Antibiotic inhibition of the Plasmodium apicoplast decreases haemoglobin degradation and antagonises dihydroartemisinin action The World Health Organisation (WHO) recommends artemisinin (ART) combinations for treatment of uncomplicated Plasmodium falciparum malaria. Understanding the interaction between co-administered drugs within combination therapies is clinically important to prevent unintended consequences. The WHO guidelines recommend second line treatments that combine artesunate with tetracycline, doxycycline, or clindamycin--antibiotics that target the Plasmodium relict plastid, the apicoplast. In addition, antibiotics can be used simultaneously against other infectious diseases, leading to their inadvertent combination with ARTs. One consequence of apicoplast inhibition is a perturbation to haemoglobin uptake and trafficking--a pathway required for activation of ART derivatives. Here, we show that apicoplast-targeting antibiotics reduce the abundance of the catalyst of ART activation (free haem) in P. falciparum, likely through diminished haemoglobin digestion. We demonstrate antagonism between ART and these antibiotics, suggesting that apicoplast inhibitors reduce ART activation. These data have potential clinical implications due to the reliance on--and widespread use of--both ARTs and these antibiotics in malaria endemic regions.
A Facile Alternative Sustainable Process for the Extraction and Purification of Microbial Melanin Conventional techniques for the extraction of the melanin encounter several disadvantages such as poor extraction efficiency, excessive use of acid and alkali, and lengthy time duration. To extract melanin in a more sustainable manner, herein suitability of an aqueous solution of quaternary ammonium hydroxide known as tetrabutylammonium hydroxide (40% w/w TBAOH in water) to extract and purify the pigment from the endophyte Streptomyces hyderabadensis 7VPT5-5R was investigated. The solvent could extract 5.54{+/-}0.03 g/L of pure melanin against 3.31{+/-} 0.02 g/L of pure melanin by conventional method from bacterial culture resulting 66% increase in the yield of the pigment. The quaternary ammonium based solvent was further recycled for 5 consecutive cycles and production of melanin with yield ranging from 5.54{+/-}0.03 g/L to 5.47{+/-} 0.02 g/L was obtained. The yield of the pigment was 38-40% higher in the recycled solvent in comparison to the melanin yield extracted by conventional method. Considering the efficiency of the solvent for effective extraction and purification of melanin and its recyclability potential, it can be considered as a sustainable solvent for the extraction of the pigments from bacterial culture. Furthermore, the developed method for extraction of melanin from microorganisms as a sustainable source would help in reducing the cost of melanin leading to its efficient utilization in diverse fields.
Catchet-MS identifies IKZF1-targeting Thalidomide analogues as novel HIV-1 latency reversal agents A major pharmacological strategy toward HIV cure aims to reverse latency in infected cells as a first step leading to their elimination. While the unbiased identification of molecular targets physically associated with the latent HIV-1 provirus would be highly valuable to unravel the molecular determinants of HIV-1 transcriptional repression and latency reversal, due to technical limitations, this has not been possible. Here we use a dCas9 targeted chromatin and histone enrichment strategy coupled to mass spectrometry (Catchet-MS) to describe the protein composition of the latent and activated HIV-1 5LTR. Catchet-MS identified known and novel latent 5LTR-associated host factors. Among these, IKZF1 is a novel HIV-1 transcriptional repressor, required for Polycomb Repressive Complex 2 recruitment to the LTR. We find the clinically advanced thalidomide analogue iberdomide, and the FDA approved analogues lenalidomide and pomalidomide, to be novel LRAs that, by targeting IKZF1 for degradation, reverse HIV-1 latency in CD4+T-cells isolated from virally suppressed people living with HIV-1.

One Sentence SummarydCas9 targeted chromatin and histone enrichment for mass spectrometry (Catchet-MS) led to the identification of IKZF1-targeting thalidomide analogues as novel HIV-1 latency reversal agents Development and Characterization of PAR-Trackers: New Tools for Detecting Poly(ADP-ribose) In Vitro and In Vivo AO_SCPLOWBSTRACTC_SCPLOWADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally-occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as "readers" for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe and characterize a set of poly(ADP-ribose) (PAR) Trackers (PAR-Ts) - optimized dimerization-dependent or split-protein reassembly PAR sensors in which a naturally occurring PAR binding domain, WWE, was fused to both halves of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc), respectively. We demonstrate that these new tools allow the detection and quantification of PAR levels in extracts, living cells, and living tissues with greater sensitivity, as well as temporal and spatial precision. Importantly, these sensors detect changes in cellular ADPR levels in response to physiological cues (e.g., hormone-dependent induction of adipogenesis without DNA damage), as well as xenograft tumor tissues in living mice. Our results indicate that PAR Trackers have broad utility for detecting ADPR in many different experimental and biological systems. Standardized genome-wide function prediction enables comparative functional genomics: a new application area for Gene Ontologies in plants BackgroundGenome-wide gene function annotations are useful for hypothesis generation and for prioritizing candidate genes potentially responsible for phenotypes of interest. We functionally annotated the genes of 18 crop plant genomes across 14 species using the GOMAP pipeline.

ResultsBy comparison to existing GO annotation datasets, GOMAP-generated datasets cover more genes, contain more GO terms, and produce datasets similar in quality (based on precision and recall metrics using existing gold standards as the basis for comparison). From there, we sought to determine whether the datasets across multiple species could be used together to carry out comparative functional genomics analyses in plants. To test the idea and as a proof of concept, we created dendrograms of functional relatedness based on terms assigned for all 18 genomes. These dendrograms were compared to well-established species-level evolutionary phylogenies to determine whether trees derived were in agreement with known evolutionary relationships, which they largely are. Where discrepancies were observed, we determined branch support based on jack-knifing then removed individual annotation sets by genome to identify the annotation sets causing unexpected relationships.

ConclusionsGOMAP-derived functional annotations used together across multiple species generally retain sufficient biological signal to recover known phylogenetic relationships based on genome-wide functional similarities, indicating that comparative functional genomics across species based on GO data hold promise for generating novel hypotheses about comparative gene function and traits. Cellular Fitness Phenotypes of Cancer Target Genes from Oncobiology to Cancer Therapeutics To define the growing significance of cellular targets of cancer drugs, we examined the fitness dependency of cellular targets or effectors of cancer drug targets across human cancer cells from 19 cancer types. We observed that the deletion of 35 out of 47 cellular mediators or targets of oncology drugs did not result in the expected loss of cell fitness in appropriate cancer types for which drugs targeting or utilizing these molecules were approved. Additionally, our analysis recognized 43 cellular targets as fitness genes in several cancer types in which these drugs were not approved, and thus, providing clues repurposing approved oncology drugs in cancer types. For example, we found the widespread upregulation and fitness dependency of the components of the mevalonate and purine biosynthesis pathways (currently targeted by bisphosphonates, statins, and pemetrexed in certain cancers) and an association between the overexpression of these targets and reduction in the overall survival duration of patients with breast and other hard-to-treat cancers, for which such drugs are not approved. In brief, the present analysis raised cautions about off-target and undesirable effects of certain oncology drugs in a subset of cancers where the in-tended cellular effectors of drug might not be fitness genes and offers a potential rationale for repurposing certain approved oncology drugs for targeted therapeutics in additional cancer types. Pulsatile contractions and pattern formation in excitable actomyosin cortex The actin cortex is an active adaptive material, embedded with complex regulatory networks that can sense, generate, and transmit mechanical forces. The cortex exhibits a wide range of dynamic behaviours, from generating pulsatory contractions and travelling waves to forming organised structures. Despite the progress in characterising the biochemical and mechanical components of the actin cortex, the emergent dynamics of this mechanochemical system is poorly understood. Here we develop a reaction-diffusion model for the RhoA signalling network, the upstream regulator for actomyosin assembly and contractility, coupled to an active actomyosin gel, to investigate how the interplay between chemical signalling and mechanical forces regulate stresses and patterns in the cortex. We demonstrate that mechanochemical feedback in the cortex acts to destabilise homogeneous states and robustly generate pulsatile contractions. By tuning active stress in the system, we show that the cortex can generate propagating contraction pulses, form network structures, or exhibit topological turbulence. Pre- and post-oviposition behavioural strategies to protect eggs against extreme winter cold in an insect with maternal care AO_SCPLOWBSTRACTC_SCPLOWDepositing eggs in an area with adequate temperature is often crucial for mothers and their offspring, as the eggs are immobile and therefore cannot avoid exposure to sub-optimal temperatures. However, the importance of temperature on oviposition site selection is less clear when mothers can avoid these potential adverse effects by both moving their eggs after oviposition and providing other forms of egg care. In this study, we addressed this question in the European earwig, an insect in which mothers care for the eggs during several months in winter, frequently moving them during this period. We set up 60 females from two random natural populations (as this species often exhibits population-specific life-history traits and behaviours) under controlled thermal gradients, and recorded the temperature at which they built their nests, tested whether they moved their eggs after an experimental temperature change, and measured the effects on egg development and hatching rate. Our results demonstrate that females indeed select oviposition sites according to temperature, and can move their eggs to reach warmer temperatures. We also show that these warmer temperatures are necessary to ensure egg hatching. Although this set of behavioural thermoregulations is present in the two tested populations, we found a population-specific modality of expression. These included the range of temperatures explored before oviposition, temperature selected at oviposition and dynamics of egg transport following a temperature change. Overall, our study sheds light on a new post-oviposition strategy in female insects that overwinter with their eggs for coping with temperature changes. More generally, it also reveals that egg care and/or egg transport do not prevent behavioural thermoregulation via oviposition site selection and highlights the diversity of behaviours that insects can adopt to enhance their tolerance to global climate change. Modular evolution of secretion systems and virulence plasmids in a bacterial species complex BackgroundMany bacterial taxa are species complexes and uncertainties regarding the organization of their genetic diversity challenge research efforts. We utilized Agrobacterium tumefaciens, a taxon known for its phytopathogenicity and applications in transformation, as a study system and devised strategies for investigating genome diversity and evolution of species complexes.

ResultsWe utilized 35 genome assemblies to achieve a comprehensive and balanced sampling of A. tumefaciens. Our confident inference of gene content and core-genome phylogeny supported a quantitative guideline for delineating 12 species and allowed for robust investigations of genes critical in fitness and ecology. For the type VI secretion system (T6SS) involved in interbacterial competition and thought to be conserved, we detected multiple losses and one horizontal gene transfer. For the tumor-inducing plasmids (pTi) and pTi-encoded type IV secretion system (T4SS) that are essential for agrobacterial phytopathogenicity, we uncovered novel diversity and hypothesized their involvement in shaping this species complex. Intriguingly, for both T6SS and T4SS, genes encoding structural components are highly conserved, whereas extensive diversity exists for genes encoding effectors and other proteins.

ConclusionsWe demonstrated that the combination of a phylogeny-guided sampling scheme and an emphasis on high-quality assemblies provides a cost-effective approach for robust analysis in evolutionary genomics. Our strategies for multi-level investigations at scales that range from whole-genomes to intragenic domains and phylogenetic depths of between- and within-species are applicable to other bacteria. Finally, modularity observed in the molecular evolution of genes and domains is useful for inferring functional constraints and informing experimental works. Optical regulation of endogenous RhoA reveals selection of cellular responses by signal amplitude How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, the small GTPase RhoA regulates both focal adhesion (FA) growth or disassembly, but whether RhoA serves as a switch selecting between cellular outcomes, or if outcomes are simply modulated by additional factors in the cell, is not clear. Here, we develop a photoswitchable RhoA guanine exchange factor, psRhoGEF, to precisely control endogenous RhoA activity. We also develop a FRET-based biosensor to allow visualization of RhoA activity together with psRhoGEF control. Using these new optical tools, we discover that low levels of RhoA activation preferentially induce FA disassembly in a Src-dependent manner, while high levels induce both FA growth and disassembly in a ROCK-dependent manner. Thus, rheostatic control of RhoA activation with photoswitchable RhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Analysis of science journalism reveals gender and regional disparities in coverage Science journalism is a critical way in which the public can remain informed and benefit from new scientific findings. Such journalism also shapes the publics view of the current state of scientific findings and legitimizes experts. Those covering science can only cite and quote a limited number of sources. Sources may be identified by the journalists research or by recommendations by other scientists. In both cases, biases may influence who is identified and ultimately included as an expert. To analyze possible biases in science journalism, we analyzed 22,001 non-research articles published by Nature. We chose to analyze Nature non-research articles since its research articles provide a natural comparator. Our analysis considered two possible sources of disparity: gender and name origin. To explore these sources of disparity, we extracted cited authors names as well as extracted names of quoted speakers. While citations and quotations within a piece do not reflect the entire information-gathering process, they can provide insight into the demographics of visible sources. We then used the extracted names to predict gender and name origin of the cited authors and speakers.

In order to appropriately quantify the level of difference, we must identify a suitable reference set for comparison. We chose first and last authors within primary research articles in Nature and a subset of Springer Nature articles in the same time period as our comparator. In our analysis, we found a skew towards male quotation in Nature science journalism-related articles. However, quotation is trending toward equal representation at a faster rate than first and last authorship in academic publishing. Interestingly, we found that the gender disparity in Nature quotes was column-dependent, with the "Career Features" column reaching gender parity. Our name origin analysis found a significant over-representation of names with predicted Celtic/English origin and under-representation of names with a predicted East Asian origin. This finding was observed both in extracted quotes and journal citations, but dampened in citations. Impact of circadian time of dosing on cardiomyocyte-autonomous effects of glucocorticoids Mitochondrial capacity is critical to adapt the high energy demand of the heart to circadian oscillations and diseased states. Glucocorticoids regulate the circadian cycle of energy metabolism, but little is known about how circadian timing of exogenous glucocorticoid dosing directly regulates heart metabolism through cardiomyocyte-autonomous mechanisms. While chronic oncedaily intake of glucocorticoids promotes metabolic stress and heart failure, we recently discovered that intermittent once-weekly dosing of exogenous glucocorticoids promoted muscle metabolism in normal and obese skeletal muscle. However, the effects of glucocorticoid intermittence on heart metabolism and heart failure remain unknown. Here we investigated the extent to which circadian time of dosing regulates the effects of the glucocorticoid prednisone in heart metabolism and function in conditions of single pulse or chronic intermittent dosing. In WT mice, we found that prednisone improved cardiac content of NAD+ and ATP with light-phase dosing (ZT0), while the effects were blocked by dark-phase dosing (ZT12). The drug effects on mitochondrial function were cardiomyocyte-autonomous, as shown by inducible cardiomyocyte-restricted glucocorticoid receptor (GR) ablation, and depended on an intact cardiomyocyte clock, as shown by inducible cardiomyocyte-restricted ablation of Brain and Muscle ARNT-like 1 (BMAL1). Conjugating time-of-dosing with chronic intermittence, we found that once-weekly prednisone improved metabolism and function in heart after myocardial injury dependent on circadian time of intake, i.e. with lightphase but not dark-phase dosing. Our study identifies cardiac-autonomous mechanisms through which circadian-specific intermittent dosing reconverts glucocorticoid drugs to metabolic boosters for the heart. Global Constraints within the Developmental Program of the Drosophila Wing Organismal development is a complex process, involving a vast number of molecular constituents interacting on multiple spatio-temporal scales in the formation of intricate body structures. Despite this complexity, development is remarkably reproducible and displays tolerance to both genetic and environmental perturbations. This robust-ness implies the existence of hidden simplicities in developmental programs. Here, using the Drosophila wing as a model system, we develop a new quantitative strategy that enables a robust description of biologically salient phenotypic variation. Analyzing natural phenotypic variation across a highly outbred population, and variation generated by weak perturbations in genetic and environmental conditions, we observe a highly constrained set of wing phenotypes. Remarkably, the phenotypic variants can be described by a single integrated mode that corresponds to a non-intuitive combination of structural variations across the wing. This work demonstrates the presence of constraints that funnel environmental inputs and genetic variation into phenotypes stretched along a single axis in morphological space. Our results provide quantitative insights into the nature of robustness in complex forms while yet accommodating the potential for evolutionary variations. Methodologically, we introduce a general strategy for finding such invariances in other developmental contexts. Performance of three freely available methods for extracting white matter hyperintensities: FreeSurfer, UBO Detector and BIANCA White matter hyperintensities (WMH) of presumed vascular origin are frequently found in MRIs of healthy older adults. WMH are also associated with aging and cognitive decline. Here, we compared and validated three freely available algorithms for WMH extraction: FreeSurfer, UBO Detector, and BIANCA (Brain Intensity AbNormality Classification Algorithm) using a longitudinal dataset comprising MRI data of cognitively healthy older adults (baseline N = 231, age range 64-87 years). As reference we manually segmented WMH in T1w, 3D FLAIR, 2D FLAIR images. These manual segmentations were then used to assess the segmentation accuracy of the different automated algorithms. Further, we assessed the relationships of WMH volume estimates provided by the algorithms with Fazekas scores and age. FreeSurfer underestimated the WMH volumes and scored worst in Dice Similarity Coefficient (DSC = 0.434) but its WMH volumes strongly correlated with the Fazekas scores (rs = 0.73). BIANCA accomplished the highest DCS (0.602) with 3D FLAIR images. However, the relations with the Fazekas scores were only moderate, especially in the 2D FLAIR images (rs = 0.41), and many outlier WMH volumes were detected when exploring within-person trajectories (2D FLAIR: ~30%). UBO Detector performed similarly to BIANCA in DSC with both modalities and reached the best DSC in 2D FLAIR (0.531) without requiring a tailored training dataset. In addition, it achieved very high associations with the Fazekas scores (2D FLAIR: rs = 0.80).

In summary, our results emphasize the importance of carefully contemplating the choice of the WMH segmentation algorithm and MR-modality. Optogenetic dissection of actomyosin-dependent mechanics underlying tissue fluidity Rapid epithelial tissue flows are essential to building and shaping developing embryos. However, it is not well understood how the mechanical properties of tissues and the forces driving them to flow are jointly regulated to accommodate rapid tissue remodeling. To dissect the roles of actomyosin in the mechanics of epithelial tissue flows, here we use two optogenetic tools, optoGEF and optoGAP, to manipulate Rho/Rho-kinase signaling and actomyosin contractility in the germband epithelium, which flows via convergent extension movements during Drosophila body axis elongation. The ability to perturb actomyosin in the tissue allows us to analyze the effects of actomyosin on cell rearrangements, tissue tensions, and tissue mechanical properties. We find that either optogenetic activation or deactivation of Rho1 signaling and actomyosin contractility at the apical surface of the germband disrupts cell rearrangements and tissue-level flows. Rho1 activation leads to poorly oriented rearrangements that are associated with a redistribution of myosin II from the junctional to the medial-apical domain, whereas Rho1 deactivation leads to fewer, slower cell rearrangements that are associated with decreased junctional and medial myosin. By probing mechanical tensions in the tissue using laser ablation and inferring tissue mechanical properties from cell packings, we find that actomyosin influences both the anisotropic forces that drive tissue flow and the mechanical properties of the tissue that resist flow, leading to complex relationships between actomyosin activity and tissue fluidity. Moreover, our results link the subcellular distribution of myosin II to tissue tension and cell packings, revealing how junctional and medial myosin have differential roles in promoting and orienting cell rearrangements during tissue flow. Large Bi-ethnic Study of Plasma Proteome Leads to Comprehensive Mapping of cis-pQTL and Models for Proteome-wide Association Studies Improved understanding of genetic regulation of proteome can facilitate the identification of causal mechanisms for complex traits. We analyzed data on 4,657 plasma proteins from 7,213 European American (EA) and 1,871 African American (AA) individuals from the ARIC study, and further replicated findings on 467 AA individuals from the AASK study. Here we identified 2,004 proteins in EA and 1,618 in AA, with majority overlapping, which showed associations with common variants in cis-regions. Availability of AA samples led to smaller credible sets and significant number of population-specific cis-pQTLs. Elastic-net produced powerful models for protein prediction in both populations. An application of proteome-wide association studies (PWAS) to serum urate and gout, implicated several proteins, including IL1RN, revealing the promise of the drug anakinra to treat acute gout flares. Our study demonstrates the value of large and diverse ancestry study for genetic mechanisms of molecular phenotypes and their relationship with complex traits. Sound localization in world and head-centered space in ferrets The location of sounds can be described in multiple coordinate systems that are defined relative to ourselves, or the world around us. Evidence from neural recordings in animals point towards the existence of both head-centered and world-centered representations of sound location in the brain; however, it is unclear whether such neural representations have perceptual correlates in the sound localization abilities of non-human listeners. Here, we establish novel behavioral tests to determine the coordinate systems in which ferrets can localize sounds. We found that ferrets could learn to discriminate between sound locations that were fixed in either world-centered or head-centered space, across wide variations in sound location in the alternative coordinate system. Using probe sounds to assess broader generalization of spatial hearing, we demonstrated that in both head and world-centered tasks, animals used continuous maps of auditory space to guide behavior. Single trial responses of individual animals were sufficiently informative that we could then model sound localization using speaker position in specific coordinate systems and accurately predict ferrets actions in held-out data. Our results indicate that an animal model in which neurons are known to be tuned to sound location in egocentric and allocentric reference frames can also localize sounds in multiple head and world-centered spaces.

Significance StatementHumans can describe the location of sounds either relative to themselves, or in the world, independent of their momentary position. These different spaces are also represented in the activity of neurons in animals, but its not clear whether non-human listeners also perceive both head and world-centered sound location. Here, we designed two behavioral tasks in which ferrets had to discriminate between two sounds using their position in the world, or relative to the head. Subjects learnt to solve both problems and showed the ability to generalize sound location in each space when presented with infrequent probe sounds. These findings reveal a perceptual correlate of neural sensitivity previously observed in the ferret brain and establish that, like humans, ferrets can access an auditory map of their local environment. CIITA induces expression of MHC-I and MHC-II in transmissible cancers MHC-I and MHC-II molecules are critical components of antigen presentation and T cell immunity to pathogens and cancer. The two monoclonal transmissible devil facial tumours (DFT1, DFT2) exploit MHC-I pathways to overcome immunological anti-tumour and allogeneic barriers. This exploitation underpins the ongoing transmission of DFT cells across the wild Tasmanian devil population. We have previously shown that constitutive expression of NLRC5 can induce stable upregulation of MHC-I on DFT1 and DFT2 cells, but unlike IFNG-treated cells, NLRC5 does not upregulate PDL1. MHC-II expression is crucial for CD4+ T cell activation and is primarily confined to haematopoietic antigen presenting cells. Transcriptomic analysis of DFT1 and DFT2 cell lines showed that several genes of the MHC-I and MHC-II pathways were upregulated in response to constitutive overexpression of the class II transactivator (CIITA) gene. This was further supported by upregulation of MHC-I protein on DFT1 and DFT2 cells, but interestingly MHC-II protein was upregulated only on DFT1 cells. The functional significance of the MHC upregulation on DFT cells was shown using serum from devils with natural or immunotherapy-induced DFT1 regressions; binding of serum IgG was stronger in CIITA-transfected cells than wild type cells, but was less than binding to NLRC5 transfected cells. This new insight into regulation of MHC-I and MHC-II in cells that naturally overcome allogeneic barriers can inform vaccine, immunotherapy, and tissue transplant strategies for human and veterinary medicine. Artificial seawater based long-term culture of colonial ascidians Tunicates are highly diverse marine invertebrate filter-feeders that are vertebrates closest relatives. These organisms, despite a drastically different body plan during their adulthood, have a tissue complexity related to that of vertebrates. Ascidians, which compose most of the Tunicata, are benthic sessile hermaphrodites that reproduce sexually through a motile tadpole larval stage. Over half of the known ascidians species are able to reproduce asexually by budding, typically leading to the formation of colonies where animals, called zooids, are interconnected through an external vascular system. In addition, colonial ascidians are established models for important biological processes including allorecognition, immunobiology, aging, angiogenesis and whole-body regeneration. However, the current paucity in breeding infrastructures limits the study of these animals to coastal regions.

To promote a wider scientific spreading and popularity of colonial ascidians, we have developed a flexible recirculating husbandry setup for their long-term in-lab culture. Our system is inspired both by the flow-through aquariums used by coastal ascidian labs, as well as by the recirculating in-lab systems used for zebrafish research. Our hybrid system thus combines colony breeding, water filtering and food culturing in a semi-automated system where specimens develop on hanging microscopy glass slides. Temperature, light/dark cycles, flow speed and feeding rates can be controlled independently in four different breeding environments to provide room for species-specific optimization as well as for running experiments. This setup is complemented with a quarantine for the acclimatization of wild isolates.

Herein we present our success in breeding Botrylloides diegensis, a species of colonial ascidians, for more than 3 years in recirculating artificial seawater over 600 km away from their natural habitat. We show that colonies adapt well to in-lab culturing provided that a suitable marine microbiome is present, and that a specific strain can be isolated, propagated and efficiently used for research over prolonged periods of time. The flexible and modular structure of our system can be scaled and adapted to the needs of specific species, such as Botryllus schlosseri, as well as of particular laboratory spaces. Overall, we show that Botrylloides diegensis can be proficiently bred in-land and suggest that our results can be extended to other species of colonial ascidians to promote research on these fascinating animals.

HighlightsO_LIFirst artificial seawater based recirculating aquaculture for colonial ascidians
C_LIO_LIOver 3 years of continuous breeding
C_LIO_LISemi-automated setup with minimized maintenance
C_LIO_LIGood biomass production for strain propagation
C_LIO_LI4 different culture conditions for optimized breeding for species of interest
C_LI Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using electron cryo-microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using HDX-MS, we observed dynamic behavior of the major five-fold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

TeaserCryo-EM and HDX-MS analysis of an encapsulin nanocompartment shows that the pores at the five-fold icosahedral vertex of the shell are flexible. Inter-microscope comparability of dental microwear texture data obtained from different optical profilometers. Dental microwear texture analysis has become a well-established approach for dietary inference and reconstruction of mammals and other tetrapods, both extant and extinct. As the amount of available data grows immensely, researchers could benefit from combining data gathered by others to perform meta-analyses. However, different devices used to capture three-dimensional surface scans for DMTA are known to produce variation even when measuring the same surface. Here we compare DMTA data of 36 guinea pigs that received different diets in a controlled feeding experiment, measured on a confocal disc-scanning and a confocal laser-scanning microscope. We are testing different pre-analysis filtering protocols to mitigate differences. We find inter-microscopes and filter-related differences for the majority of analysed 41 DMTA parameters. Certain microscope-specific filter routines resulted in less differences than other pre-analysis protocols. We further identify DMTA parameters which were stable regardless of microscope or pre-analysis treatment. Overall, the results obtained on both microscopes show the same dietary differentiation between guinea pig feeding groups, which supports that DMTA is a suitable method to obtain repeatable, objective dietary inferences. We finally propose a roadmap to enhance data exchange and inter-lab comparability and collaboration in the future. PIN-FORMED1 polarity in the shoot is insensitive to the polarity of neighbouring cells In plants, epidermal cells are planar-polarized along an axis marked by the asymmetric localization patterns of several proteins including PIN-FORMED1 (PIN1), which facilitates the directional efflux of the plant hormone auxin to pattern phyllotaxis (Heisler et al., 2010; Mansfield et al., 2018; Reinhardt et al., 2003). While PIN1 polarity is known to be regulated non-cell autonomously via the MONOPTEROS (MP) transcription factor, how this occurs has not been determined (Bhatia et al., 2016). Here we use mosaic expression of the serine threonine kinase PINOID (PID) to test whether PIN1 polarizes according to the polarity of neighbouring cells. Our findings reveal that PIN1 is insensitive to the polarity of PIN1 in neighbouring cells arguing against auxin flux or extracellular auxin concentrations acting as a polarity cue, in contrast to previous model proposals (Abley et al., 2016; Stoma et al., 2008). Co-infection of Candidatus Piscichlamydia trichopodus (order Chlamydiales) and Henneguya sp (Myxosporea, Myxobolidae) in snakeskin gourami Trichopodus pectoralis (Regan 1910) The present study describes a simultaneous infection of a novel Chlamydia-like organism (CLO) with a Myxozoa parasite, Henneguya sp. in snakeskin gourami Trichopodus pectoralis in Thailand. A new CLO is proposed "Candidatus Piscichlamydia trichopodus" (CPT) based on 16S rRNA phylogenetic analysis. Systemic intracellular CPT infection was confirmed by histological examination, in situ hybridization, PCR assay, and sequencing of 16S rRNA. This novel pathogen belongs to the order Chlamydiales but differs in certain aspects from other species. The histopathological changes associated with CPT infection were different from the typical pathological lesions of epitheliocystis caused by previously known CLO. Unlike other CLO, CPT localized in the connective tissue rather than in the epithelial cells and formed smaller clumps of intracellular bacteria that stained dark blue with hematoxylin. On the other hand, typical myxospores of the genus Henneguya with tails were observed in the gill sections. Infection with Henneguya sp. resulted in extensive destruction of the gill filaments, most likely leading to respiratory distress. Due to the frequency of co-infections and the unavailability of culture methods for CLO and Henneguya sp, it was difficult to determine which pathogens were directly responsible for the associated mortality. However, co-infections may increase the negative impact on the host and the severity of the disease. Given the commercial importance of the snakeskin gourami and its significant aquaculture potential, the findings of this study are important for further studies on disease prevention. Stress alters the activity of Leydig cells dependently on the diurnal time BackgroundThe increasing amount of data points to the circadian timing system as an essential part of processes regulating androgen homeostasis. However, the relationship between stress response, timekeeping-, and steroidogenesis-related systems is unexplored.

ObjectiveThe purpose of the study was to analyze the stress-response of the testosterone-producing Leydig cells depending on the stressful events time.

Materials and methodsThe study was designed to follow the effects of 3-hour immobilization (IMO) applied at different periods during the day. The IMO performed once (1xIMO) or repeated in 10 consecutive days (10xIMO). Principal-component-analysis (PCA) followed the expression study of the clock and steroidogenic-related genes in Leydig cells.

ResultsBoth types of IMO in all investigated periods increased corticosterone and decreased testosterone blood level. Transcriptional analysis revealed different sensitivity to IMO events depending on the circadian time. The majority of steroidogenesis-related genes (Lhcgr, Cyp11a1, Cyp17a1, Hsd3b1/2) were down-regulated in the inactive but unchanged or even up-regulated in the active phase of the day. Both types of IMO potentiated the expression of clock elements Bmal1/BMAL1, Per1/PER1 regardless of the days stage and reduced Reverba in the inactive phase. The PCA confirmed a major shift, for both IMO-types, in the transcription of steroidogenesis and clock genes across the inactive/active phase. Further, the diurnal pattern of the glucocorticoid receptor (Nr3c1/GR) expression in Leydig cells was increased in the inactive phase due to 10xIMO. The observed time-dependent IMO-response of the Leydig cells correlated with different GR engagements.

DiscussionStress- and the circadian-system coordinatively shape Leydig cells physiology, assuming diverse GR engagement as a possible factor in mediating the diurnal effect of stress.

ConclusionThe Leydig cells stress-response depends on the time of the stressful situation, emphasizing the importance of circadian activity in supporting androgen homeostasis and male fertility. Comparative toxicogenomics of glyphosate and Roundup herbicides by mammalian stem cell-based genotoxicity assays and molecular profiling in Sprague-Dawley rats Whether glyphosate-based herbicides (GBHs) are more potent than glyphosate alone at activating cellular mechanisms, which drive carcinogenesis remains controversial. As GBHs are more cytotoxic that glyphosate, we reasoned they may also be more capable of activating carcinogenic pathways. We tested this hypothesis by comparing the effects of glyphosate with Roundup GBHs both in vitro and in vivo. First, glyphosate was compared with representative GBHs namely MON 52276 (EU), MON 76473 (UK) and MON 76207 (USA) using the mammalian stem cell-based ToxTracker system. Here, MON 52276 and MON 76473, but not glyphosate and MON 76207, activated oxidative stress and unfolded protein responses. Second, molecular profiling of liver was performed in female Sprague-Dawley rats exposed to glyphosate or MON 52276 (both at 0.5, 50, 175 mg/kg bw/day glyphosate) for 90 days. MON 52276 but not glyphosate increased hepatic steatosis and necrosis. MON 52276 and glyphosate altered the expression of genes in liver reflecting TP53 activation by DNA damage and circadian rhythm regulation. Genes most affected in liver were similarly altered in kidneys. Small RNA profiling in liver showed decreased amounts of miR-22 and miR-17 from MON 52276 ingestion. Glyphosate decreased mir-30 while miR-10 levels were increased. DNA methylation profiling of liver revealed 5,727 and 4,496 differentially methylated CpG sites between the control and glyphosate and MON 52276 exposed animals respectively. Apurinic/apyrimidinic DNA damage formation in liver was increased with glyphosate exposure. Altogether, our results show that Roundup formulations cause more biological changes linked with carcinogenesis than glyphosate.