadenylation in Viscaceae.


title: Aminoglycoside-induced premature termination codon readthrough of COL4A5 nonsense mutations that cause Alport syndrome

abstract: Alport syndrome (AS) is characterized by glomerular basement membrane (GBM) abnormalities leading to progressive glomerulosclerosis. Mutations in the COL4A3, COL4A4 or COL4A5 genes encoding type IV collagen 345 cause AS. Truncated 3, 4, and 5 chains lacking an intact COOH-terminal noncollagenous domain due to a premature termination codon (PTC) cannot assemble into heterotrimers or incorporate into the GBM. Therefore, achieving full-length protein expression is a potential therapy for AS caused by truncating nonsense mutations. Small molecule-based PTC readthrough (PTC-RT) therapy has been well studied in other genetic diseases, but whether PTC-RT is applicable to AS is unexplored. To investigate the feasibility of PTC-RT therapy in AS, we made a cDNA to express COL4A5 fused to a C-terminal NanoLuc luciferase (NLuc) to monitor full-length translation. Full-length COL4A5-NLuc produces luminescence, but mutants truncated due to a PTC do not. To screen for COL4A5 nonsense mutants susceptible to PTC-RT, we introduced 49 individual nonsense mutations found in AS patients into the COL4A5-NLuc cDNA. Luciferase assays revealed that 11 mutations (C29X, S36X, E130X, C1521X, R1563X, C1567X, W1594X, S1632X, R1683X, C1684X and K1689X) were susceptible to PTC-RT induced by G418, which is known to have high readthrough activity. Moreover, we found that some next-generation "designer" PTC-RT drugs induced RT, and RT enhancer compounds increased the efficacy of PTC-RT in a G418-susceptible PTC mutant. These results suggest that PTC-RT therapy is a feasible approach for some patients with AS. Our luciferase-based COL4A5 translation reporter system will contribute to further development of PTC-RT therapies in a personalized medicine approach to treating AS.


title: The YcnI protein from Bacillus subtilis contains a copper-binding domain

abstract: Bacteria require a precise balance of copper ions to ensure that essential cuproproteins are fully metalated while also avoiding copper-induced toxicity. The Gram positive bacterium Bacillus subtilis maintains appropriate copper homeostasis in part through its ycn operon. The ycn operon comprises genes encoding three proteins: the putative copper importer YcnJ, the copper-dependent transcriptional repressor YcnK, and the uncharacterized DUF1775 domain-containing YcnI. DUF1775 domains are found across bacterial phylogeny and bioinformatics analyses indicate that they frequently neighbor domains implicated in copper homeostasis and transport. Here, we investigated whether YcnI can interact with copper and, using electron paramagnetic resonance (EPR) and inductively-coupled plasma-mass spectrometry (ICP-MS) find that it can bind a single Cu(II) ion. We determine the structure of both the apo and copper-bound forms of the protein by X-ray crystallography, uncovering a copper binding site featuring a unique mono-histidine brace ligand set that is highly conserved among DUF1775 domains. These data suggest a possible role for YcnI as a copper chaperone and that DUF1775 domains in other bacterial species may also function in copper homeostasis.


title: Glioblastoma cell fate is differentially regulated by the microenvironments of the tumour bulk and infiltrative margin

abstract: Glioblastoma recurrence originates from invasive cells at the tumour margin that escape surgical debulking, but their biology remains poorly understood. Here we generated three somatic mouse models recapitulating the main glioblastoma driver mutations to characterise margin cells. We find that, regardless of genetics, tumours converge on a common set of neural- like cellular states. However, bulk and margin display distinct neurogenic patterns and immune microenvironments. The margin is immune-cold and preferentially follows developmental-like trajectories to produce astrocyte-like cells. In contrast, injury-like programmes dominate in the bulk, are associated with immune infiltration and generate lowly-proliferative injured neural progenitor-like (iNPCs) cells. In vivo label-retention approaches further demonstrate that iNPCs account for a significant proportion of dormant glioblastoma cells and are induced by interferon signalling within T-cell niches. These findings indicate that tumour region is a major determinant of glioblastoma cell fate and therapeutic vulnerabilities identified in bulk may not extend to the margin residuum.

category: cancer biology


title: HOXB8 counteracts MAPK/ERK oncogenic signaling in a chicken embryo model of neoplasia

abstract:
HOX transcription factors are members of an evolutionarily conserved family of proteins required for the establishment of the anteroposterior body axis during bilaterian development. Although they are often deregulated in cancers, the molecular mechanisms by which they act as oncogenes or tumor suppressor genes are only partially understood. Since the MAPK/ERK signaling pathway is deregulated in most cancers, we aimed at apprehending if and how Hox proteins interact with ERK oncogenicity.

Using an in vivo neoplasia model in the chicken embryo that we have developed, consisting in the overactivation of the ERK1/2 kinases in the trunk neural tube, we analyzed the consequences of HOXB8 gain of function at morphological and transcriptional level in this model. We found that HOXB8 acts as a tumor suppressor, counteracting ERK-induced neoplasia. HOXB8 tumor suppressor function in this model relies on a large reversion of the oncogenic transcriptome induced by ERK. In addition to showing that HOXB8 protein controls the transcriptional responsiveness to ERK oncogenic signaling, our study identified new downstream targets of ERK oncogenic activation in an in vivo context that could provide clues for therapeutic strategies.


title: Self-assembled nanoparticles enable delivery of VCP inhibitor, resulting in enhanced photoreceptor protection in retinal explants

abstract: 1.Mutations in rhodopsin lead to its misfolding resulting in autosomal dominant retinitis pigmentosa (adRP). Pharmacological inhibition of the ATP-driven chaperone valosin- containing protein (VCP), a molecular checkpoint for protein quality control, slows down retinal degeneration in animal models. However, poor water-solubility of VCP inhibitors poses a challenge to their clinical translation as intravitreal injections for retinal treatment. In order to enable the delivery of VCP inhibitors, we have developed and investigated two formulations for the VCP inhibitor ML240. Nanoformulations of ML240 were obtained by using amphiphilic polymers methoxy-poly (ethylene glycol)5kDa-cholane (mPEG5kDa-cholane) and methoxy-poly (ethylene glycol)5kDa- cholesterol (mPEG5kDa-cholesterol). Both formulations increased the water-solubility of ML240 by two orders of magnitude and prolonged the drug released over ten days. Encapsulation of ML240 in mPEG5kDa-cholane showed superior photoreceptor protection at lower drug concentrations, normalized rhodopsin localization, and alleviated inflammatory microglial responses in an ex vivo rat model of retinal degeneration. The study demonstrates the potential of VCP inhibitor nanoformulations to treat adRP, a pharmacologically orphan disease.


title: Phytochemical Nrf2 activator attenuates skeletal muscle mitochondrial dysfunction and impaired proteostasis in a preclinical model of musculoskeletal aging

abstract: Musculoskeletal dysfunction is an age-related syndrome associated with impaired mitochondrial function and proteostasis. However, few interventions have tested targeting two drivers of musculoskeletal decline. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that stimulates transcription of cytoprotective genes and improves mitochondrial function. We hypothesized daily treatment with a Nrf2 activator in Hartley guinea pigs, a model of age-related musculoskeletal dysfunction, attenuates the progression of skeletal muscle mitochondrial dysfunction and impaired proteostasis, preserving musculoskeletal function. We treated 2-month- and 5-month-old male and female Hartley guinea pigs for 3 and 10 months, respectively, with the phytochemical Nrf2 activator PB125 (Nrf2a). Longitudinal assessments of voluntary mobility were measured using Any-Maze open-field enclosure monitoring. Cumulative skeletal muscle protein synthesis rates were measured using deuterium oxide over the final 30 days of treatment. Mitochondrial oxygen consumption in permeabilized soleus muscles was measured using ex vivo high resolution respirometry. In both sexes, Nrf2a 1) increased electron transfer system capacity; 2) attenuated the disease/age-related decline in coupled and uncoupled mitochondrial respiration; and 3) attenuated declines in protein synthesis in the myofibrillar, mitochondrial, and cytosolic subfractions of the soleus. These improvements were not associated with statistically significant prolonged maintenance of voluntary mobility in guinea pigs. Collectively, these results demonstrate that treatment with an oral Nrf2 activator contributes to maintenance of skeletal muscle mitochondrial function and proteostasis in a pre-clinical model of musculoskeletal decline. Further investigation is necessary to determine if these improvements are also accompanied by slowed progression of other aspects of musculoskeletal decline.


title: Proximity-based labeling reveals DNA damage-induced N-terminal phosphorylation of fused in sarcoma (FUS) leads to distinct changes in the FUS protein interactome.

abstract: Cytoplasmic accumulation of the RNA/DNA binding protein, fused in sarcoma (FUS), into inclusions is a common hallmark of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) pathology. We have previously shown that DNA damage can trigger the cytoplasmic accumulation of an N-terminally phosphorylated FUS. However, the functional consequences of N-terminal FUS phosphorylation are unknown. To gain insight into this question, we utilized proximity-dependent biotin labeling via ascorbate peroxidase 2 (APEX2) paired with mass-spectrometry (MS) to investigate whether N-terminal phosphorylation shifts the FUS protein-protein interaction network (interactome), and subsequently, its function. We report the first comparative analysis of the interactomes for three FUS variants: homeostatic wild-type FUS (FUS WT), a phosphomimetic variant of FUS (a proxy for N-terminally phosphorylated FUS, FUS PM), and a toxic FUS P525L mutant (a mutation that causes juvenile ALS, FUS P525L). Data are available via ProteomeXchange with identifier PXD026578. We demonstrate that compared to FUS WT and FUS P525L, the FUS PM interactome uniquely enriches for a set of cytoplasmic proteins that mediate mRNA metabolism and translation and nuclear proteins involved in spliceosome and DNA repair functions, respectively. We further identify and validate three proteins, VPS35, MOV10, and CLTA, as novel interacting partners of all three FUS variants. Lastly, we provide functional evidence that N-terminally phosphorylated FUS may disrupt homeostatic translation and steady state levels of specific mRNA transcripts. Taken together, these results highlight phosphorylation as a unique modulator of the FUS interactome and function.

category: neuroscience


title: Reduced Hippocampal Inhibition and Enhanced Autism-Epilepsy Comorbidity in Mice Lacking Neuropilin 2

abstract: The neuropilin receptors and their secreted semaphorin ligands play key roles in brain circuit development by regulating numerous crucial neuronal processes, including the maturation of synapses and migration of GABAergic interneurons. Consistent with its developmental roles, the neuropilin 2 (Nrp2) locus contains polymorphisms in patients with autism spectrum disorder (ASD). Nrp2 deficient mice show autism-like behavioral deficits and propensity to develop seizures. In order to determine the pathophysiology in Nrp2 deficiency, we examined the hippocampal numbers of interneuron subtypes and inhibitory regulation of hippocampal CA1 pyramidal neurons in mice lacking one or both copies of Nrp2. Immunostaining for interneuron subtypes revealed that Nrp2-/- mice have reduced number of parvalbumin, somatostatin and Neuropeptide Y cells, mainly in CA1. Whole cell recordings identified reduced firing and hyperpolarized shift in resting membrane potential in CA1 pyramidal neurons from Nrp2+/- and Nrp2-/- mice compared to age-matched wild-type controls indicating decrease in intrinsic excitability. Simultaneously, the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) are reduced in Nrp2 deficient mice. A convulsive dose of kainic acid evoked electrographic and behavioral seizures with significantly shorter latency, longer duration and higher severity in Nrp2-/- compared to Nrp2+/+ animals. Finally, Nrp2+/- and Nrp2-/-, but not Nrp2+/+, mice have impaired cognitive flexibility demonstrated by reward-based reversal learning, a task associated with hippocampal circuit function. Together these data demonstrate a broad reduction in interneuron subtypes and compromised inhibition in CA1 of Nrp2-/- mice, which could contribute to the heightened seizure susceptibility and behavioral deficits consistent with an ASD/epilepsy phenotype.


title: Oncomodulin Delays Age-Related Hearing Loss in C57 and CBA Mice

abstract: Ca2+ signaling is a major contributor to sensory hair cell function in the cochlea. Oncomodulin (OCM) is a Ca2+ binding protein preferentially expressed in outer hair cells of the cochlea and few other specialized cell types. Here, we expand on our previous reports and show that OCM prevents early progressive hearing loss in mice of two different genetic backgrounds: CBA/CaJ and C57BI/6J. In both backgrounds, genetic disruption of Ocm leads to early progressive hearing loss as measured by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE). In both strains, loss of Ocm reduced hearing across lifetime (hearing span) by more than 50% relative to wild type (WT). Even though the two WT strains have very different hearing spans, OCM plays a considerable and similar role within their genetic environment to regulate hearing function. The accelerated ARHL of the Ocm KO illustrates the importance of Ca2+ signaling in maintaining hearing health.


title: Holosteans contextualize the role of the teleost genome duplication in promoting the rise of evolutionary novelties in the ray-finned fish innate immune system

abstract: Over 99% of ray-finned fishes (Actinopterygii) are teleosts, a clade that comprises half of all living vertebrates that have diversified across virtually all fresh and saltwater ecosystems. This ecological diversity raises the question of how the immunogenetic diversity required to persist under heterogeneous pathogen pressures evolved. The teleost genome duplication (TGD) has been hypothesized as the evolutionary event that provided the genomic substrate for rapid genomic evolution and innovation. However, studies of putative teleost-specific innate immune receptors have been largely limited to comparisons either among teleosts or between teleosts and distantly related vertebrate clades such as tetrapods. Here we describe and characterize the receptor diversity of two clustered innate immune gene families in the teleost sister lineage: Holostei (bowfin and gars). Using genomic and transcriptomic data, we provide a detailed investigation of the phylogenetic history and conserved synteny of gene clusters encoding diverse immunoglobulin domain-containing proteins (DICPs) and novel immune-type receptors (NITRs). These data demonstrate an ancient linkage of DICPs to the major histocompatibility complex (MHC) and reveal an evolutionary origin of NITR variable-joining (VJ) exons that predate the TGD by at least 50 million years. Further characterizing the receptor diversity of Holostean DICPs and NITRs illuminates a sequence diversity that rivals the diversity of these innate immune receptor families in many teleosts. Taken together, our findings provide important historical context for the evolution of these gene families that challenge prevailing expectations concerning the consequences of the TGD during actinopterygiian evolution.

category: immunology


title: In situ treatment of H. pylori infection in mice stomach with bioengineered probiotic bacteria releasing guided Antimicrobial peptides

abstract:
ObjectivesTargeted therapies seek to selectively eliminate a pathogen without disrupting the resident microbial community. This is even more important when a pathogen like H. pylori resides in stomach, a sensitive microbial ecosystem. Using a probiotic like Lactococcus lactis and bioengineering it to release a guided Antimicrobial Peptide (AMP) targeted towards the pathogen offers a pathway to specifically knock-out the deleterious species and not disturbing the stomach microbiome.

ResultsThree AMPs, Alyteserin, CRAMP and Laterosporulin, were genetically fused to a guiding peptide MM1, which selectively binds to Vacuolating Toxin A (VacA) of H. pylori and cloned into an excretory vector pTKR inside L. lactis. When cultured together in vitro, the L. lactis bioengineered with guided AMPs selectively killed H. pylori when compared to E. coli or Lactobacillus plantarum, as determined by qPCR. Chemically synthesized Alyteserin and MM1-Alyteserin showed similar preferential inhibition of H. pylori when compared against E. coli, with the MIC of MM1-Alyteserin becoming significantly higher for E. coli than Alytserin whereas no such effet was observed against H. pylori.

ConclusionsProbiotics bioengineered to excrete guided AMPs can be a novel and useful approach for combating pathogens without endangering the natural microbial flora. Given the wealth of AMPs and guiding ligands, both natural and synthetic, this approach can be adapted to develop a diverse array of chimeric guided AMPs and can be cloned into probiotics to create a safe and effective alternative to conventional chemical antibiotics.


title: An interactive deep learning-based approach reveals mitochondrial cristae topologies

abstract: Outer and inner mitochondrial membranes are highly specialized structures with distinct functional properties. Reconstructing complex 3D ultrastructural features of mitochondrial membranes at the nanoscale requires analysis of large volumes of serial scanning electron tomography data. While deep-learning-based methods improved in sophistication recently, time-consuming human intervention processes remain major roadblocks for efficient and accurate analysis of organelle ultrastructure. In order to overcome this limitation, we developed a deep-learning image analysis platform called Python-based Human-In-the-LOop Workflows (PHILOW). Our implementation of an iterative segmentation algorithm and Three-Axis-Prediction method not only improved segmentation speed, but also provided unprecedented ultrastructural detail of whole mitochondria and cristae. Using PHILOW, we found that 42% of cristae surface exhibits tubular structures that are not recognizable in light microscopy and 2D electron microscopy. Furthermore, we unraveled a fundamental new regulatory function for the dynamin-related GTPase Optic Atrophy 1 (OPA1) in controlling the balance between lamellar versus tubular cristae subdomains.

category: cell biology


title: Cell-matrix adhesion contributes to permeability control in human colon organoids

abstract:
Background and aimsCell-cell adhesion structures (desmosomes and, especially, tight junctions) are known to play important roles in control of transepithelial permeability in the colon. The involvement of cell-matrix interactions in permeability control is less clear. The goals of the present study were to: i) determine if disruption of colon epithelial cell interactions with the extracellular matrix alters permeability control and ii) determine if increasing the elaboration of protein components of cell-matrix adhesion complexes improves permeability control and mitigates the effects of cell-matrix disruption.

MethodsHuman colon organoids were interrogated for transepithelial electrical resistance (TEER) under control conditions (0.25 mM calcium) and in the presence of Aquamin(R), a multi-mineral product, at a level providing 1.5 mM calcium. The effects of Aquamin(R) on cell-matrix adhesion protein expression were determined in a proteomic screen and by Western blotting. In parallel, TEER was assessed in the presence of a function-blocking antibody directed at an epitope in the C-terminal region of laminin 3 chain.

ResultsTreatment of colon organoids with Aquamin(R) increased the expression of multiple basement membrane and hemidesmosomal proteins as well as keratin 8 and 18. TEER values were higher in the presence of Aquamin(R) than they were under control conditions. Anti-laminin antibody reduced TEER values under all conditions but was most effective in the absence of Aquamin(R), where laminin expression was low and TEER values were lower to begin with.

ConclusionsThese findings provide evidence that cell-matrix interactions contribute to permeability control in the colon. They suggest that the elaboration of proteins important to cell-matrix interactions can be increased in human colon organoids by exposure to a multi-mineral natural product. Increasing the elaboration of such proteins may help to mitigate the consequences of disrupting cell-matrix interactions on permeability control.


title: Mutations in SIX1 associated with Branchio-oto-renal Syndrome (BOR) differentially affect otic expression of putative target genes

abstract: Single nucleotide mutations in SIX1 are causative in some individuals diagnosed with branchio-otic/branchio-oto-renal (BOR) syndrome. To test whether these mutations have differential effects on otic gene expression, we engineered four BOR mutations in Xenopus six1 and targeted mutant protein expression to the neural crest and cranial placode precursor cells in wild-type embryos. Changes in the otic expression of putative Six1 targets and/or co-factors were monitored by qRT-PCR and in situ hybridization. We found that each mutant had a different combination of effects. The V17E mutant reduced eya2, tspan13, zbtb16 and pa2g4 otic vesicle expression at a frequency indistinguishable from wild-type Six1, but reduced prdm1 more and spry1 less compared to wild-type Six1. For most of these genes, the R110W, W122R and Y129C mutants were significantly less repressive compared to wild-type Six1. Their individual effects varied according to the level at which they were expressed. The R110W, W122R and Y129C mutants also often expanded prdm1 otic expression. Since previous studies showed that all four mutants are transcriptionally deficient and differ in their ability to interact with co-factors such as Eya1, we propose that altered co-factor interactions at the mutated sites differentially interfere with their ability to drive otic gene expression.


title: Structure of ABCB1/P-glycoprotein bound to the CFTR potentiator ivacaftor.

abstract: ABCB1 (P-glycoprotein) is an ATP binding cassette transporter that is involved in the clearance of xenobiotics and it affects the disposition of many drugs in the body. Here we have studied ABCB1 in the drug-bound and drug-free states, simultaneously, using high contrast cryo-electron microscopy imaging and a Volta phase plate. The binding of the potent CFTR potentiator, ivacaftor, at a site in the central aqueous cavity is mediated by transmembrane -helices 3,6,10,11 & 12. Binding is associated with a wider separation of the two halves of the transporter in the inward-facing state. Induced-fit changes the nucleotide binding domains in a way that may explain their increased affinity for ATP when drug is bound. Comparison of ivacaftor-bound structures of CFTR and ABCB1 suggests common features in the binding modes.

category: biochemistry


title: Reduced-Order Modeling and the Physics Governing Flapping Wing Fluid-Structure Interaction

abstract: Flapping, flexible insect wings deform during flight from aerodynamic and inertial forces. This deformation is believed to enhance aerodynamic and energetic performance. However, the predictive models used to describe flapping wing fluid-structure interaction (FSI) often rely on high fidelity computational solvers such as computational fluid dynamics (CFD) and finite element analysis (FEA). Such models require lengthy solution times and may obscure the physical insights available to analytical models. In this work, we develop a reduced order model (ROM) of a wing experiencing single-degree-of-freedom flapping. The ROM is based on deformable blade element theory and the assumed mode method. We compare the ROM to a high-fidelity CFD/FEA model and a simple experiment comprised of a mechanical flapper actuating a paper wing. Across a range of flapping-to-natural frequency ratios relevant to flying insects, the ROM predicts wingtip deflection five orders of magnitude faster than the CFD/FEA model. Both models are resolved to predict wingtip deflection within 30% of experimentally measured values. The ROM is then used to identify how the physical forces acting on the wing scale relative to one another. We show that, in addition to inertial and aerodynamic forces, added mass and aerodynamic damping influence wing deformation nontrivially.


title: Principles of ecDNA random inheritance drive rapid genome change and therapy resistance in human cancers

abstract: The foundational principles of Darwinian evolution are variation, selection, and identity by descent. Oncogene amplification on extrachromosomal DNA (ecDNA) is a common event, driving aggressive tumour growth, drug resistance, and shorter survival in patients1-4. Currently, the impact of non-chromosomal oncogene inheritance--random identity by descent--is not well understood. Neither is the impact of ecDNA on variation and selection. Here, integrating mathematical modeling, unbiased image analysis, CRISPR-based ecDNA tagging, and live-cell imaging, we identify a set of basic "rules" for how random ecDNA inheritance drives oncogene copy number and distribution, resulting in extensive intratumoural ecDNA copy number heterogeneity and rapid adaptation to metabolic stress and targeted cancer treatment. Observed ecDNAs obligatorily benefit host cell survival or growth and can change within a single cell cycle. In studies ranging from well-curated, patient-derived cancer cell cultures to clinical tumour samples from patients with glioblastoma and neuroblastoma treated with oncogene-targeted drugs, we show how these ecDNA inheritance "rules" can predict, a priori, some of the aggressive features of ecDNA-containing cancers. These properties are entailed by their ability to rapidly change their genomes in a way that is not possible for cancers driven by chromosomal oncogene amplification. These results shed new light on how the non-chromosomal random inheritance pattern of ecDNA underlies poor outcomes for cancer patients.


title: p53 mutations exhibit sex specific gain-of-function activity in gliomagenesis

abstract: The tumor suppressor TP53 is the most frequently mutated gene in cancer. Most TP53 mutations are missense mutations in the DNA-binding domain, which in addition to loss of canonical p53 activity, frequently confer gain-of-function (GOF) aberrant transcriptional activity through mutant p53 localization to non-canonical genes. GOF phenotypes differ by mutation and cell identity and are reported to include increased proliferation, migration, metabolic reprogramming, and therapy resistance. We found that several recurring p53 mutations exhibit a sex-bias in patients with glioblastoma (GBM). In vitro and in vivo analysis of three mutations, p53R172H, p53Y202C, and p53Y217C revealed sex differences in each mutations ability to transform primary mouse astrocytes. p53R172H exhibited a far greater ability to transform female astrocytes than males, p53Y202C transformed both male and female astrocytes with a small male bias, and p53Y217C only exhibited GOF transformation effects in male astrocytes. These phenotypic differences reflect an interaction between sex and GOF mutation to drive unique gene expression patterns in cancer pathways. We found that mutant p53 exhibits sex and mutation specific aberrant genomic localization to the transcriptional start sites of upregulated genes, whose promoter regions were enriched for different sets of transcription factor DNA-binding motifs. Together, our data establish a novel paradigm for sex specific mutant p53 GOF activity in GBM with implications for all cancer.


title: A quantitative gibberellin signalling biosensor reveals a role for gibberellins in internode specification at the shoot apical meristem

abstract: Growth at the shoot apical meristem (SAM) is essential for shoot architecture construction. The phytohormones gibberellins (GA) play a pivotal role in coordinating plant growth, but their role in the SAM remains mostly unknown. Here, we developed a ratiometric GA signalling biosensor by engineering one of the DELLA repressors, to suppress its master regulatory function in GA transcriptional responses while preserving its degradation upon GA sensing. We demonstrate that this novel degradation-based biosensor accurately reports on cellular changes in GA levels and perception during development. We used this biosensor to map GA signalling activity in the SAM. We show that high GA signalling is found primarily in cells located between organ primordia that are the precursors of internodes. By gain- and loss-of-function approaches, we further demonstrate that GAs regulate cell division plane orientation to establish the typical cellular organisation of internodes, thus contributing to internode specification in the SAM.


title: Control of SARS-CoV-2 infection after Spike DNA or Spike DNA+Proteinco-immunization in rhesus macaques

abstract:
The speed of development, versatility and efficacy of mRNA-based vaccines have been amply demonstrated in the case of SARS-CoV-2. DNA vaccines represent an important alternative since they induce both humoral and cellular immune responses in animal models and in human trials. We tested the immunogenicity and protective efficacy of DNA-based vaccine regimens expressing different prefusion-stabilized SARS-CoV-2 Spike antigens upon intramuscular injection followed by electroporation in rhesus macaques. Different Spike DNA vaccine regimens induced antibodies that potently neutralized SARS-CoV-2 in vitro and elicited robust T cell responses. The DNA-only vaccine regimens were compared to a regimen that included co- immunization of Spike DNA and protein in the same anatomical site, the latter of which showed significant higher antibody responses. All vaccine regimens led to control of SARS-CoV-2 intranasal/intratracheal challenge and absence of virus dissemination to the lower respiratory tract. Vaccine-induced binding and neutralizing antibody titers and antibody-dependent cellular phagocytosis inversely correlated with transient virus levels in the nasal mucosa. Importantly, the Spike DNA+Protein co-immunization regimen induced the highest binding and neutralizing antibodies and showed the strongest control against SARS-CoV-2 challenge in rhesus macaques.

Author summaryAnti-Spike neutralizing antibodies provide strong protection against SARS-CoV-2 infection in animal models, and correlate with protection in humans, supporting the notion that induction of strong humoral immunity is key to protection. We show induction of robust antibody and T cell responses by different Spike DNA-based vaccine regimens able to effectively mediate protection and to control SARS-CoV-2 infection in the rhesus macaque model. This study provides the opportunity to compare vaccines able to induce different humoral and cellular immune responses in an effort to develop durable immunity against the SARS-CoV-2. A vaccine regimen comprising simultaneous co-immunization of DNA and Protein at the same anatomical site showed best neutralizing abilities and was more effective than DNA alone in inducing protective immune responses and controlling SARS-CoV-2 infection. Thus, an expansion of the DNA vaccine regimen to include co-immunization with Spike protein may be of advantage also for SARS-CoV-2.


title: Interferon-α subtype treatment induces the repression of SRSF1 in HIV-1 target cells and affects HIV-1 post integration steps

abstract:
Efficient replication of HIV-1 depends on balanced levels of host cell components, including cellular splicing factors. Type I interferons (IFN-I), playing a crucial role in the innate immune defense against viral infections, are well known to induce the transcription of IFN-stimulated genes (ISGs) including potent host restriction factors. Not so well known is, that IFN-repressed genes (IRepGs) also affect viral infections by downregulating host dependency factors that are essential for viral replication. So far, knowledge about IRepGs involved in HIV-1 infection is very limited. Here, we demonstrate that expression levels of the serine/arginine-rich splicing factor 1 (SRSF1) were repressed upon treatment with IFN subtypes in HIV-1 susceptible cell lines as well as primary cells. Furthermore, we could demonstrate in two independent patient cohorts that HIV-1 infection and the concomitant inflammation during the acute and chronic phase, resulted in the strong induction of ISGs, but at the same time significantly repressed SRSF1. 4sU-labeling of newly transcribed mRNAs revealed that IFN-mediated repression of SRSF1 originated from a transcriptional shutdown. Experimental downregulation as well as overexpression of SRSF1 expression levels resulted in crucial changes in HIV-1 LTR-transcription, alternative splice site usage and virus production. While lower SRSF1 levels resulted in low vif mRNA levels and thus severely reduced viral infectivity, higher levels of SRSF1 impaired LTR-Tat-activity and HIV-1 particle production.

Our data highlight the so far undescribed role of SRSF1 acting as an IFN-repressed cellular dependency factor decisively regulating HIV-1 post integration steps.

Author SummaryIFN-I play a central role in the innate immune defense against viral infections by regulating the expression of interferon stimulated genes (ISGs) and interferon repressed genes (IRepGs). The stimulation of host restriction factors and the reduction of host dependency factors decisively affects the efficiency of HIV-1 replication. After the stable integration of the provirus into the host chromosome, HIV-1 exploits the host cell transcription and splicing machinery for its replication. A network of conserved splice sites and splicing regulatory elements maintain balanced levels of viral transcripts essential for virus production and immune evasion.

We demonstrate the so far undescribed role of the splicing factor SRSF1 as an IRepG crucially involved in HIV-1 RNA processing. In HIV-1 infected individuals, we observed inversely proportional expression of high ISG15 and low SRSF1 levels, which were restored in ART treated patients. We could demonstrate, that IFN-I stimulation of HIV-1 target cells resulted in a significant repression of SRSF1 RNA and protein levels. Since low SRSF1 expression decisively reduced HIV-1 vif mRNA levels, a severe impairment of viral replication was observed in APOBEC3G expressing cells. As overexpression negatively affected HIV-1 LTR transcription and virus production, balanced levels of SRSF1 are indispensable for efficient replication.


title: Litter characteristics and helping context during early life shape the responsiveness of the stress axis in a wild cooperative breeder

abstract: Stress responses have evolved to quickly and appropriately deal with environmental stressors in order to secure or restore homeostasis. Since the regulation of stress hormones plays a key adaptive role, the regulatory processes controlling stress hormones levels may be under high selective pressure. The social environment during early life (parents and litter characteristics) strongly affects ontogeny of the hypothalamic-pituitary-adrenal (HPA) axis. In cooperative breeders, offspring are also confronted with helpers but whether and how variation in the helping context can affect HPA axis responsiveness of offspring remains unanswered. Combining dexamethasone suppression and adrenocorticotropic hormone stimulation tests, we investigated the link between the social environment and the characteristics of the HPA axis at the early stages of life in wild Alpine marmots. We show that when raised in the presence of helpers, marmot pups exhibit a greater capacity not only to mount, but also to turn off a stress response. The capacity to mount a stress response was also higher as the pups were raised in large litters. Determining impacts of such social modulation of the HPA axis functioning on individual fitness would make an important contribution to our understanding of the evolution of cooperative breeding.

category: evolutionary biology


title: Illuminating the transposon insertion landscape in plants using Cas9-targeted Nanopore sequencing and a novel pipeline

abstract: Transposable elements (TEs) are a major source of genomic novelty, contributing to plant adaptation, speciation and new cultivar production. The often large, complex genomes of plants make identifying TE insertions from short reads challenging, while whole-genome sequencing remains expensive. In addition, rare somatic insertions reflecting mobilome dynamic are difficult to track using short reads. To overcome these challenges, we applied the Cas9-targeted Nanopore sequencing (CANS) approach and a novel pipeline, NanoCasTE, to trace both genetically inherited and somatic TE insertions in plants. We performed CANS of EVD retrotransposon in wild-type Arabidopsis thaliana and obtained up to 40x coverage of the targets after only a few hours of sequencing on a MinION sequencer. To estimate the ability to detect new TE and T-DNA insertions, we exploited the A. thaliana ddm1 mutant, which has elevated TE activity. Using CANS, we detect the hemizygous T-DNA insertion site and genetically inherited EVD insertions. CANS also detected >800 somatic TE insertions allowing monitoring of TE insertions in real time. By this, we built a high-density physical map of EVD insertion preferences in the A. thaliana ddm1 and found that EVD insertions are biased toward pericentromeric regions in ddm1 that differ from wild-type A. thaliana ecotypes suggesting a pivotal role of DNA methylation in the shaping of EVD insertion preferences. Using CANS, we were able to detect transposition events of ONSEN transposon family that occurred in response to heat stress and found hotspots of ONSEN insertions. Our results indicate that CANS and NanoCasTE are effective tools for the detection of TEIs in real time, opening an avenue for obtaining a deeper understanding of mobilome organization in response to stress and in different genetic backgrounds. The ability of CANS to detect genetically inherited TEIs and T-DNA integration sites make it useful for screening insertion mutants and transgenic plants.

category: genomics


title: Illuminating the plant transposon insertion landscape in real time using Cas9-targeted Nanopore sequencing and a novel pipeline


title: Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors.

abstract:
Beta human papillomavirus ({beta}-HPV) are hypothesized to make DNA damage more mutagenic and potentially more carcinogenic. Double strand breaks in DNA (DSBs) are the most deleterious DNA lesion. They are typically repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs after DNA replication while NHEJ can occur at any point in the cell cycle. They are not thought to occur in the same cell at the same time. By destabilizing p300, {beta}-HPV type 8 protein E6 ({beta}-HPV8 E6) attenuates both repair pathways. However, {beta}-HPV8 E6 delays rather than abrogates DSB repair. Thus, {beta}-HPV8 E6 may cause DSBs to be repaired through a more mutagenic process. To evaluate this, immunofluorescence microscopy was used to detect colocalization, formation, and resolution of DSB repair complexes following damage. Flow cytometry and immunofluorescence microscopy were used to determine the cell cycle distribution of repair complexes. The resulting data show that {beta}-HPV8 E6 causes HR factors (RPA70 and RAD51) to colocalize with a persistent NHEJ factor (pDNA-PKcs). RPA70 complexes gave way to RAD51 complexes as in canonical HR, but RAD51 and pDNA-PKcs colocalization did not resolve within 32 hours of damage. The persistent RAD51 foci occur in G1 phase, consistent with recruitment after NHEJ fails. Chemical inhibition of p300, p300 knockout cells, and an {beta}-HPV8 E6 mutant demonstrate that these phenotypes are the result of {beta}-HPV8 E6-meidated p300 destabilization. Mutations associated with DSB repair were identified using next generation sequencing after a CAS9-induced DSB. {beta}-HPV8 E6 increases the frequency of mutations (>15 fold) and deletions (>20 fold) associated with DSB repair. These data suggest that {beta}-HPV8 E6 causes abnormal DSB repair where both NHEJ and HR occur at the same lesion and that his leads to deletions as the single stranded DNA produced during HR is removed by NHEJ.

Author SummaryOur previous work shows that a master transcription regulator, p300, is required for two major DNA double strand break (DSB) repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). By degrading p300, beta genus Human Papillomavirus 8 protein E6 ({beta}-HPV8 E6) hinders DNA-PKcs activity, which is a key factor of NHEJ. {beta}-HPV8 E6 also stalls HR via p300 degradation resulting in the persistence of a core factor, RAD51. NHEJ and HR are known competitive to each other and only one pathway can be initiated to repair a DSB. Particularly, NHEJ tends to be used in G1 phase and HR occurs in S/G2 phase. Here, we show that {beta}-HPV8 E6 allows NHEJ and HR to occur at the same break site. This is expected to be mutagenic because HR generates overhangs while NHEJ removes them. Further, we show that {beta}-HPV8 E6 allows HR to occur in G1, which cannot be finished due to the lack of homologous templates. Finally, our sequencing results show that {beta}-HPV8 E6 significantly increases genomic variations including deletions and insertions following CAS9 induced DSB. This study supports the hypothesis that {beta}-HPV8 infections increases genomic instability.


title: Structural Basis for Cytoplasmic Dynein-1 Regulation by Lis1

abstract: The lissencephaly 1 gene, LIS1, is mutated in patients with the neurodevelopmental disease lissencephaly. The Lis1 protein is conserved from fungi to mammals and is a key regulator of cytoplasmic dynein-1, the major minus-end-directed microtubule motor in many eukaryotes. Lis1 is the only dynein regulator that binds directly to dyneins motor domain, and by doing so alters dyneins mechanochemistry. Lis1 is required for the formation of fully active dynein complexes, which also contain essential cofactors: dynactin and an activating adaptor. Here, we report the first high-resolution structure of the yeast dynein-Lis1 complex. Our 3.1[A] structure reveals, in molecular detail, the major contacts between dynein and Lis1 and between Lis1s {beta}-propellers. Structure-guided mutations in Lis1 and dynein show that these contacts are required for Lis1s ability to form fully active human dynein complexes and to regulate yeast dyneins mechanochemistry and in vivo function. We present a model for the conserved role of Lis1 in regulating dynein from yeast to humans.


title: Analysis of the Mechanosensor Channel Functionality of TACAN

abstract: Mechanosensitive ion channels mediate transmembrane ion currents activated by mechanical forces. A mechanosensitive ion channel called TACAN was recently reported. We began to study TACAN with the intent to understand how it senses mechanical forces and functions as an ion channel. Using cellular patch-recording methods we failed to identify mechanosensitive ion channel activity. Using membrane reconstitution methods we found that TACAN, at high protein concentrations, produces non-selective, heterogeneous conduction levels that are not mechanosensitive and are most consistent with disruptions of the lipid bilayer. We determined the structure of TACAN using single particle cryo-EM and observe that it forms a symmetrical dimeric transmembrane protein. Each protomer contains an intracellular-facing cleft with a coenzyme-A co-factor, confirmed by mass spectrometry. The TACAN protomers are related in 3-dimensional structure to a fatty acid elongase, ELOVL. Whilst its physiological function remains unclear, we anticipate that TACAN is not a mechanosensitive ion channel.

category: biophysics


title: Developmental Regulation of Homeostatic Plasticity in Mouse Primary Visual Cortex

abstract:
Homeostatic plasticity maintains network stability by adjusting excitation, inhibition, or the intrinsic excitability of neurons, but the developmental regulation and coordination of these distinct forms of homeostatic plasticity remains poorly understood. A major contributor to this information gap is the lack of a uniform paradigm for chronically manipulating activity at different developmental stages. To overcome this limitation, we utilized Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to directly suppress neuronal activity in layer (L) 2/3 of mouse primary visual cortex (V1) at two important developmental timepoints: the classic visual system critical period (CP, P24-29), and adulthood (P45-55). We show that 24 hours of DREADD-mediated activity suppression simultaneously induces excitatory synaptic scaling up and intrinsic homeostatic plasticity in L2/3 pyramidal neurons during the CP, consistent with previous observations using prolonged visual deprivation. Importantly, manipulations known to block these forms of homeostatic plasticity when induced pharmacologically or via visual deprivation also prevented DREADD-induced homeostatic plasticity. We next used the same paradigm to suppress activity in adult animals. Surprisingly, while excitatory synaptic scaling persisted into adulthood, intrinsic homeostatic plasticity was completely absent. Finally, we found that homeostatic changes in quantal inhibitory input onto L2/3 pyramidal neurons were absent during the CP but present in adults. Thus, the same population of neurons can express distinct sets of homeostatic plasticity mechanisms at different development stages. Our findings suggest that homeostatic forms of plasticity can be recruited in a modular manner according to the evolving needs of a developing neural circuit.

Significance statementDeveloping brain circuits are subject to dramatic changes in inputs that could destabilize activity if left uncompensated. This compensation is achieved through a set of homeostatic plasticity mechanisms that provide slow, negative feedback adjustments to excitability. Given that circuits are subject to very different destabilizing forces during distinct developmental stages, the forms of homeostatic plasticity present in the network must be tuned to these evolving needs. Here we developed a method to induce comparable homeostatic compensation during distinct developmental windows, and found that neurons in the juvenile and mature brain engage strikingly different forms of homeostatic plasticity. Thus, homeostatic mechanisms can be recruited in a modular manner according to the developmental needs of the circuit.


title: Chemical Profiling of Antimicrobial Metabolites from Halophilic Actinomycete Nocardiopsis_sp. Al-H10-1 (KF384482) Isolated from Alang, Gulf of Khambhat, India

abstract: The overuse of antibiotics has resulted in the development of drug resistant, a major problem in disease curing processes i.e. development of drug resistance. The World Health Organization (WHO) released its first list of the most concerning pathogens for human health in 2017 which suggested that there are total 12 bacterial families which have developed multiple drug resistance and for which novel antibiotics are required immediately (WHO 2017). There is a requirement to explore some novel compounds to overcome this issue. Thus our study aimed at exploration of marine actinomycetes as a valuable resource for novel products with antimicrobial properties. The halophilic actinomycete Nocardiopsis_sp. Al-H10-1 (KF384482) was isolated from saline water (20 m away from shore) of Alang coast (Gulf of Khambhat), Bhavnagar, Gujarat, India. The isolate Al-H10-1 was identified as Nocardiopsis sp. through rigorous morphological and cultural characteristics; the species was confirmed through 16s rRNA phylogenetic analysis. The antimicrobial potential of Nocardiopsis sp. Al-H10-1 was assessed against a range of Gram-positive and Gram-negative bacteria as well as three fungi, there it demonstrated antimicrobial activity against four Gram negative bacteria and one Gram positive bacteria. Further active antimicrobial compounds present in ethyl acetate extract was identified using Gas Chromatography-Mass Spectroscopy (GC-MS). GC-MS analysis showed the presence of 17 compounds which included antimicrobial compounds like 2, 4-bis (1, 1-dimethylethyl)-Phenol, Dibutyl phthalate as well as various types of alkanes and their derivatives.


title: Sustained replication of synthetic canine distemper virus defective genomes in vitro and in vivo

abstract:
Defective interfering (DI) genomes restrict viral replication and induce type-I interferon. Since DI genomes have been proposed as vaccine adjuvants or therapeutic antiviral agents, it is important to understand their generation, delineate their mechanism of action, develop robust production capacities, assess their safety and in vivo longevity and determine their long-term effects. To address this, we generated a recombinant (r) canine distemper virus (CDV) from an entirely synthetic molecular clone designed using the genomic sequence from a clinical isolate obtained from a free-ranging raccoon with distemper. rCDV was serially passaged in vitro to identify DI genomes that naturally arise during rCDV replication. Defective genomes were identified by Sanger and next-generation sequencing techniques and predominant genomes were synthetically generated and cloned into T7-driven plasmids. Fully encapsidated DI particles (DIPs) were then generated using a rationally attenuated rCDV as a producer virus to drive DI genome replication. We demonstrate these DIPs interfere with rCDV replication in a dose-dependent manner in vitro. Finally, we show sustained replication of a fluorescent DIP in experimentally infected ferrets over a period of 14 days. Most importantly, DIPs were isolated from the lymphoid tissues which are a major site of CDV replication. Our established pipeline for detection, generation and assaying DIPs is transferable to highly pathogenic paramyxoviruses and will allow qualitative and quantitative assessment of the therapeutic effects of DIP administration on disease outcome.

ImportanceDefective interfering (DI) genomes have long been considered inconvenient artifacts that suppressed viral replication in vitro. However, advances in sequencing technologies have led to DI genomes being identified in clinical samples, implicating them in disease progression and outcome. It has been suggested that DI genomes could be harnessed therapeutically. Negative strand RNA virus research has provided a rich pool of natural DI genomes over many years and they are probably the best understood in vitro. Here, we demonstrate identification, synthesis, production and experimental inoculation of novel CDV DI genomes in highly susceptible ferrets. These results provide important evidence that rationally designed and packaged DI genomes can survive the course of a wild-type virus infection.


title: A secondary metabolite drives intraspecies antagonism in a gut symbiont that is inhibited by peptidoglycan acetylation

abstract: The mammalian microbiome encodes numerous secondary metabolite biosynthetic gene clusters, yet their role in microbe-microbe interactions is unclear. Here, we characterized two polyketide synthase gene clusters (fun and pks) in the gut symbiont Limosilactobacillus reuteri. The pks, but not the fun cluster, encodes antimicrobial activity. Forty-one out of 51 L. reuteri strains tested are sensitive to Pks products, which was independent of strains host origin. The sensitivity to Pks was also established in intraspecies competition experiments in gnotobiotic mice. Comparative genome analyses between Pks-resistant and sensitive strains identified an acyltransferase gene (act) that is unique to Pks-resistant strains. Subsequent cell wall analysis of the wild-type and the act mutant strains showed that Act acetylates cell wall components. The pks mutants lost their competitive advantage and act mutants lost their Pks resistance in vivo. Thus, our findings provide insight into how closely related gut symbionts can compete and co- exist in the gastrointestinal tract.


title: A secondary metabolite drives intraspecies antagonism in a gut symbiont that is inhibited by cell wall acetylation


title: Photosynthesis from stolen chloroplasts increases sea slug reproductive fitness

abstract:
Some sea slugs are able to steal functional chloroplasts (kleptoplasts) from their algal food sources, but the role and relevance of photosynthesis to the animal host remain controversial. While some researchers claim that kleptoplasts are slowly digestible  snacks, others advocate that they enhance the overall fitness of sea slugs much more profoundly. Our analysis show light-dependent incorporation of 13C and 15N in the albumen gland and gonadal follicles of the sea slug Elysia timida, representing translocation of photosynthates to kleptoplast-free reproductive organs. Long-chain polyunsaturated fatty acids with reported roles in reproduction were produced in the sea slug cells using labelled precursors translocated from the kleptoplasts. Finally, we report reduced fecundity of E. timida by suppressing kleptoplast photosynthesis. The present study provides the first thorough experimental evidence that photosynthesis enhances the reproductive fitness of kleptoplast-bearing sea slugs, confirming the biological relevance of this remarkable association between a metazoan and an algal-derived organelle.

TeaserSea slugs incorporate functional chloroplasts from algae and use products of photosynthesis to maximize reproductive output.

category: ecology


title: CRISPRa screen on a genetic risk locus shared by multiple autoimmune diseases identifies a dysfunctional enhancer that affects IRF8 expression through cooperative lncRNA and DNA methylation machinery

abstract: Dysregulated transcription factors represent a major class of drug targets that mediate the abnormal expression of many critical genes involved in SLE and other autoimmune diseases. Although strong evidence suggests that natural human genetic variation affects basal and inducible gene expression, it is still a considerable challenge to establish a biological link between GWAS-identified non-coding genetic risk variants and their regulated gene targets. Here, we combine genetic data, epigenomic data, and CRISPR activation (CRISPRa) assays to screen for functional variants regulating IRF8 expression. Using CRISPR-mediated deletion and 3D chromatin structure analysis, we demonstrate that the locus containing rs2280381 is a cell-type-specific distal enhancer for IRF8 that spatially interacts with the IRF8 promoter. Further, rs2280381 mediates IRF8 expression through enhancer RNA AC092723.1, which recruits TET1 to the IRF8 promoter to modulate IRF8 expression by affecting methylation levels. The alleles of rs2280381 modulate PU.1 binding and chromatin state to differentially regulate AC092723.1 and IRF8 expression. Our work illustrates a strategy to define the functional genetic variants modulating transcription factor gene expression levels and identifies the biologic mechanism by which autoimmune diseases risk genetic variants contribute to the pathogenesis of disease.


title: Enviromic-based Kernels Optimize Resource Allocation with Multi-trait Multi-environment Genomic Prediction for Tropical Maize

abstract: Genomic prediction (GP) success is directly dependent on establishing a training population, where incorporating envirotyping data and correlated traits may increase the GP accuracy. Therefore, we aimed to design optimized training sets for multi-trait for multi-environment trials (MTMET). For that, we evaluated the predictive ability of five GP models using the genomic best linear unbiased predictor model (GBLUP) with additive + dominance effects (M1) as the baseline and then adding genotype by environment interaction (G x E) (M2), enviromic data (W) (M3), W+G x E (M4), and finally W+G x W (M5), where G x W denotes the genotype by enviromic interaction. Moreover, we considered single-trait multi-environment trials (STMET) and MTMET for three traits: grain yield (GY), plant height (PH), and ear height (EH), with two datasets and two cross-validation schemes. Afterward, we built two kernels for genotype by environment by trait interaction (GET) and genotype by enviromic by trait interaction (GWT) to apply genetic algorithms to select genotype:environment:trait combinations that represent 98% of the variation of the whole dataset and composed the optimized training set (OTS). Using OTS based on enviromic data, it was possible to increase the response to selection per amount invested by 142%. Consequently, our results suggested that genetic algorithms of optimization associated with genomic and enviromic data efficiently design optimized training sets for genomic prediction and improve the genetic gains per dollar invested.