title: Concurrent TMS-fMRI to determine adaptive brain changes to virtual lesions interfering with visual processing

abstract:
IntroductionUnderstanding the causal relationship between a focal lesion and brain network (re)organization is a crucial step in order to accurately predict the resulting symptoms of the lesion and implement personalized rehabilitation strategies. Transcranial magnetic stimulation (TMS) can be used to create local and transient neural perturbations, the so-called "virtual lesion" approach. In this study, we tested how a virtual lesion applied to the Early Visual Areas (EVA) or the extrastriate, motion-sensitive, medio-temporal area (hMT+/V5) in different contexts affects behavior and changes neuronal network activity and organisation, assessed with functional magnetic resonance imaging (fMRI).

MethodsWe applied short trains of 10 Hz TMS to healthy participants over the EVA or hMT+/MT. This was done both at rest and at relevant times during a motion discrimination task, while concurrent fMRI was performed. Regional BOLD activity was analysed using a general linear model (GLM) and functional brain networks were assessed using independent component analysis (ICA). Motion direction discrimination and motion awareness were related to the imaging data.

ResultsTMS applied over the EVA and hMT+/V5 induced transient modulation of motion perception and discrimination. Comparing resting versus active states, both TMS sites showed a common suppression of local and remote brain activity at rest while an over-activation of the stimulated areas and related networks were found during the task. More subtly, distinct dynamic and topological TMS-induced networks properties could be revealed depending on the exact visual processing stages TMS was applied at. In particular, brain networks associated with EVA stimulation showed a clear context-dependency and were spatially more restricted than for hMT+/V5 stimulation.

DiscussionThe present findings highlight the possibility of interfering with distinct visual processing stages and the possibility of imaging the local and remote neural correlates of the behavioral impact. They also confirm the complexity of TMS effects on BOLD activity, switching from signal suppression at rest to irrelevant neural noise addition during active visual processing, even differing throughout the time course of information processing. Moreover, the networks analyses suggest that the EVA might be more resilient to focal perturbations by means of a "virtual lesion" than hMT+/V5. As a critical processing center, the EVA may be important for maintaining stability in the visual network, whereas the perturbation of a more specialized region such as hMT+/V5 has greater impact on local and network activity, as well as on behavior. These findings add to the understanding of visual motion processing and especially to the impact and potential mechanism of focal lesions in this system.


title: EEG Responses to Auditory Figure-Ground Perception

abstract: Speech-in-noise difficulty is commonly reported among hearing-impaired individuals. Recent work has established generic behavioural measures of sound segregation and grouping that are related to speech-in-noise processing but do not require language. In this study, we assessed potential clinical electroencephalographic (EEG) measures of central auditory grouping (stochastic figure-ground test) and speech-in-noise perception (speech-in-babble test) with and without relevant tasks. Auditory targets were presented within background noise (16 talker-babble or randomly generated pure-tones) in 50% of the trials and composed either a figure (pure-tone frequency chords repeating over time) or speech (English names). EEG was recorded while participants were presented with the target stimuli (figure or speech) under different attentional states (relevant task or visual-distractor task). EEG time-domain analysis demonstrated enhanced negative responses during detection of both types of auditory targets within the time window 650-850 ms but only figure detection produced significantly enhanced responses under the distracted condition. Further single-channel analysis showed that simple vertex-to-mastoid acquisition defines a very similar response to more complex arrays based on multiple channels. Evoked-potentials to the generic figure-ground task therefore represent a potential clinical measure of grouping relevant to real-world listening that can be assessed irrespective of language knowledge and expertise even without a relevant task.

category: neuroscience


title: An innovative hematopoietic stem cell gene therapy approach benefits CLN1 disease in the mouse model

abstract: Hematopoietic stem and progenitor cells (HSPCs) can lead to the establishment of a long-lasting microglia-like progeny in the brain of properly myeloablated hosts. We exploited this approach to treat the severe CLN1 neurodegenerative disorder, which is the most aggressive form of neuronal ceroid lipofuscinoses, due to deficiency of palmitoyl-protein thioesterase 1 (hPPT1). We here provide first evidence that: i) transplantation of wild type HSPCs exerts a partial but long-lasting mitigation of the symptoms; ii) transplantation of HSPCs over-expressing hPPT1 by lentiviral gene transfer enhances therapeutic benefit as compared to wild type cell transplant, with first demonstration of such a dose-effect benefit for a purely neurodegenerative condition like CLN1 disease; iii) transplantation of hPPT1 over-expressing HSPCs by a novel intracerebroventricular (ICV) approach is sufficient to transiently ameliorate CLN1 disease symptomatology in the absence of hematopoietic tissue engraftment of the transduced cells; and iv) the combinatorial transplantation of transduced HSPCs intravenously and ICV results in the most robust therapeutic benefit among the tested approaches on both pre-symptomatic as well as symptomatic animals. Overall, these findings provide first evidence of the efficacy and feasibility of this novel approach to treat CLN1 disease and possibly other neurodegenerative conditions, paving the way for its future clinical application.


title: An Arrayed Genome-Wide Perturbation Screen Identifies the Ribonucleoprotein hnRNP K As Rate-Limiting for Prion Propagation

abstract: A defining characteristic of mammalian prions is their capacity for self-sustained propagation. Theoretical considerations and experimental evidence suggest that prion propagation is modulated by cell-autonomous and non-autonomous modifiers. Using a novel quantitative phospholipase protection assay (QUIPPER) for high-throughput prion measurements, we performed an arrayed genome-wide RNA interference (RNAi) screen aimed at detecting modifiers of prion propagation. We exposed prion-infected cells in high-density microplates to 35364 ternary pools of 52746 siRNAs targeting 17582 genes representing the mouse protein-coding transcriptome. We identified 1191 modulators of prion propagation. While 1151 of these modified the expression of both the pathological prion protein, PrPSc, and its cellular counterpart PrPC, 40 genes affected selectively PrPSc. Of the latter, 20 genes augmented prion production when suppressed. A prominent limiter of prion propagation was the heterogeneous nuclear ribonucleoprotein Hnrnpk. Psammaplysene A (PSA), which binds Hnrnpk, reduced prion levels in cultured cells and protected them from cytotoxicity. PSA also reduced prion levels in infected cerebellar organotypic slices and alleviated locomotor deficits in prion-infected Drosophila melanogaster expressing ovine PrPC. Hence, genome-wide QUIPPER-based perturbations can discover actionable cellular pathways involved in prion propagation. Finally, the unexpected identification of a prioncontrolling ribonucleoprotein suggests a role for RNA in the generation of infectious prions.


title: Inhibition is a prevalent mode of activity in the neocortex around awake hippocampal ripples

abstract: Coordinated peri-ripple activity in the hippocampal-neocortical network is essential for mnemonic information processing in the brain. Hippocampal ripples likely serve different functions in sleep and awake states. Thus, the corresponding neocortical activity patterns may differ in important ways. We addressed this possibility by conducting voltage and glutamate wide-field imaging of the neocortex with concurrent hippocampal electrophysiology in awake mice. Contrary to our previously published sleep results, deactivation and activation were dominant in post-ripple neocortical voltage and glutamate activity, respectively, especially in the agranular retrosplenial cortex (aRSC). Additionally, the spiking activity of aRSC neurons, estimated by two-photon calcium imaging, revealed the existence of two subpopulations of excitatory neurons with opposite peri-ripple modulation patterns: one increases and the other decreases firing rate. These differences in peri-ripple spatiotemporal patterns of neocortical activity in sleep versus awake states might underlie the reported differences in the function of sleep versus awake ripples.


title: Reconstructed human pigmented skin/epidermis models achieve epidermal pigmentation through melanocore transfer.

abstract:
The skin acts as a barrier to environmental insults and provides many vital functions. One of these is to shield DNA from harmful UV radiation, which is achieved by skin pigmentation arising as melanin is produced and dispersed within the epidermal layer. This is a crucial defence against DNA damage, photo-ageing and skin cancer. The mechanisms and regulation of melanogenesis and melanin transfer involve extensive crosstalk between melanocytes and keratinocytes in the epidermis, as well as fibroblasts in the dermal layer. Although the predominant mechanism of melanin transfer continues to be debated and several plausible models have been proposed, we and others previously provided evidence for a coupled exo/phagocytosis of melanocore model. Herein, we performed histology and immunohistochemistry analyses and demonstrated that a newly developed full-thickness 3D reconstructed human pigmented skin model and an epidermis-only model exhibit dispersed pigment throughout keratinocytes in the epidermis. Transmission electron microscopy revealed melanocores between melanocytes and keratinocytes, suggesting that melanin is transferred through coupled exocytosis/phagocytosis of the melanosome core similar to our previous observations in human skin biopsies. We therefore present evidence that our in vitro models of pigmented human skin show epidermal pigmentation comparable to human skin. These findings have a high value for studies of skin pigmentation mechanisms and pigmentary disorders, whilst reducing the reliance on animal models and human skin biopsies.

SignificanceCurrently, many approaches to study skin pigmentation and pigmentary disorders use 2D cultures or co-culture of melanocytes and/or keratinocytes, as well as skin biopsies obtained from consenting donors. However, challenges are faced when it is necessary to mimic the tissue microenvironment as there is a limited availability of skin biopsies, especially for the case of rare pigmentary disorders. Through this study, we show the potential of 3D in vitro reconstructed pigmented skin/epidermis models for both general and specific pigmentary research questions, which could reduce reliance on animal models and human skin biopsies.

category: cell biology


title: Koisio Technology-Modulated Cell Culture Media can Significantly Increase the Antioxidant Capacity of Mouse Fibroblasts

abstract: Oxidative stress is not only a critical common pathological factor of numerous diseases, but also an important factor in the aging process. It is highly valuable to discover novel and economic substances for enhancing the antioxidant capacity of human body. In this study we used a cell culture model to determine the antioxidant capacity of Koisio technology-modulated solutions without additions of any external substances. Our study has obtained the following findings: First, the cells cultured in Koisio technology-modulated cell culture media (KM) showed significantly greater capacity to decrease H2O2-induced oxidative stress, compared with the cells cultured in regular cell culture media; second, the cells cultured in KM showed significantly greater capacity to resist H2O2-induced decreases in cell number, compared with the cells cultured in regular cell culture media; and third, the H2O2 prepared in Koisio technology-modulated MEM produced significantly lower levels of decreases in cell number, compared with the H2O2 prepared in MEM. Collectively, our study has indicated that Koisio technology can increase the antioxidant capacity of both solutions and cells without additions of any external substances. Based on our previous findings, we proposed that Koisio technology-modulated solutions increase cellular antioxidant capacity at least partially by increasing intracellular ATP levels.


title: AMP-activated protein kinase (AMPK) is required for recovery from metabolic stress induced by ultrasound microbubble treatment

abstract: Ultrasound and microbubbles (USMB) is a promising strategy for cancer therapy. USMB can induce a variety of effects on cells including transient formation of plasma membrane pores (sonoporation) and enhanced endocytosis, which enhance drug delivery, and can also lead to enhanced cell death. However, the outcomes of USMB on cell physiology are heterogeneous, in that USMB elicits cell death in a proportion of cells while exerting minimal effects on others. This suggests that mechanisms of adaptation following USMB allow some cells to survive and/or proliferate. The molecular mechanisms of adaptation to USMB-induced stress remain poorly understood, thus potentially hindering broad therapeutic applications of USMB. Herein, we used several triple negative breast cancer cells to study the effect of USMB-induced metabolite stress and the role of AMPK as a response to this stress. We found that USMB alters steady-state levels of amino acids, glycolytic intermediates, and citric acid cycle intermediates. USMB treatment acutely reduces ATP levels and stimulates AMP-activated protein kinase (AMPK) phosphorylation and activation. Further, AMPK is required to restore ATP levels in cells that survived the initial insult and support cell proliferation post-USMB treatment. These results suggest that AMPK and metabolic perturbations are likely determinants of the anti-neoplastic efficacy of USMB treatment.


title: Focal adhesion-based cell migration is differentially regulated in vivo versus in vitro by Paxillin phosphorylation

abstract: Focal adhesions are important subcellular structures that physically link the cell to the extracellular matrix (ECM), thus facilitating efficient cell migration. Although in vitro cell culture studies have provided a wealth of information regarding focal adhesion biology, it is critical to understand how focal adhesions are dynamically regulated in their native environment. We developed a zebrafish transplantation system in which we could efficiently visualize focal adhesion structures during single cell migration in vivo with high-resolution live cell imaging. By comparing focal adhesions between this in vivo system and the traditional in vitro cell culture model, we show differential regulation of a core focal adhesion protein, Paxillin. We find that a key site of phosphoregulation on Paxillin, tyrosine 118 (Y118), exhibits reduced phosphorylation in migrating cells in vivo in both zebrafish and mouse melanoma models, contrary to the pivotal role for this phosphorylation event in cell culture studies. Furthermore, direct modulation of this residue by site directed mutagenesis leads to opposite cell migration phenotypes in vivo versus in vitro in both migrating cancer cells and macrophages. Unexpectedly, expression of a non-phosphorylatable version of Y118-Paxillin promotes cell migration in vivo, despite inhibiting cell migration in the in vitro cell culture conditions. To further understand the mechanism of this regulation, we find that the upstream kinase, focal adhesion kinase (FAK), is downregulated in cells in vivo, and that cells expressing non-phosphorylatable Y118-Paxillin exhibit increased interactions between Paxillin and CRKII, an adaptor protein known to promote cell migration signaling. Collectively, our findings provide significant new insight into how focal adhesions are regulated in cells migrating in their native environment.


title: A cell non-autonomous FOXO/DAF-16-mediated germline quality assurance program that responds to somatic DNA damage

abstract:
Germline integrity is critical for progeny fitness. Organisms deploy the DNA damage response (DDR) signalling to protect germline from genotoxic stress, facilitating cell-cycle arrest of germ cells and DNA repair or their apoptosis. Cell-autonomous regulation of germline quality is well-studied; however, how quality is enforced cell non-autonomously on sensing somatic DNA damage is less known. Using Caenorhabditis elegans, we show that DDR disruption, only in the uterus, when insulin-IGF-1 signalling (IIS) is low, arrests germline development and induces sterility in a FOXO/DAF-16 transcription factor (TF)-dependent manner. Without FOXO/DAF-16, germ cells of the IIS mutant escape arrest to produce poor quality oocytes, showing that the TF imposes strict quality control during low IIS. In response to low IIS in neurons, FOXO/DAF-16 works cell autonomously as well as non-autonomously to facilitate the arrest. Activated FOXO/DAF-16 promotes transcription of checkpoint and DDR genes, protecting germline integrity. However, on reducing DDR during low IIS, the TF decreases ERK/MPK-1 signaling below a threshold, and transcriptionally downregulates genes involved in spermatogenesis-to-oogenesis switch as well as cdk-1/Cyclin B to promote germline arrest. Altogether, our study reveals how cell non-autonomous function of FOXO/DAF-16 promotes germline quality and progeny fitness in response to somatic DNA damage.

Significance StatementReproductive decisions are supervised processes that take into account various inputs like cellular energy availability and status of damage repair in order to ensure healthy progeny. In this study, we show that the absence of optimal DNA damage repair in the somatic uterine tissues prevents oocyte development by the cell-autonomous as well non-autonomous function of activated FOXO transcription factor DAF-16. Thus, this study elucidates a new surveillance role of FOXO/DAF-16 in somatic tissues that ensures progeny fitness.

category: developmental biology


title: Apical constriction induces tissue rupture in a proliferative epithelium

abstract: Apical-basal polarity is an essential epithelial trait controlled by the evolutionarily conserved PAR-aPKC polarity network. Deregulation of polarity proteins disrupts tissue organization during development and in disease, but the underlying mechanisms are unclear due to the broad implications of polarity loss. Here, we uncovered how Drosophila aPKC maintains epithelial architecture by directly observing tissue disorganization after fast optogenetic inactivation in living adult flies and ovaries cultured ex vivo. We show that fast aPKC perturbation in the proliferative follicular epithelium produces large epithelial gaps that result from increased apical constriction, rather than loss of apical-basal polarity. Accordingly, we could modulate the incidence of epithelial gaps by increasing and decreasing actomyosin-driven contractility. We traced the origin of epithelial gaps to tissue rupture next to dividing cells. Live imaging shows that aPKC perturbation rapidly induces apical constriction in non-mitotic cells, producing pulling forces that ultimately detach dividing and neighbouring cells. We further demonstrate that epithelial rupture requires a global increase of apical constriction, since it was prevented by the presence of non-constricting cells. Conversely, a global induction of apical tension through light-induced recruitment of RhoGEF2 to the apical side was sufficient to produce tissue rupture. Hence, our work reveals that the roles of aPKC in polarity and actomyosin regulation are separable and provides the first in vivo evidence that excessive tissue stress can break the epithelial barrier during proliferation.


title: Unifying adaptive stress and adaptive habitat selection hypotheses through movement ecology

abstract:
O_LIGlucocorticoid production is often indicative of internal state that underpins movement and resulting habitat selection. Habitat selection is adaptive behaviour, but habitats where animals exhibit atypical glucocorticoid production are often associated with low fitness. Discrepancies between poor fitness outcomes and purported adaptive behaviour have contributed to a lack of consensus about the ecological and evolutionary implications of glucocorticoid production.
C_LIO_LIWe aim to demonstrate the adaptive ecological and evolutionary roles of glucocorticoids by integrating stress physiology with habitat selection theory.
C_LIO_LIFirst, we show how habitat selection theory can benefit from a mechanistic understanding of the relationships between stressors, internal state, and habitat selection. We show that glucocorticoids drive everything from short-term locomotion to large-scale movements. In turn, these movements produce familiar patterns of adaptive habitat selection like habitat specialization and migration. Populations arrive at these evolutionarily stable habitat selection strategies by responding to stressors like predation and food availability. To quantify responses to stressors, we show how to parameterize glucocorticoid data in habitat selection models. Modelling the ecological and evolutionary patterns that make habitat selection adaptive also help clarify relationships between glucocorticoids, stress, and fitness.
C_LIO_LIOur approach delivers a practical framework for testing existing hypotheses about habitat selection and stress physiology. Our framework can also be applied to new questions that integrate both, with the potential to understand adaptations of populations to their environments, and implications of stressors and habitat selection for ecosystem functioning.
C_LI

category: ecology


title: Genomic compatibility excels possible 'good genes' effects of sexual selection in lake char

abstract: Mating is rarely random in nature, but the effects of mate selection on offspring performance are still poorly understood, even in well-established models such as salmonid fish. We sampled wild lake char (Salvelinus umbla) and used their gametes to investigate the genetic consequences of different mating scenarios. In a first study, we used full-factorial breeding to experimentally separate additive genetic from maternal environmental effects. This led to 60 families and in total 1,073 embryos that were raised singly after sublethal exposures to the pathogen Aeromonas salmonicida, the common pollutant ethinylestradiol, or water only. Contrary to predictions of  good genes sexual selection, offspring of more yellow males were less tolerant to the pathogen than offspring of pale males, while male coloration did not predict offspring tolerance to ethinylestradiol. However, increased kinship between the parents had strong negative effects on embryo development in all treatment groups. In a second experiment, we monitored 1,464 singly-raised embryos that resulted from 70 pair-wise sperm competition trials. These embryos were raised in an environment that supports the growth of symbiotic microbes (sublethal stress) or in a clean environment. Offspring of yellow males were again less stress tolerant than those of pale males, and embryo development was again slowed down with increasing genetic similarity between the parents. We conclude that genetic benefits of mate selection would be strongest if females avoided genetic similarity during mate selection, for example based on MHC-linked signals, while male breeding colors seem more relevant in intra-sexual selection.

category: evolutionary biology


title: A selective bottleneck during host entry drives the evolution of new legume symbionts

abstract: During the emergence of new host-microbe symbioses, multiple selective pressures-acting at the different steps of the microbial life cycle-shape the phenotypic traits that jointly determine microbial fitness. However, the relative contribution of these different selective pressures to the adaptive trajectories of microbial symbionts are still poorly known. Here we characterized the dynamics of phenotypic adaptation and its underlying genetic bases during the experimental evolution of a plant pathogenic bacterium into a legume symbiont. We observed that fast adaptation was predominantly driven by selection acting on competitiveness for host entry, which outweighed selection acting on within-host proliferation. Whole-population sequencing of evolved bacteria revealed that phenotypic adaptation was supported by the continuous accumulation of new mutations and the sequential sweeps of cohorts of mutations with similar temporal trajectories. The identification of adaptive mutations within the fixed mutational cohorts showed that all of them improved competitiveness for host entry, while only a subset of those also improved within host proliferation. Computer simulations predict that this effect emerges from the presence of a strong selective bottleneck at host entry. Together, these results show how selective bottlenecks can alter the relative influence of selective pressures acting during bacterial adaptation to multistep infection processes.


title: Compositionally constrained sites drive long branch attraction

abstract: Accurate phylogenies are fundamental to our understanding of the pattern and process of evolution. Yet, phylogenies at deep evolutionary timescales, with correspondingly long branches, have been fraught with controversy resulting from conflicting estimates from models with varying complexity and goodness of fit. Analyses of historical as well as current empirical datasets, such as alignments including Microsporidia, Nematoda or Platyhelminthes, have demonstrated that inadequate modeling of across-site compositional heterogeneity, which is the result of biochemical constraints that lead to varying patterns of accepted amino acid along sequences, can lead to erroneous topologies that are strongly supported. Unfortunately, models that adequately account for across-site compositional heterogeneity remain computationally challenging or intractable for an increasing fraction of contemporary datasets. Here, we introduce "compositional constraint analysis", a method to investigate the effect of site-specific amino acid diversity on phylogenetic inference, and show that more constrained sites with lower diversity and less constrained sites with higher diversity exhibit ostensibly conflicting signal for models ignoring across-site compositional heterogeneity. We demonstrate that more complex models accounting for across-site compositional heterogeneity can ameliorate this bias. We present CAT-PMSF, a pipeline for diagnosing and resolving phylogenetic bias resulting from inadequate modeling of across-site compositional heterogeneity based on the CAT model. Our analyses indicate that CAT-PMSF is unbiased. We suggest using CAT-PMSF when convergence of the CAT model cannot be assured. We find evidence that compositional constrained sites are driving long branch attraction in two metazoan datasets and recover evidence for Porifera as the sister group to all other animals.


title: Cophylogeny and specificity between cryptic coral species (Pocillopora spp.) at Moorea and their symbionts (Symbiodiniaceae)

abstract: The congruence between phylogenies of tightly associated groups of organisms (cophylogeny) reflects evolutionary links between ecologically important interactions. However, despite being a classic example of an obligate symbiosis, tests of cophylogeny between scleractinian corals and their photosynthetic algal symbionts have been hampered in the past because both corals and algae contain genetically unresolved and morphologically cryptic species. Here, we studied co-occurring, cryptic Pocillopora species from Moorea, French Polynesia, that differ in their relative abundance across depth. We constructed new phylogenies of the host Pocillopora (using genomic loci, complete mitochondrial genomes, and thousands of single nucleotide polymorphisms) and their Symbiodiniaceae symbionts (using ITS2 and psbAncr markers) and tested for cophylogeny. The analysis supported the presence of five Pocillopora species on the fore-reef at Moorea that mostly hosted either Cladocopium latusorum or C. pacificum. Only Pocillopora species hosting C. latusorum, and that have similar relative abundances across depths, also hosted taxa from Symbiodinium and Durusdinium. In general, the Cladocopium phylogeny mirrored the Pocillopora phylogeny. Within Cladocopium species, lineages also differed in their associations with Pocillopora haplotypes, except those showing evidence of nuclear introgression, and with depth in the two most common Pocillopora species. We also found evidence for a new Pocillopora species (haplotype 10), that has so far only been sampled from French Polynesia, that warrants formal identification. The linked phylogenies of these Pocillopora and Cladocopium species and lineages suggest that symbiont speciation is driven by niche diversification in the host, but there is still evidence for symbiont flexibility in rare cases.


title: Parametric effects of light acting via multiple photoreceptor types contribute significantly to circadian entrainment in Drosophila melanogaster.

abstract: Circadian rhythms in physiology and behavior have near 24-hour periodicities that must adjust to the exact 24-hour geophysical cycles on earth to ensure stable and adaptive daily timing, a phenomenon referred to as entrainment. Entrainment can either occur via instantaneous shifts in the phase of the clock, a process referred to as non-parametric entrainment, or by light acting continuously to alter the speed of the clock, a process referred to as parametric entrainment. Though typically studied in isolation, real-world entrainment involves both parametric and non-parametric processes. While Drosophila melanogaster has been a powerful model organism for understanding the molecular and neuronal underpinnings of entrainment, the vast majority of the work on entrainment in this species has focused on non-parametric light effects. Though parametric effects of light have been described for D. melanogaster, there have been few studies examining the mechanisms underlying such effects. This has been due, in large part, to the fact that the flys circadian system does not free-run strongly under constant light conditions, which is required to experimentally address parametric effects of light. In this study, we demonstrate that light has robust parametric effects on the Drosophila circadian clock and that the timing of daily activity is strongly influenced by these effects, even under the standard step-function light/dark cycles employed by the field. We also show that parametric effects of light depend on external photoreceptors and, to a surprising extent, on the blue light photoreceptor cryptochrome. Furthermore, we introduce ramped light cycles as an entrainment paradigm that produces entrainment features that are similar to those seen in mammalian systems, in that they eliminate the confounding effects of the large startle responses displayed by flies under step function light/dark cycles and require multiple days for re-entrainment to shifted cycles. These features make possible a new experimental approach to understanding the mechanisms underlying entrainment in the fly.

category: genetics


title: Engineering chimeric antigen receptor neutrophils from human pluripotent stem cells for targeted cancer immunotherapy

abstract: Neutrophils, the most abundant white blood cells in the circulation, are closely related to cancer development and progression. Primary neutrophils from healthy donors present potent cytotoxicity against different human cancer cell lines through direct contact and via the generation of reactive oxygen species (ROS). However, due to their short half-life and resistance to genetic modification, neutrophils have not yet been engineered with widely used chimeric antigen receptors (CARs) to enhance their anti-tumor cytotoxicity for targeted immunotherapy. Here, we genetically engineered human pluripotent stem cells (hPSCs) with different synthetic CARs and successfully differentiated them into functional neutrophils by implementing a novel chemically-defined differentiation platform. Neutrophils expressing the chlorotoxin (CLTX)-T-CAR presented specific cytotoxicity against glioblastoma (GBM) cells both in monolayer and 3D cultures. In a GBM xenograft mouse model, systematically-administered CLTX-T-CAR neutrophils also displayed enhanced anti-tumor activity and prolonged animal survival compared with peripheral blood-neutrophils, hPSC-neutrophils and CLTX-NK-CAR natural killer (NK) cells. Collectively, we established a new platform for production of CAR-neutrophils, paving the way to myeloid cell-based therapeutic strategies that would complement and boost current cancer treatment approaches.

category: bioengineering


title: Assessment of binocular fixational eye movements including cyclotorsion with split-field binocular scanning laser ophthalmoscopy

abstract: Fixational eye motion is a hallmark of human gaze behavior, yet little is known about how they interact between fellow eyes. Here, we designed, build and validated a split-field binocular scanning laser ophthalmoscope (bSLO) to record high-resolution eye motion traces from both eyes of six observers during fixation at different binocular vergence conditions. In addition to microsaccades and drift, torsional eye motion could be extracted, with a spatial measurement error of less than 1 arcmin. Microsaccades were strongly coupled between fellow eyes under all conditions. No monocular microsaccade occurred and no significant delay between microsaccades onset across fellow eyes could be detected. Cyclotorsion was also firmly coupled between both eyes, occurring typically in conjugacy, with gradual changes during drift and abrupt changes during saccades.


title: A mechanistic spatiotemporal model for drug resistant infections

abstract:
When drug resistance is suspected to be in a region, patients in the region are sampled and the suspicion is confirmed. This biased sampling limits our ability to capture underlying dynamics, meaning strategies to lengthen the lifespan of drugs are reactionary, not proactive.

Testing for drug resistant infections is becoming easier and cheaper, therefore we should revisit sampling decisions. We present a hierarchical mechanistic Bayesian model, and apply it to a simulated dataset, where we sample between 5% and 30% of the population in a biased and unbiased manner. We show that unbiased spatiotemporal data on the presence of drug resistant infections, combined with our model, highlights underlying dynamics.

Our mechanistic model is more accurate than a generalised additive model with space and time components. Moreover, highlighting underlying dynamics creates novel strategies that lengthen the lifespan of drugs. In low to middle income countries, generally, drug resistance emerges into a population from hotspots such as treatment centres (perhaps the use of sub-standard drugs), or major transport hubs, and then resistance spreads throughout the population. Using our model, we rank resistance hotspots, enabling resources to be targeted - such as verifying the quality of drugs at a particular health care centre.

category: bioinformatics


title: Exhaustive Cross-linking Search with Protein Feedback

abstract: Improving the sensitivity of protein-protein interaction detection and protein structure probing is a principal challenge in cross-linking mass spectrometry (XL-MS) data analysis. In this paper, we propose an exhaustive cross-linking search method with protein feedback (ECL-PF) for cleavable XL-MS data analysis. ECL-PF adopts an optimized /{beta} mass detection scheme and establishes protein-peptide association during the identification of cross-linked peptides. Existing major scoring functions can all benefit from the ECL-PF workflow to a great extent. In comparisons using synthetic datasets and hybrid simulated datasets, ECL-PF achieved three-fold higher sensitivity over standard techniques. In experiments using real datasets, it also identified 91.6% more cross-link spectrum matches and 52.6% more unique cross-links.


title: PtRNAdb: A web resource of Plant tRNA genes from a wide range of plant species

abstract:
tRNA, as well as their derived products such as short interspersed nuclear elements (SINEs), pseudogenes and transfer-RNA, derived fragments (tRFs) has now been shown to be vital for cellular life, functioning and adaptation during different stress conditions in all diverse life forms. In this study, we have developed PtRNAdb (www.nipgr.ac.in/PtRNAdb), a plant exclusive tRNA database containing 113849 tRNA gene sequences from phylogenetically diverse plant species. We have analysed a total of 106 nuclear, 89 plastidial and 38 mitochondrial genomes of plants by tRNAscan-SE software package, and after careful curation of the output data, we developed this database and integrated the data. The information about the tRNA gene sequences obtained, were further enriched with consensus sequence based study of tRNA genes based on their isoacceptors and isodecoders. We have also built covariance models based on the isoacceptors and isodecoders of all the tRNA sequences using infernal tool. The user can also perform BLAST not only against PtRNAdb entries but also against all the tRNA sequences stored in PlantRNA databases; and annotated tRNA genes across the plant kingdom available at NCBI. For the users ease, we have also incorporated the tRNAscan-SE tool for tRNA gene prediction, and ViennaRNA package for structural analysis on the home page of PtRNAdb. This resource is believed to be of high utility for plant researchers as well as molecular biologists to carry out further exploration of plant tRNAome on a wider spectrum, as well as for performing comparative and evolutionary studies related to tRNAs and their derivatives across all domains of life.

Database URLhttp://www.nipgr.ac.in/PtRNAdb/


title: Confluence and tight junction dependence of volume regulation in epithelial tissue

abstract: Epithelial cell volume regulation is a key component to tissue stability and dynamics. In particular, how cells respond to osmotic stresses is of significant physiological interest in kidney epithelial tissue. For individual mammalian cells, it is well established that Na-K-2Cl cotransporter (NKCC) channels mediate cell volume homeostasis in response to hyperosmotic stress. However, whether mature epithelium respond similarly is not well known. Here we show that while small colonies of MDCK epithelial cells behave similarly to single cells and exhibit volume homeostasis that is dependent on the NKCC channel function, mature epithelial tissue does not. Instead, the cell volume decreases by 33% when confluent monolayers or acini formed from MDCK are subjected to hyperosmotic stress. We show that the tight junction protein, ZO-1, and Rho-associated kinase (ROCK) are essential for osmotic regulation of cell volume in mature epithelium. Since these both are known to be essential for tight junction assembly, this strongly suggest a role for tight junctions in changing volume response in mature epithelium. Thus, tight junctions act either directly or indirectly in osmotic pressure response of epithelial tissue to suppress volume homeostasis common to isolated epithelial cells.

category: biophysics


title: Exploring the Neoantigen burden in Breast Carcinoma Patients

abstract: In this study we performed a multi-omics analysis comprising whole-exome sequencing (WES) and RNA sequencing (RNA-Seq) on seven breast cancer patients, consisting of three Estrogen receptor (ER) positive and four Triple negative breast cancer (TNBC) subtypes to understand the neoantigen burden in breast cancer tumor samples. We predicted both class-I and class-II human leukocyte antigen (HLA) bound neoantigens by analyzing matched tumor-normal pair of exomes. Across all the patients, we predicted 434 unique neoantigens (NeoFil) in total, affecting 237 different genes and 87% of them (n = 378) are expressed at RNA level (Neoexp). The missense mutations (87%) are the major contributor in neoantigen (Neoexp) generation, followed by frameshift (11%) and indels (2%). The neoantigens (NeoFil) were found to be positively correlated with the somatic mutations (R2 = 0.89). We also noted that the vast majority (99.98%) of the predicted neoantigens are patient specific. Overall, the current study offers significant insight into the neoantigen profile in tumor types with intermediate/low mutation burdens like breast cancer.

category: cancer biology


title: Bioluminescent zebrafish transplantation model for drug discovery

abstract: In the last decade, zebrafish have accompanied the mouse as a robust animal model for cancer research. The possibility of screening small-molecule inhibitors in a large number of zebrafish embryos makes this model particularly valuable. However, the dynamic visualization of fluorescently labeled tumor cells needs to be complemented by a more sensitive, easy, and rapid mode for evaluating tumor growth in vivo to enable high-throughput screening of clinically relevant drugs. In this study we proposed and validated a pre-clinical screening model for drug discovery by utilizing bioluminescence as our readout for the determination of transplanted cancer cell growth and inhibition in zebrafish embryos. For this purpose, we used NanoLuc luciferase, which ensured rapid cancer cell growth quantification in vivo with high sensitivity and low background when compared to conventional fluorescence measurements. This allowed us large-scale evaluation of in vivo drug responses of 180 kinase inhibitors in zebrafish. Our bioluminescent screening platform could facilitate identification of new small-molecules for targeted cancer therapy as well as for drug repurposing.

category: pharmacology and toxicology


title: Aeration Strategy at Birth Does Not Impact Carotid Blood Flow and Oxygen Delivery in Preterm Lambs

abstract:
ObjectiveTo examine the effect of three different ventilation strategies at birth on markers of cerebral haemodynamics and oxygenation.

DesignAnimal research.

SettingAnimal research facility.

Subjects124-127 day gestation apnoeic preterm lambs (n=68) born via caesarean section.

InterventionsLambs were randomised to either positive pressure ventilation with positive end-expiratory pressure (PEEP) 8 cmH2O (No-RM; n=12), sustained inflation (SI; n=15) or a dynamic PEEP strategy (DynPEEP; n=41) at birth, followed by 90 minutes of standardised ventilation. Prior to birth a carotid artery flow probe was placed, with occlusive cannulation of the contralateral carotid artery. Carotid blood flow, ventilation data, peripheral oxygen saturation (SpO2), heart rate and arterial blood pressure were continuously recorded, with intermittent arterial blood gas analysis at set time points.

Main outcome measuresCarotid blood flow, carotid artery oxygen content and carotid oxygen delivery (product of carotid artery blood flow and oxygen content).

ResultsOverall carotid blood flow measures were comparable between strategies. The exception being mean carotid blood flow during the first 3 minutes after birth, which was significantly lower for the SI group compared to the No-RM and DynPEEP groups respectively (p<0.0001; repeated-measure mixed effect ANOVA). Carotid arterial oxygen content (p=0.19) and oxygen delivery (p=0.50) were similar between strategies. Maximum PEEP did not alter cerebral haemodynamic measures.

ConclusionsThis is the first study to compare cerebral haemodynamics during different PEEP strategies with SI at birth. Although there were some short-term variations, there were no sustained differences.

What is already known on this topicFacilitating the respiratory transition at birth for preterm infants is essential for the lungs as well as the brain. Whilst our understanding of the impact of different respiratory strategies on the lungs is well understood, preclinical data involving other organs is sparse. Concerns have been raised that aggressive lung recruitment may impede cerebral haemodynamics in the preterm infant.

What this study addsTo our knowledge, this is the first study that assesses the effect of the three different aeration strategies currently being considered in clinical practice; sustained inflation, dynamic and static positive end-expiratory pressure (PEEP), on cerebral haemodynamics. We found that differing ventilation strategies did not alter carotid blood flow, carotid oxygen content or carotid oxygen delivery. Specifically, there was no difference between sustained inflation (to 35 cmH2O) and maximum PEEP levels of 8, 14 and 20 cmll.O applied during the first three minutes after birth.

How this study might affect research, practice or policyFollowing preterm birth it is important to consider the effect of ventilation strategies on other organs, especially the brain. This preclinical study provides some reassurance that respiratory strategies designed to focus on lung aeration at birth may not impact cerebral haemodynamics in preterm neonates.

category: physiology


title: Morpho-functional analysis of the head glands in three Auchenorrhyncha species and their possible biological significance

abstract: The Cicadomorpha Philaenus spumarius, Neophilaenus campestris and Cicadella viridis are known transmitters of the bacterium Xylella fastidiosa. Here, we studied the ultrastructural organization of their cephalic glands. Our investigations with scanning, transmission, FIB-SEM electron microscope and light microscope revealed for the first time in Auchenorrhyncha the presence of two types of cephalic glands. Both belonged to the class III epidermal glands, according to Noirot and Quennedey classification. Type I glands were the most common, being mainly located around antennae, lorum and gena. Moreover, these glands were observed also on the abdomen and thorax, always in association with sensilla trichoidea. The second type of glands were located exclusively at the apical part of the postclypeus in P. spumarius and N. campestris. The ultrastructural organization was similar in both types, being composed of a secretory cell and a conducting canal. Differences were observed in the width of the cuticular opening, being smaller in the type II glands. In addition, we have recorded the presence of a maxillary sensory pit in all species and described sensilla trichoidea ultrastructural organization. Finally, we discussed the ultrastructural organization of the glands and their potential biological role.

category: zoology


title: Cardiac cycle affects the asymmetric value updating in instrumental reward learning

abstract: This study aimed to investigate whether instrumental reward learning is affected by the cardiac cycle. To this end, we examined the effects of the cardiac cycle (systole or diastole) on the computational processes underlying the participants choices in the instrumental learning task. In the instrumental learning task, participants were required to select one of two discriminative stimuli (neutral visual stimuli) and immediately receive reward/punishment feedback depending on the probability assigned to the chosen stimuli. To manipulate the cardiac cycle, the presentation of discriminative stimuli was timed to coincide with either cardiac systole or diastole. We fitted the participants choices in the task with reinforcement learning (RL) models and estimated parameters involving instrumental learning (i.e., learning rate and inverse temperature) separately in the systole and diastole trials. Model-based analysis revealed that the learning rate for positive prediction errors was higher than that for negative prediction errors in the systole trials; however, learning rates did not differ between positive and negative prediction errors in the diastole trials. These results demonstrate that the natural fluctuation of cardiac afferent signals can affect asymmetric value updating in instrumental reward learning.


title: Simulating semantic dementia in a brain-constrained model of action and object words learning

abstract:
The nature of semantic knowledge - conceptual information stored in the brain - is highly debated in the field of cognitive science. Experimental and clinical data specify various cortical regions involved in the processing of meaning. Those include semantic hubs that take part in semantic processing in general as well as sensorimotor areas that process specific conceptual categories according to their modality. Biologically inspired neurocomputational models can help adjudicate between different theories about the exact roles of those regions in the functioning of the semantic system.

Here, we used an existing neuroanatomically constrained model of frontotemporal brain areas implicated in language acquisition and grounding. We adapted it to replicate and explain the effects of semantic dementia on word processing abilities. Semantic dementia is a disease characterized by semantic knowledge deterioration that correlates with neural damage in the anterior temporal lobe. The behavior of our model is in accordance with clinical data - namely, word recognition performance decreases as SD lesions progress, whereas word repetition abilities remain preserved, or are less affected. Furthermore, our model makes novel predictions about category-specific effects of SD - namely, our simulation results indicate that word processing should be more impaired for object-than for action-related words, and that white matter degradation should lead to more severe consequences than the same proportion of grey matter degradation.

The present results provide a mechanistic, cortical-level explanatory account of a range of language impairments as observed in clinical populations during the onset and progress of semantic dementia.


title: MiR-182-5p regulates Nogo-A expression and promotes neurite outgrowth of hippocampal neurons in vitro

abstract:
Nogo-A protein is a key myelin-associated inhibitor for axonal growth, regeneration, and plasticity in the central nervous system (CNS). Regulation of the Nogo-A/NgR1 pathway facilitates functional recovery and neural repair after spinal cord trauma and ischemic stroke. MicroRNAs are described as effective tools for the regulation of important processes in CNS such as neuronal differentiation, neuritogenesis, and plasticity. Our results showed that miR-182-5p mimic specifically downregulates the expression of the luciferase reporter gene fused to the mouse Nogo-A 3UTR, and Nogo-A protein expression in Neuro-2a and C6 cells. Finally, we observed that when rat primary hippocampal neurons are co-cultured with C6 cells transfected with miR-182-5p mimic, there is a promotion of the outgrowth of neuronal neurites in length. From all these data we suggest that miR-182-5p may be a potential therapeutic tool for the promotion of axonal regeneration in different diseases of the CNS.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/482803v1_ufig1.gif" ALT="Figure 1">
View larger version (17K):
org.highwire.dtl.DTLVardef@e0979forg.highwire.dtl.DTLVardef@10265eborg.highwire.dtl.DTLVardef@71603dorg.highwire.dtl.DTLVardef@1654818_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIBioinformatics analyses show that miR-182-5p targets Nogo-A 3UTR.
C_LIO_LIMiR-182-5p downregulates Nogo-A protein expression in murine cell lines.
C_LIO_LIMiR-182-5p promotes neurite outgrowth of rat primary hippocampal neurons in vitro.
C_LIO_LIMiR-182-5p is suggested as a potential therapeutic tool for the promotion of axonal regeneration in different pathologies/diseases of the central nervous system.
C_LI


title: The predictive power of mRNA mapping for Cannabinoid 1 receptor protein in the human brain

abstract:
Type 1 cannabinoid (CB1) receptor is expressed in cortex, hippocampus, amygdala, basal ganglia, and cerebellum. With the help of the Allen Human Brain Atlas, genomic maps visualize not only the gene expression across whole brain regions, but also the functional profile of brain structures. Therefore, it is more timely than ever to integrate genomic mapping from brain mRNA atlas with the protein expression from positron emission tomography (PET) scans for better understanding of CB1 receptor of the human brain. F18-FMPEP-d2 PET scans were retrieved from the AIVO neuroinformatics project. Autoradiography data were based on the study with H3-CP55940. mRNA expressions of CNR1 gene (Cannabinoid receptor 1) were downloaded from the Allen Human Brain Atlas. Volume of distribution (VT) from F18-FMPEP-d2 PET scans, CNR1 gene expression, and H3-CP55940 binding were calculated and Spearman correlation analysis was performed. Also, a meta-analysis was done to investigate the association between protein expression from PET and mRNA expression from the Allen Human Brain Atlas. Between VT of F18-FMPEP-d2 PET scans and CNR1 mRNA expression, moderate strength of correlation was observed (rho = 0.5026, p = 0.0354). Strong positive correlation was also found between CNR1 mRNA expression and H3-CP55940 binding (rho = 0.6727, p = 0.0281), validating the finding between F18-FMPEP-d2 PET scans and CNR1 mRNA. From the meta-analysis, the correlation coefficient ranged from -0.46 to 0.99, with a pooled effect of 0.58. In conclusion, the moderate to strong associations between gene and protein expression for CB1 receptor in the human brain were observed that CNR1 mRNA mapping might have the predictive power for in vivo CB1 receptor protein expression. From the meta-analysis, the moderate to strong correlation was observed between mRNA expression and protein expressions across multiple genes, showing the predictive power of genes to estimate protein levels of human brains.

HIGHLIGHTSO_LIWe investigated the association between CNR1 gene expression from the Allen Human Brain Atlas and type 1 cannabinoid (CB1) receptor expression from F18-FMPEP-d2 PET scans.
C_LIO_LIThe moderate to strong associations between gene and protein expression for CB1 receptor in the human brain were observed that CNR1 mRNA mapping might have the predictive power for in vivo CB1 receptor protein expression.
C_LIO_LIFrom the meta-analysis, the moderate to strong correlation was observed between mRNA expression and protein expressions across multiple genes, showing the predictive power of genes to estimate protein levels of human brains.
C_LI


title: Emx1-Cre is expressed in vagal afferent nerves that regulate central cardiorespiratory functions

abstract:
The Emx1-IRES-Cre transgenic mouse is commonly used to direct genetic recombination in forebrain excitatory neurons. However the original study reported that Emx1-Cre is also expressed in peripheral autonomic ganglia, which could potentially affect the interpretation of targeted circuitry contributing to systemic phenotypes. Here, we report that Emx1-Cre is expressed in nodose ganglion cells whose axon travel within afferent vagal nerves and terminate in the nucleus tractus solitarius (NTS), a critical central autonomic regulatory pathway located in the dorsal medulla. Optogenetic stimulation of Emx1-Cre+ fibers reliably evoked excitatory postsynaptic currents in the NTS in vitro and evoked a parasympathetic bradycardia reflex occasionally with irregular respirations in anesthetized mice in vivo. Our study clearly demonstrates that Emx1-IRES-Cre is expressed in the vagal nerve fibers traveling within the central autonomic system involving autonomic cardiorespiratory regulation. These results raise caution when interpreting the systemic phenotypes of Emx1-IRES-Cre conditional recombinant mice, and also suggest the utility of this line to investigate the modulators of afferent vagal system.

Significance StatementEmx1-IRES-Cre mice are widely used to dissect critical circuitry underlying neurological disorders such as epilepsy. These studies often assume the Cre is expressed selectively only in forebrain excitatory neurons. However, earlier works reported that Emx1 is expressed in several peripheral tissues of the developing embryo and thus gene recombination may affect these peripheral structures. In this study, we characterized the expression and physiological functions of Emx1-Cre expressed in the afferent vagus nerve. Optogenetic stimulation of these Emx1-Cre+ vagus nerves activates the nucleus tractus solitarius neurons within the brainstem in vitro and induces brief bradycardia in vivo. This study confirmed that peripheral Emx1-Cre+ cells are involved in autonomic regulation and potentially affect transgenic mouse phenotypes.


title: p73 controls cell junction dynamics during sprouting angiogenesis and acts via Angiomotin

abstract:
Vascular development comprises vasculogenesis, sprouting and non-sprouting angiogenesis and vessel assembly and remodeling. These processes require the coordinated regulation of intercellular junctions to maintain vascular architecture and vessel barrier. Here we demonstrate a key function for p73 transcription factor orchestrating transcriptional programs involved in the regulation of endothelial cell junction morphology during sprouting angiogenesis. TP73 is required for vessel integrity and stability to maintain the endothelial barrier. p73-downregulation leads to hyperpermeability in vitro, while total-p73 or TAp73-specific knockout results in hemorrhages in vivo. We identified AMOT as a p73 direct transcriptional target. Lack of p73 affects expression, localization, and function of Angiomotin. In turn, stabilization of Angiomotin levels in p73-deficent cells can rescue their junctional defects, altogether demonstrating that AMOT is one of p73-downstream mediators during the regulation of endothelial junction dynamics. These results redefine p73 role in angiogenesis by revealing p73 as a vascular architect that regulates endothelial cell junctions, thereby organizing and stabilizing the endothelial barrier.

SummaryWe explore p73 role in vascular morphogenesis and uncover that it orchestrates gene regulation to organize and stabilize the endothelial barrier during sprouting angiogenesis. Mechanistically, TAp73 isoform modulates transcriptional programs involved in vascular architecture including endothelial junction dynamics via AMOT.


title: Oxidative stress profiles of lymphoblasts from Amyotrophic Lateral Sclerosis patients with or without known SOD1 Mutations

abstract:
Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing neurodegenerative disease that affects motor neurons. This disease is associated with oxidative stress especially in mutant superoxide dismutase 1 (mutSOD1) patients. However, less is known for the most prevalent sporadic ALS due to a lack of disease models. Here, we studied oxidative the stress profiles in lymphoblasts from ALS patients with mutSOD1 or unknown (undSOD1) mutations.

mutSOD1 and undSOD1 lymphoblasts, as well as sex/age-matched controls (3/group) were obtained from Coriell and divided in 46 years-old-men (C1), 46 years-old-women (C2) or 26/27 years-old-men (C3) cohorts. Growth curves were performed, and several parameters associated with redox homeostasis were evaluated, including SOD activity and expression, general oxidative stress levels, lipid peroxidation, response to oxidative stimulus, glutathione redox cycle, catalase expression, and activity, and Nrf2 transcripts. Pooled (all cohorts) and paired (intra-cohort) statistical analyses were performed, followed by clustering and principal component analyses (PCA).

Although a high heterogeneity among lymphoblast redox profiles was found between cohorts, clustering analysis based on 7 parameters with high chi-square ranking (total SOD activity, oxidative stress levels, catalase transcripts, SOD1 protein levels, metabolic response to mM concentrations of tert-butyl hydroperoxide, glutathione reductase activity, and Nrf2 transcript levels) provided a perfect separation between samples from healthy controls and ALS (undSOD1 and mutSOD1), also visualized in the PCA analysis.

Our results show distinct redox signatures in lymphoblasts from mutSOD1, undSOD1, and healthy controls that can be used as therapeutic targets for ALS drug development.

Highlights Lymphoblasts from ALS patients present altered redox properties
 Redox profiling evidenced total SOD and glutathione reductase activities, SOD1 protein levels, DCF fluorescence, catalase and Nrf2 transcripts, and tert-butyl hydroperoxide cytotoxicity, as discriminant features for experimental groups.
 Lymphoblast redox profiles may be helpful for patient stratification and precision medicine


title: SUMOylation of ABCD3 restricts bile acid synthesis and regulates metabolic homeostasis

abstract: Mitochondrial anchored protein ligase (MAPL) has been shown to function as both a SUMO and ubiquitin ligase with multiple roles in mitochondrial quality control, cell death pathways and inflammation. To examine the global function of MAPL we generated a knock-out mouse model and sought functional insight through unbiased BioID, transcriptomics and metabolic analysis. MAPL KO mice are lean and highly insulin sensitive, ultimately developing fully penetrant, spontaneous hepatocellular carcinoma after 18 months. BioID revealed the peroxisomal bile acid transporter ABCD3 as a primary MAPL interacting partner, which we show is SUMOylated in a MAPL-dependent manner. MAPL KO animals showed increased bile acid secretion in vivo and in isolated primary hepatocytes, along with robust compensatory changes in the expression of enzymes synthesizing and detoxifying bile acid. In addition, MAPL KO livers showed signs of ER stress and secreted high levels of Fgf21, the starvation hormone known to drive the reduction of white fat stores and promote insulin sensitivity. Lastly, during aging all MAPL KO mice developed hepatocellular carcinomas. These data reveal a major function for MAPL in the regulation of bile acid synthesis leading to profound changes in whole body metabolism and the ultimate generation of liver cancer when MAPL is lost.


title: The molecular chaperone Cysteine String Protein is required to stabilize trans-SNARE complexes during human sperm acrosomal exocytosis

abstract:
Membrane fusion in sperm cells is crucial for acrosomal exocytosis and must be preserved to assure fertilizing capacity. Evolutionarily conserved protein machinery regulates acrosomal exocytosis. Molecular chaperones play a vital role in spermatogenesis and post-testicular maturation. Cysteine String Protein (CSP) is a member of the Hsp40 co-chaperones, and for more than 20 years, most research published focused on CSPs role in synapsis. However, the participation of molecular chaperones in acrosomal exocytosis is poorly understood. Using western blot and indirect immunofluorescence, we showed that CSP is present in human sperm, is predominantly bound to membranes, and is palmitoylated. Moreover, using electron microscopy and functional assays, we reported that sequestration of CSP avoided the assembly of trans-complexes and inhibited exocytosis. In summary, our data demonstrated that CSP is necessary and mediates the trans-SNARE complex assembly between the outer acrosomal and plasma membranes, thereby regulating human sperm acrosomal exocytosis. Understanding CSPs role is critical in identifying new biomarkers and generating new rational-based approaches to treating male infertility.

Summary statementCysteine String Protein is necessary and mediates the trans-SNARE complex assembly between the outer acrosomal and plasma membranes, thereby regulating human sperm acrosomal exocytosis.


title: Helminth parasites decrease survival probability in young red deer

abstract: Helminths are common parasites of wild ungulates that can have substantial costs for growth, mortality, and reproduction. While these costs are relatively well documented for mature animals, knowledge of helminths impacts on juveniles is more limited. Identifying these effects is important because young individuals are often heavily infected, and juvenile mortality is an important process regulating wild populations. Here, we investigated associations between helminth infection and overwinter survival in juvenile wild red deer (Cervus elaphus) on the Isle of Rum, Scotland. We collected faecal samples non-invasively from known individuals and used them to count propagules of three helminth taxa (strongyle nematodes, Fasciola hepatica, and Elaphostrongylus cervi). Using generalised linear models, we investigated associations between parasite counts and overwinter survival for calves and yearlings. Strongyles were associated with reduced survival in both age class, and F. hepatica was associated with reduced survival in yearlings, while E. cervi infection showed no association with survival in either age class. This study provides observational evidence for fitness costs of helminth infection in juveniles of a wild mammal, and suggests that these parasites could play a role in regulating population dynamics.


title: Effect of larval diet on adult immune function is contingent upon selection history and host sex in Drosophila melanogaster

abstract: Mounting an immune response requires a considerable energy investment by the host. This makes expression of immune phenotypes susceptible to changes in availability of resources. There is ample evidence in scientific literature to suggest that hosts become more vulnerable to infection by pathogens and parasites when access to nutrition is limited. Using populations of Drosophila melanogaster experimentally evolved to better survive bacterial infections, we explore if host selection history influences host response to resource deprivation in terms of immune function. We find that when reared on a suboptimal diet (both in larval and adult stages), adult flies from evolved populations are still more immune to infections compared to flies from control populations. Furthermore, we observe a sex-dependent effect of interaction between selection history and diet on immune function. We thus conclude that immune function of hosts adapted to pathogen challenge is less affected by resource limitation compared to non-adapted hosts.


title: Recent Reconfiguration Of An Ancient Developmental Gene Regulatory Network In Heliocidaris Sea Urchins

abstract: Changes in developmental gene regulatory networks (dGRNs) underlie much of the diversity of life1, but the evolutionary mechanisms that operate on interactions with these networks remain poorly understood. Closely related species with extreme phenotypic divergence provide a valuable window into the genetic and molecular basis for changes in dGRNs and their relationship to adaptive changes in organismal traits. Here we analyze genomes, epigenomes, and transcriptomes during early development in two sea urchin species in the genus Heliocidaris that exhibit highly divergent life histories and in an outgroup species. Signatures of positive selection and changes in chromatin status within putative gene regulatory elements are both enriched on the branch leading to the derived life history, and particularly so near core dGRN genes; in contrast, positive selection within protein-coding regions have at most a modest enrichment in branch and function. Single-cell transcriptomes reveal a dramatic delay in cell fate specification in the derived state, which also has far fewer open chromatin regions, especially near dGRN genes with conserved roles in cell fate specification. Experimentally perturbing the function of three key transcription factors reveals profound evolutionary changes in the earliest events that pattern the embryo, disrupting regulatory interactions previously conserved for [~]225 million years. Together, these results demonstrate that natural selection can rapidly reshape developmental gene expression on a broad scale when selective regimes abruptly change and that even highly conserved dGRNs and patterning mechanisms in the early embryo remain evolvable under appropriate ecological circumstances.


title: CDK4 deletion in mice prevents fat accumulation and increases endurance capacity through activation of estrogen related receptor (ERR)driven oxidative metabolism in skeletal muscle

abstract: Cyclin-dependent kinase 4 (CDK4) canonical role is to control cell cycle progression from G1 to S phases. However, recent studies reported that CDK4 regulates energy metabolism in non-proliferating cells such as hepatocytes or adipocytes. The objective of our work is to study CDK4 function in skeletal muscle using a model of mice lacking CDK4 (cdk4-/-). By coupling treadmill running to indirect calorimetry, we show that cdk4-/- mice display improved endurance and higher capacity to use fat as fuel during exercise. Isolated muscles lacking CDK4 are more resistant to fatigue in response to repeated contractions and have increased oxidative capacity and mitochondrial content compared to cdk4+/+ muscles. Transcriptomic analysis reveals upregulation of genes controlled by the nuclear receptors estrogen-related receptors (ERRs) in cdk4-/- skeletal muscle, associated with elevated levels of the ERR co-activator PGC1a. Supporting in vivo results, C2C12 myotubes treated with a CDK4 inhibitor have increased mitochondrial oxygen consumption, PGC1 expression and ERR transcriptional activity measured by a luciferase reporter. In normal housing conditions, cdk4-/- mice show an increased basal metabolic rate and are resistant to weight gain and fat accumulation. In conclusion, our study uncovers a role for CDK4 in the control of skeletal muscle metabolism. Moreover, CDK4 inhibition may be an alternative strategy against obesity-associated metabolic disorders.


title: Validation of carbon isotopologue distribution measurements by GC-MS and application to 13C-metabolic flux analysis of plant tricarboxylic acid cycle

abstract: The estimation of metabolic fluxes in photosynthetic organisms represents an important challenge that has gained interest over the last decade with the development of 13C-Metabolic Flux Analysis at isotopically non-stationary steady-state. This approach requires a high level of accuracy for the measurement of Carbon Isotopologue Distribution in plant metabolites, which has still not been accurately evaluated at the isotopoloque level for GC-MS. Thus, uncertainties remain about the accuracy of the CID measured and subsequently, the metabolic fluxes associated. Moreover, a significant part of our knowledge about the functioning and the regulation of plant TCA cycle is solely based on 13C-labeling experiments analyzed with Nuclear Magnetic Resonnance. But this method is not very sensitive at low 13C abundance and can hardly fully resolve isotopologue dynamics inherent to the TCA cycle compared to MS. Here, we developed a workflow to validate the measurements of CIDs from plant metabolites with GC-MS by producing tailor-made E. coli standard extracts harboring a predictable binomial CID for all metabolites. Most of our TMS-derivatives mass fragments were validated with these standards and at natural isotope abundance in plant matrices. Then, we applied this validated MS method to investigate the light/dark regulation of plant TCA cycle metabolic fluxes by incorporating U-13C-pyruvate to Brassica napus leaf discs. We took advantage of pathway-specific isotopologues/isotopomers to show that the TCA cycle has a nearly four-fold higher contribution to Citrate and Malate production than phosphoenolpyruvate carboxylase (PEPc). Overall, the dynamic of 13C-enrichment in Citrate, Glutamate, Succinate and Malate ruled out the hypothesis of the stored citrate contribution to TCA cycle and highlithed the major contribution of the forward plant TCA cycle to malate biosynthesis in both dark and light conditions. Interestingly, the light-dependent 13C-incorporation into Glycine and Serine showed that nearly 5% of TCA cycle-derived CO2 was actively reassimilated by the photosynthesis. We also observed an increase of PEPc contribution to malate production in the light while there was a higher involvement of PEPc-derived carbons to citrate production in dark condition. The results suggested an important role for Malate import into mitochondria in sustaining TCA cycle in dark conditions.

category: plant biology


title: A tissue boundary orchestrates the segregation of inner ear sensory organs

abstract: The inner ear contains distinct sensory organs, produced sequentially by segregation from a large sensory-competent domain in the developing otic vesicle. To understand the mechanistic basis of this process, we investigated the changes in prosensory cell patterning, proliferation and character during the segregation of some of the vestibular organs in the mouse and chicken otic vesicle. We discovered a specialized boundary domain, located at the interface of segregating organs. It is composed of prosensory cells that gradually enlarge, elongate and are ultimately diverted from a prosensory fate. Strikingly, the boundary cells align their apical borders and constrict basally at the interface of cells expressing or not the Lmx1a transcription factor, an orthologue of drosophila Apterous. The boundary domain is absent in Lmx1a-deficient mice, which exhibit defects in sensory organ segregation, and is disrupted by the inhibition of ROCK-dependent actomyosin contractility. Altogether, our results suggest that actomyosin-dependent tissue boundaries ensure the proper separation of inner ear sensory organs and uncover striking homologies between this process and the compartmentalization of the drosophila wing disc by lineage-restricted boundaries.


title: ACCUMULATION OF MICROPLASTIC AND MICRORUBBER PARTICLES IN STORMWATER POND FISH AND INVERTEBRATES

abstract:
Stormwater ponds serve as best management practices to trap or remove both physical and chemical pollutants before further discharge of stormwater into receiving natural waterbodies. Stormwater ponds can therefore accumulate high levels of pollutants, including microplastics (MP) and microrubber (MR) from road runoff. It was hypothesized that biota in stormwater ponds that receive large amounts of road runoff, or are in proximity to roadways and major developments, would contain significant amounts of MP per individual, with high abundances of tire particles (TP). Fish and invertebrates were collected from five stormwater ponds and their adjacent tidal creeks in Mt. Pleasant, SC, USA. Whole organisms were digested using KOH and digested contents were filtered and analyzed by visual microscopy to identify and count MPs.

The majority (>80%) of MP recovered from biota across all sites were suspected TP. The average number of MP per individual ranged from 0.3 to 71 MP and the average number of suspected tire particles per individual ranged from 0 to 57.7 tire particles.

There were significant differences in MP per individual observed between sites and between species. A combination of factors such as availability of MPs based on surrounding land use, pond hydrodynamics, organism size, and species feeding habitat likely influenced the total MP observed among the different sites and species analyzed. These data provide preliminary examination into the fate and transport of MP and MR in stormwater ponds and an evaluation of MP and MR abundance in organisms from stormwater ponds in the coastal zone.


title: Computational Assessment of Different Structural Models for Claudin-5 Complexes in Blood-Brain-Barrier Tight-Junctions

abstract: The blood-brain barrier (BBB) strictly regulates the exchange of ions and molecules between the blood and the central nervous system. Tight junctions (TJs) are multimeric structures that control the transport through the paracellular spaces between adjacent brain endothelial cells of the BBB. Claudin-5 (Cldn5) proteins are essential for the TJ formation and assemble into multi-protein complexes via cis-interactions within the same cell membrane and trans-interactions across two contiguous cells. Despite the relevant biological function of Cldn5 proteins and their role as targets of brain drug delivery strategies, the molecular details of their assembly within TJs are still unclear. Two different structural models have been recently introduced, in which Cldn5 dimers belonging to opposite cells join to generate paracellular pores. However, a comparison of these models in terms of ionic transport features is still lacking. In this work, we used molecular dynamics simulations and free energy (FE) calculations to assess the two Cldn5 pore models and investigate the thermodynamic properties of water and physiological ions permeating through them. Despite different FE profiles, both structures present single/multiple FE barriers to ionic permeation, while being permissive to water flux. These results reveal that both models are compatible with the physiological role of Cldn5 TJ strands. By identifying the protein-protein surface at the core of TJ Cldn5 assemblies, our computational investigation provides a basis for the rational design of synthetic peptides and other molecules capable of opening paracellular pores in the BBB.


title: Maternal immune activation and adolescent alcohol exposure increases alcohol drinking and disrupts cortical-striatal-hippocampal oscillations in adult offspring

abstract: Maternal immune activation (MIA) is strongly associated with an increased risk of developing mental illness in adulthood, which often co-occurs with alcohol misuse. The current study aimed to begin to determine whether MIA, combined with adolescent alcohol exposure (AE), could be used as a model with which we could study the neurobiological mechanisms behind such co-occurring disorders. Pregnant Sprague-Dawley rats were treated with PolyI:C or saline on gestational day 15. Half of the offspring were given continuous access to alcohol during adolescence, leading to four experimental groups: controls, MIA, AE, and Dual (MIA + AE). We then evaluated whether MIA and/or AE alters: 1) alcohol consumption; and 2) cortical-striatal-hippocampal oscillations in adult offspring. Dual rats, particularly females, drank significantly more alcohol in adulthood compared to all other groups. Using machine learning to build predictive models from oscillations, we were able to differentiate Dual rats from control rats and AE rats in both sexes, and Dual rats from MIA rats in females. The current data suggest that MIA+AE (Dual "hits") is a valuable model that we can use to study the neurobiological underpinnings of co-occurring disorders. Our future work aims to extend these findings to other addictive substances to enhance the translational relevance of this model.


title: The tail domain of neurofilament light chain accumulates in neuronal nuclei during oxidative injury

abstract: Neurofilament light chain (NFL), one of the major subunits of neuron-specific intermediate filaments, is an important component of the neuronal cytoskeleton. Mutations in the NEFL gene and disturbances of NFL turnover or transport are associated with the pathophysiology of various diseases of the nervous system. Also, NFL is released from neurons as a result of neuroaxonal damage and NFL levels in cerebrospinal fluid and blood have emerged as a biomarker of the progression of numerous neurological diseases. One common underlying factor in these diseases is oxidative stress. Although both NFL and oxidative injury play a role in the development of neurological diseases, the effect of oxidative stress on NFL at the cellular level has not been extensively studied. Here, by investigating the response of neurofilaments to in vitro oxidative injury, we discovered that NFL localizes in the nuclei of primary neurons. We show that this depends on calpain activity and determine that a proteolytic fragment of the tail domain, not the full-length NFL, is that which accumulates in the nuclei. Furthermore, we demonstrate that the recombinant NFL tail domain accumulates in the nuclei of neurons and neuroblastoma cells, even in the absence of oxidative injury. Finally, we show that the tail domain of NFL interacts with the DNA in the nuclei of living cells, suggesting that NFL plays a role in the regulation of gene expression in response to oxidative injury.


title: What the flip? What the P-N flip can tell us about proactive suppression

abstract: It has been debated whether salient distractors in visual search can be proactively suppressed to completely prevent attentional capture, as the occurrence of proactive suppression implies that the initial shift of attention is not entirely driven by physical salience. While the presence of a Pd component in the EEG (associated with suppression) without a preceding N2pc component (associated with selection) has been used as evidence for proactive suppression, the link between these ERPs and the underlying mechanisms is not always clear. This is exemplified in two recent papers that observed the same waveform pattern, where an early Pd-like component flipped to a N2pc-like component, but provided very different interpretations (Drisdelle & Eimer, Psychophysiology, 58(9), 1-12, 2021; Kerzel & Burra, Journal of Cognitive Neuroscience, 32(6), 1170-1183, 2020). Using RAGNAROC (Wyble et al., Psychological Review, 127(6), 1163-1198, 2020), a computational model of reflexive attention, we successfully simulated this ERP pattern with minimal changes to its existing architecture, providing a parsimonious and mechanistic explanation for this flip in the EEG that is unique from both of the previous interpretations. Our account supports the occurrence of proactive suppression and demonstrates the benefits of incorporating computational modelling into theory building.


title: Loss of NF1 causes tactile hypersensitivity and impaired synaptic transmission in a Drosophila model of autism spectrum disorder

abstract: Autism Spectrum Disorder (ASD) is a neurodevelopmental condition in which the mechanisms underlying its core symptomatology are largely unknown. Studying animal models of monogenic syndromes associated with ASD, such as neurofibromatosis type 1 (NF1), can offer insights into its aetiology. Here, we show that loss of function of the Drosophila NF1 ortholog results in larval tactile hypersensitivity, paralleling the sensory abnormalities observed in individuals with ASD. Mutant larvae also exhibit synaptic transmission deficits at the glutamatergic neuromuscular junction (NMJ), with increased spontaneous but reduced evoked release. Diminished expression of NF1 specifically within central cholinergic neurons induces both excessive neuronal firing and tactile hypersensitivity, suggesting the two may be linked. Furthermore, both impaired synaptic transmission and behavioural deficits are fully rescued via knockdown of Ras proteins. These findings validate NF1-/- Drosophila as a tractable model of ASD with the potential to elucidate important pathophysiological mechanisms.


title: Germinal center block exacerbates extrafollicular responses and accelerates autoimmune disease progression in a murine lupus model

abstract:
Systemic lupus erythematosus and numerous other autoimmune diseases are characterized by affinity-matured, class-switched autoantibodies to nuclear antigens. Such antibodies are generally thought to arise in germinal centers (GCs). Several strategies to block GC formation and progression are currently being explored clinically. However, recent studies have suggested a key role for extrafollicular responses in driving the early events in autoimmune development. To investigate the relative contribution of these two pathways in autoimmune disease development, we leveraged a lupus murine model, where we could genetically block the GC pathway. We find that a B cell intrinsic block in GC formation accelerates extrafollicular responses and exacerbates autoimmune progression. The manifestations included higher levels of circulating, class-switched autoantibodies, as well as antibody- and complement-deposition in the kidney glomeruli. GC B cell cultures in vitro showed that loss of the GC transcription factor Bcl-6 prevents cellular expansion and accelerates plasma cell differentiation. This suggests that the in vivo phenotype was a direct consequence of rewiring of B cell intrinsic transcriptional programming. In a competitive scenario in vivo, in autoreactive mixed bone marrow chimeras, B cells harboring the genetic GC block contributed disproportionately highly to the plasma cell output. Taken together, this emphasizes the extrafollicular pathway as a key contributor to autoimmune pathogenesis and suggests that strategies aimed at blocking GCs should simultaneously target this pathway to avoid rerouting the pathogenic response.

Highlights- Genetic GC block exacerbates autoimmune progression in a lupus model
- An intrinsic GC block drives B cell differentiation into terminally differentiated plasma cells in vitro
- B cells harboring a GC block competitively contribute to the plasma cell compartment in an autoreactive setting in vivo
- Lupus mice with a GC block display immune complex deposition in kidney glomeruli that is indistinguishable from their wild-type counterparts

category: immunology


title: Development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of the potato wart pathogen Synchytrium endobioticum

abstract: Potato (Solanum tuberosum) wart, caused by the biotrophic fungal pathogen Synchytrium endobioticum, is a serious disease of cultivated potatoes. A rapid and accurate method for wart diagnosis is needed. In this study, we described the development and evaluation of a loop-mediated isothermal amplification (LAMP) method for the detection of S. endobioticum. Four sets of LAMP primers were designed. Their sensitivity was tested on a gBlock and the specificity of the most sensitive primer set (LAMP1) was verified by in silico analysis and on DNA of non-target species. The LAMP1 primer set showed a detection limit at 7-8 gBlock DNA molecules per reaction. On sensitivity, the LAMP was comparable to qPCR (SYBR green- and probe-based) and about 10 times more sensitive than conventional PCR. Considering the convenience of operation, the LAMP protocol is accurate and robust and should be recommended for the routine lab testing for potato wart.

category: microbiology


title: Myo1e knockout in the adult podocytes leads to proteinuria but has less severe consequences for kidney function than Myo1e loss during renal development

abstract: Myosin 1e (Myo1e) is expressed in specialized epithelial cells in the kidney (podocytes) and plays an important role in renal filtration. Knockout of Myo1e in mice and mutations in the MYO1E gene in humans cause proteinuria and disrupt the ultrastructure of glomeruli, the initial segments of renal nephrons that are responsible for selective excretion of waste products without the loss of proteins from the bloodstream. Previous studies have demonstrated that the loss of Myo1e early in development results in severe defects in renal function in mice and may lead to end-stage renal disease in patients homozygous for MYO1E mutations. However, little is known about the effects of Myo1e loss later in life. In this study, we used inducible knockout of Myo1e in mouse podocytes to examine the effects of Myo1e loss from the adult kidneys. We have found that Myo1e loss after the completion of renal development causes proteinuria and podocyte defects but the effects are milder and more variable compared to the effects of Myo1e knockout in developing podocytes. These findings indicate that Myo1e plays an important role in podocyte development and differentiation but may also contribute to the maintenance of the glomerular barrier in the adult kidneys.


title: Host control and species interactions jointly determine microbiome community structure

abstract:
The host microbiome can be considered as an ecological community where microbial species interact with each other in a highly complex and dynamic host environment. The host is under selective pressure to ensure the microbiome remains beneficial. Hosts can impose a range of ecological filters including host immune response that can influence the assembly and composition of the microbial community. How the host immune control and its interactions with the intrinsic within-microbiome community dynamics affect the assembly of the microbiome has been largely unexplored. We present a mathematical framework to elucidate the role of host immune control and its interaction with the balance of ecological interactions types within the microbiome community. We find that highly mutualistic microbial communities characteristic of high community population sizes are most susceptible to changes in immune control. Such communities can become invasion prone as host immune control strength is increased whereas communities prevalent with competitive interactions remain relatively stable in resisting invasion to changing host immune control. Our model reveals that the host immune control can interact in unexpected ways with a microbial community depending on the prevalent ecological interactions types for that community. We stress that understanding microbial community assembly and stability is incomplete without considering the role of host-control mechanisms on the microbial community.

1 SignificanceHost-associated microbial communities, microbiomes, are complex ecological systems whose dynamics are jointly determined by interactions happening between the microbes and host control through mechanisms such as nutritional resource supply and immune responses. To understand the assembly of microbiomes, it is crucial to consider the joint effects of both their intrinsic dynamics and control by hosts. We present here a community assembly model of the microbiome, incorporating diversity of between-species interactions and the host immune control. Our results reveal that host immune control operates differently according to the balance of interactions within the microbial community. We also find that host immune control weakens the influence of the species interactions on the properties of the microbial community.


title: Morph-dependent effect of nematode infection on host movement in the land snail Cepaea nemoralis (Mollusca, Gastropoda)

abstract: Host behaviour can be influenced by parasitic risk and infection through a variety of direct and indirect mechanisms. We can expect individuals expressing different phenotypes to also differ in the ways their behaviour is altered by parasites. We used the land snail Cepaea nemoralis, a species with various shell colour morphs differing in behaviour and physiology, as a model to study the link between parasite response and individual behaviour variation. We analysed metazoan parasite abundance and its relation to behaviour (movement and food intake) in snails from three shell morphs (from light unbanded to darker five-banded) and from two neighbouring populations from contrasted environments. Snails were parasitized by mites, trematodes and nematodes, from rarest to most frequent. We confirm that terrestrial gastropods can defend against infection by trapping parasitic nematodes in their shell. We show that nematode encapsulation can uncover past infections even when a population is currently nematode-free, possibly due to parasite seasonality. We present new observations suggesting that not only nematodes but also mites might be encapsulated in shells. Vulnerability to parasites was morph-dependent, with darker snails harbouring fewer nematodes. Behaviour (movement and food intake) was dependent on nematode, but not trematode infection. Individuals with higher nematode load ate less, irrespective of morph and population. The morph that was most vulnerable to parasitism (unbanded snails) showed reduced activity in the nematode-infected population compared to the one that was nematode-free at sampling time. As a result, parasite risk may cancel previously described behavioural differences between morphs. We discuss the possible mechanisms explaining morph-dependent responses to parasites, and how parasite risk may be an unseen force shaping Cepaea nemoralis morph variation in space and time. We conclude on the possible ecological consequences of the link, mediated by shell colour, between thermal and immune responses.


title: Effects of somatic mutations on cellular differentiation in iPSC models of neurodevelopment

abstract: The use of induced pluripotent stem cells (iPSC) as models for development and human disease has enabled the study of otherwise inaccessible tissues. A remaining challenge in developing reliable models is our limited understanding of the factors driving irregular in vitro differentiation of iPSCs, particularly the impact of acquired somatic mutations. We leveraged data from a pooled dopaminergic neuron differentiation experiment of 238 iPSC lines profiled with single-cell and whole-exome sequencing to study how somatic mutations affect differentiation outcomes. Differentiation was tracked at three time points corresponding to neural progenitors, early neurons and mature neurons. We found that deleterious somatic mutations in key developmental genes, notably the BCOR gene, are strongly associated with failure in dopaminergic neuron differentiation, with lines carrying such mutations also showing larger proliferation rate in culture. We further identified broad differences in cell type composition between failed and successfully differentiating lines, as well as significant changes in gene expression contributing to the inhibition of neurogenesis, a functional process also targeted by deleterious mutations in failed lines. Our work highlights the need to routinely measure the burden of deleterious mutations in iPSC lines and calls for caution in interpreting differentiation-related phenotypes in disease-modelling experiments.

category: genomics


title: Zika virus persistence in the male macaque reproductive tract

abstract:
Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in that it is also vertically and sexually transmitted by humans. The male reproductive tract is thought to be a ZIKV reservoir; however, the reported magnitude and duration of viral persistence in male genital tissues varies widely in humans and non-human primate models. ZIKV tissue and cellular tropism and potential effects on male fertility also remain unclear. The objective of this study was to resolve these questions by analyzing archived genital tissues from 51 ZIKV-inoculated male macaques and correlating data on plasma viral kinetics, tissue tropism, and ZIKV-induced pathological changes in the reproductive tract. We hypothesized that ZIKV would persist in the male macaque genital tract for longer than there was detectable viremia, where it would localize to germ and epithelial cells and associate with lesions. We detected ZIKV RNA and infectious virus in testis, epididymis, seminal vesicle, and prostate gland. In contrast to prepubertal males, sexually mature macaques were significantly more likely to harbor persistent ZIKV RNA or infectious virus somewhere in the genital tract, with detection as late as 60 days post-inoculation. ZIKV RNA localized primarily to testicular stem cells/sperm precursors and epithelial cells, including Sertoli cells, epididymal duct epithelium, and glandular epithelia of the seminal vesicle and prostate gland. ZIKV infection was associated with microscopic evidence of inflammation in the epididymis and prostate gland of sexually mature males, which could have significant effects on male fertility. The findings from this study increase our understanding of persistent ZIKV infection which can inform risk of sexual transmission during assisted reproductive therapies as well as potential impacts on male fertility.

Author SummaryZika virus (ZIKV) spread since 2015 led to establishment of urban epidemic cycles involving humans and Aedes mosquitoes. ZIKV is also sexually and vertically transmitted and causes congenital Zika syndrome. Together, these features show that ZIKV poses significant global public health risks. By virtue of similar reproductive anatomy and physiology to humans, macaques serve as a useful model for ZIKV infection. However, macaque studies to date have been limited by small sample size, typically 1 to 5 animals. Although mounting evidence identifies the male reproductive tract as a significant ZIKV reservoir, data regarding the duration of ZIKV persistence, potential for sexual transmission, and male genitourinary sequelae remain sparse. Here, we analyzed archived genital tissues from more than 50 ZIKV-inoculated male macaques. Our results show that ZIKV can persist in the male macaque reproductive tract after the resolution of viremia, with virus localization to sperm precursors and epithelial cells, and microscopic evidence of inflammation in the epididymis and prostate gland. Additionally, we show that freezing is not a viable method of destroying infectious ZIKV. Our findings help explain cases of sexual transmission of ZIKV in humans, which also carries a risk for transmission via assisted fertility procedures, even after resolution of detectable viremia.

category: pathology


title: Diversity in DNA sequence, structure, and heterozygosity of nuclear rRNA gene region in Neopyropia yezoensis

abstract: DNA sequence reads of Neopyropia yezoensis (susabi-nori), its relative N. tenera (asakusa-nori), and their hybrids available in the sequence read archive produced assembled sequences of the nuclear rRNA gene region for 82 samples. Analysis of the assembled sequences revealed structural differences in the region of nuclear rRNA genes, with 17 forms depending on the presence or absence of introns and their lengths. The samples were divided into three groups based on differences in DNA sequences: Japanese N. yezoensis, Chinese N. yezoensis, and N. tenera/hybrids of N. tenera and N. yezoensis that exist in both countries. Despite genetic differentiation, the Japanese and Chinese N. yezoensis exhibit common structural forms. One sample of Chinese N. yezoensis presented almost 1:1 heterozygosity, whereas five other samples of Chinese N. yezoensis showed non-1:1 heterozygosity. In the latter case, neither the ratio of alleles nor the ratio of the number of introns was 1:1, suggesting the existence of an ongoing mechanism to eliminate the nuclear rRNA gene region on one of the homologous chromosomes in N. yezoensis.