title: Temporally extended and reversible neuronal silencing with Aion

abstract: Optogenetic silencing allows to reveal the necessity of selected neuronal populations for various neurophysiological functions ranging from specific behaviors to neuronal network activity. An ideal optogenetic silencing tool should be switchable between active and inactive states with precise timing while preserving its activity in the absence of light until switched to an inactive state. Although bistable anion-conducting channelrhodopsins (ACRs) were previously engineered to reach this goal, their conducting state life time was limited to only a few minutes and some ACRs were not fully switchable. Here we report Aion, a bistable ACR displaying a long-lasting open state with a spontaneous closing time constant close to 15 min. Moreover, Aion can be switched between the open and closed state with millisecond precision using blue and orange light, respectively. The long conducting state enables overnight silencing of neurons with minimal light exposure. We further generated trafficking-optimized versions of Aion, which show enhanced membrane localization and allow precisely timed, long-lasting all-optical control of nociceptive responses in larvae of Drosophila melanogaster.


title: Basolateral amygdala to insular cortex activity makes sign-tracking behavior insensitive to outcome value

abstract: Goal-tracking rats are sensitive to Pavlovian outcome devaluation while sign-tracking rats are devaluation insensitive. During outcome devaluation, goal-tracking (GT) rats flexibly modify responding to cues based on the current value of the associated outcome. However, sign-tracking (ST) rats rigidly respond to cues regardless of the current outcome value. Our prior study demonstrated disconnection of the basolateral amygdala (BLA) and anterior insular cortex (aIC) decreased both goal- and sign-tracking behaviors. Given the role of these regions in appetitive motivation and behavioral flexibility we predicted that disrupting BLA to aIC pathway during outcome devaluation would reduce flexibility in GT rats and reduce rigid appetitive motivation in ST rats. We inhibited the BLA to aIC pathway by infusing inhibitory DREADDs (hM4Di-mcherry) or control (mCherry) virus into the BLA and implanted cannulae into the aIC to inhibit BLA terminals using intracranial injections of clozapine N-oxide (CNO). After training, we used a within subject satiety-induced outcome devaluation procedure in which we sated rats on training pellets (devalued condition) or homecage chow (valued condition). All rats received bilateral CNO infusions into the aIC prior to brief non-reinforced test sessions. Contrary to our hypothesis, BLA-IC inhibition did not interfere with devaluation sensitivity in GT rats but did make ST behaviors sensitive to devaluation. Intermediate rats showed the opposite effect, showing rigid in responding to cues with BLA-aIC pathway inactivation. Together, these results demonstrate BLA-IC projections mediate tracking-specific Pavlovian devaluation sensitivity and highlights the importance of considering individual differences in Pavlovian approach when evaluating circuitry contributions to behavioral flexibility.

category: neuroscience


title: Fish as model systems to study epigenetic drivers in human self-domestication and neurodevelopmental cognitive disorders

abstract: Modern humans exhibit phenotypic traits that are shared across independent domestication events, suggesting the human self-domestication hypothesis. Epigenetic changes may facilitate early self-domestication in humans, since they can be the first layer of response to a novel environment. Here, we argue that fish provide model systems to study epigenetic drivers in human self-domestication. To do this, we compare genes that carry epigenetic changes in early domesticates of European sea bass with 1) anatomically modern humans and 2) neurodevelopmental cognitive disorders with abnormal self-domestication traits, i.e., schizophrenia, Williams syndrome and autism spectrum disorders. We found that genes with epigenetic changes in fish and in modern vs ancient humans were shared and were involved in processes like limb morphogenesis and phenotypes like abnormal snout morphology and hypopigmentation. Moreover, early domestication in fish and neurodevelopmental cognitive impairment in humans affected paralogue genes involved in processes such as neural crest differentiation and ectoderm differentiation. We conclude that parallel epigenetic changes may occur at the initial steps of domestication in absence of deliberate selection in phylogenetically distant vertebrates. These findings pave the way for future studies using fish as models to investigate epigenetic changes as drivers of human-self domestication and as triggers of cognitive disorders.

category: evolutionary biology


title: Biogeographical history shapes evolution of reproduction in a global warming scenario

abstract: Adaptive evolution might be critical for animal populations to thrive on the fast-changing natural environments. Ectotherms are particularly vulnerable to global warming and, although their limited coping ability has been suggested, few real-time evolution experiments have directly accessed their evolutionary potential. Here, we report a long-term experimental evolution study addressing the evolution of thermal reaction norms, after [~]30 generations under different thermal environments. We analyzed the evolutionary dynamics of Drosophila subobscura populations as a function of the thermally variable environments in which they evolved and their distinct biogeographical background. Our results showed clear differences between the historically differentiated populations: while the northern D. subobscura populations showed a temporal increase in performance at higher temperatures, their southern counterparts presented the opposite pattern. This suggests that the northern populations might be better equipped to cope with the current rising temperatures. Remarkably, no effect of thermal selection was found. The lack of a clear long-term adaptive response at higher temperatures after evolution under a global warming scenario raises concerns about the evolutionary potential of ectotherms. Our results highlight the complex nature of thermal responses in face of environmental heterogeneity and emphasize the importance of considering intra-specific variation in thermal evolution studies.


title: Temporal matches and mismatches between monarch butterfly and milkweed population changes over the past 12,000 years

abstract: In intimate ecological interactions, the interdependency of species may result in correlated demographic histories. For species of conservation concern, understanding the long-term dynamics of such interactions may shed light on the drivers of population decline. Here we address the demographic history of the monarch butterfly, Danaus plexippus, and its dominant host plant, the common milkweed Asclepias syriaca, using broad-scale sampling and genomic inference. Because genetic resources for milkweed have lagged behind those for monarchs, we first release a chromosome-level genome assembly and annotation for common milkweed. Next, we show that despite its enormous geographic range across eastern North America, A. syriaca is best characterized as a single, roughly panmictic population. Using Approximate Bayesian Computation via Random Forests (ABC-RF), a machine learning method for reconstructing demographic histories, we show that both monarchs and milkweed experienced concurrent range expansion during the most recent recession of North American glaciers [~]12,000 years ago. Our data identify an expansion of milkweed during the large-scale clearing of eastern forests ([~]200 years ago) but was inconclusive as to expansion or contraction of the monarch butterfly population during this time. Finally, our results indicate that neither species experienced a population contraction over the past 75 years. Thus, the well-documented decline of monarch abundance over the past 40 years is not visible in our genomic dataset, reflecting a possible mismatch of the overwintering census population to effective population size in this species.


title: Deep Augmentation for Electrode Shift Compensation in Transient-phase High-density Electromyographical Neural Interfaces: Towards Application in Neurorobotics

abstract: Going beyond the traditional sparse multi-channel peripheral human-machine interface that has been used widely in neurorobotics, high-density surface electromyography (HD-sEMG) has shown significant potential for decoding upper-limb motor control. We have recently proposed heterogeneous temporal dilation of LSTM-based deep neural networks for a very large number of gestures (>60), securing spatial resolution and fast convergence. However, several fundamental questions remain unanswered. One problem targeted explicitly in this paper is the issue of "electrode shift", which can happen specifically for high-density systems and during doffing and donning the sensor grid. Another real-world problem is the question of transient versus plateau classification, which connects to the temporal resolution of neural interfaces and seamless control. In this paper, for the first time, we implement gesture prediction on the transient phase of HD-sEMG data while robustifying the human-machine interface decoder to electrode shift. For this, we propose the concept of deep data augmentation for transient HD-sEMG. We show that without using the proposed augmentation, a slight shift of 10mm may drop the decoders performance to as low as 20%. Combining the proposed data augmentation with a 3D Convolutional Neural Network (CNN), we recovered the performance to 84.6% while securing a high spatiotemporal resolution, robustifying to the electrode shift, and getting closer to large-scale adoption by the end-users, enhancing resiliency.

category: bioengineering


title: Agent-based models help interpret patterns of clinical drug resistance by contextualizing competition between distinct drug failure modes.

abstract:
Structured AbstractO_ST_ABSIntroductionC_ST_ABSModern targeted cancer therapies are carefully crafted small molecules. These exquisite technologies exhibit an astonishing diversity of failure modes (drug resistance mechanisms) in the clinic. This diversity is surprising because back of the envelope calculations and classic modeling results in evolutionary dynamics suggest that the diversity in the modes of clinical drug resistance should be considerably smaller than what is observed. These same calculations suggest that known microenvironmental resistance mechanisms should not be able to compete for outgrowth with genetic resistance within a tumor, and yet evidence of microenvironmental resistance is often observed in the clinic. Quantitatively understanding the underlying biological mechanisms of failure mode diversity may improve the next generation of targeted anticancer therapies. It also provides insights into how intratumoral heterogeneity might shape interpatient diversity during clinical relapse.

Materials and MethodsWe employed spatial agent-based models to explore regimes where spatial constraints enable microenvironmental resistance to significantly compete with genetically resistant subclones. In order to parameterize a model of microenvironmental resistance, BT20 cells were cultured in the presence and absence of fibroblasts from 16 different tissues. The degree of resistance conferred by cancer associated fibroblasts (CAFs) in the tumor microenvironment was quantified by treating mono- and co-cultures with letrozole and then measuring the death rates.

Results and DiscussionOur simulations indicate that, even when a mutation is more drug resistant, its outgrowth can be delayed by abundant, low magnitude microenvironmental resistance across large regions of a tumor. These observations hold for different modes of microenvironmental resistance, including juxtacrine signaling, soluble secreted factors, and remodeled ECM. This result helps to explain the remarkable diversity of resistance mechanisms observed in solid tumors, which subverts the presumption that the failure mode that causes the quantitatively fastest growth in the presence of drug should occur most often in the clinic.

ConclusionOur model results demonstrate that spatial effects can interact with low magnitude of resistance microenvironmental effects to successfully compete against genetic resistance that is orders of magnitude larger. Clinical outcomes of solid tumors are intrinsically connected to their spatial structure, and the tractability of spatial agent-based models like the ones presented here enable us to understand this relationship more completely.


title: Information-Content-Informed Kendall-tau Correlation: Utilizing Missing Values

abstract:
Almost all correlation measures currently available are unable to handle missing values. Typically, missing values are either ignored completely by removing them or are imputed and used in the calculation of the correlation coefficient. In both cases, the correlation value will be impacted based on a perspective that missing data represents no useful information. However, missing values occur in real data sets for a variety of reasons. In omics data sets that are derived from analytical measurements, the primary reason for missing values is that a specific measurable phenomenon falls below the detection limits of the analytical instrumentation. These missing data are not missing at random, but represent some information by their "missingness." Therefore, we propose an information-content-informed Kendall-tau (ICI-Kt) correlation coefficient that allows missing values to carry explicit information in the determination of concordant and discordant pairs. With both simulated and real data sets from RNA-seq experiments, we demonstrate that the ICI-Kt allows for the inclusion of missing data values as interpretable information. Moreover, our implementation of ICI-Kt uses a mergesort-like algorithm that provides O(nlog(n)) computational performance. Finally, we show that approximate ICI-Kt correlations can be calculated using smaller feature subsets of large data sets with significant time savings, which has practical computational value when feature sizes are very large.

The ICI-Kt correlation calculation is available in an R package and Python module on GitHub at https://github.com/moseleyBionformaticsLab/ICIKendallTau and https://github.com/moseleyBionformaticsLab/icikt, respectively.

category: bioinformatics


title: Quantitative contribution of the spacer length in the supercoiling-sensitivity of bacterial promoters

abstract: DNA supercoiling acts as a global transcriptional regulator in bacteria, but the promoter sequence or structural determinants controlling its effect remain unclear. It was previously proposed to modulate the torsional angle between the -10 and -35 hexamers, and thereby regulate the formation of the closed-complex depending on the length of the "spacer" between them. Here, we develop a ther-modynamic model of this notion based on DNA elasticity, providing quantitative and parameter-free predictions on the relative activation of promoters containing a short vs long spacer when the DNA supercoiling level is varied. The model is tested through an analysis of in vitro and in vivo expression assays of mutant promoters with variable spacer lengths, confirming its accuracy for spacers ranging from 15 to 19 nucleotides, except those of 16 nucleotides where other regulatory mechanisms likely overcome the effect of this specific step. An analysis at the whole-genome scale in E. coli then demonstrates a significant effect of the spacer length on the genomic expression after transient or inheritable superhelical variations, validating the models predictions. Altogether, this study shows that the torsional constraints associated to promoter binding by RNA Polymerase underpin a basal and global regulatory mechanism independent of specific transcription factors.


title: Modulation of nuclear and cytoplasmic mRNA fluctuations by time-dependent stimuli: analytical distributions

abstract: Temporal variation of environmental stimuli leads to changes in gene expression. Since the latter is noisy and since many reaction events occur between the birth and death of an mRNA molecule, it is of interest to understand how a stimulus affects the transcript numbers measured at various sub-cellular locations. Here, we construct a stochastic model describing the dynamics of signal-dependent gene expression and its propagation downstream of transcription. For any time-dependent stimulus and assuming bursty gene expression, we devise a procedure which allows us to obtain time-dependent distributions of mRNA numbers at various stages of its life-cycle, e.g. in its nascent form at the transcription site, post-splicing in the nucleus, and after it is exported to the cytoplasm. We also derive an expression for the error in the approximation whose accuracy is verified via stochastic simulation. We find that, depending on the frequency of oscillation and the time of measurement, a stimulus can lead to cytoplasmic amplification or attenuation of transcriptional noise.

category: systems biology


title: Morphometric characters in the taxonomic recognition of the species of Dolichopus pennatus group (Diptera: Dolichopodidae)

abstract: Sexually dimorphic structures (ornaments) on the legs are important diagnostic features for Dolichopus males, whereas females of those males are often morphologically similar and their species diagnostics is difficult. To assessing the relevance and feasibility of setting diagnostic characters for five sister species of the genus Dolichopus Latreille, 1796 (D. argyrotarsis, D. lineaticornis, D. pennatus, D. popularis, D. subpennatus) analysis of morphometric trait was used: comparison of wing shape by methods of geometric morphometry, as well as comparison of the ratios of the leg segments lengths. Comparative analysis of morphometric characters with molecular data made it possible to study the phylogenetic signal of male leg ornaments. It was shown for Dolichopus species, that modifications of the males middle legs occurred independently several times in the genus. In addition, the evolutionary pattern in the formation of similar ornaments was also associated with changes in the morphometric features of the legs and wings. Based on wing and legs morphometry, new diagnostic characters were proposed for females of the studied species group.

category: zoology


title: Opioid Antagonism in Humans: A Primer on Optimal Dose and Timing for Central Mu-Opioid Receptor Blockade

abstract: Non-human animal studies outline precise mechanisms of central mu-opioid regulation of pain, stress, affiliation and reward processing. In humans, pharmacological blockade with non-selective opioid antagonists such as naloxone and naltrexone is typically used to assess involvement of the mu-opioid system in such processing. However, robust estimates of the opioid receptor blockade achieved by opioid antagonists are missing. Dose and timing schedules are highly variable and often based on single studies. Here, we provide a detailed analysis of central opioid receptor blockade after opioid antagonism based on existing positron emission tomography data. We also create models for estimating opioid receptor blockade with intravenous naloxone and oral naltrexone. We find that common doses of intravenous naloxone (0.10-0.15 mg/kg) and oral naltrexone (50 mg) are more than sufficient to produce full blockade of central MOR (>90% receptor occupancy) for the duration of a typical experimental session ([~]60 minutes), presumably due to initial super saturation of receptors. Simulations indicate that these doses also produce high KOR blockade (78-100%) and some DOR blockade (10% with naltrexone and 48-74% with naloxone). Lower doses (e.g., 0.01 mg/kg intravenous naloxone) are estimated to produce less DOR and KOR blockade while still achieving a high level of MOR blockade over [~]30 minutes. The models and simulations form the basis of two novel web applications for detailed planning and evaluation of experiments with opioid antagonists. These tools and recommendations enable selection of appropriate antagonists, doses and assessment time points, and determination of the achieved receptor blockade in previous studies.


title: TMS over dorsolateral prefrontal cortex affects the timing of motor imagery but not overt action: further support for the Motor-Cognitive model

abstract: The Motor-Cognitive model suggests a functional dissociation between motor imagery and overt action, in contrast to the traditional notion of shared processes central to the Functional Equivalence view. According to the Motor-Cognitive model, motor imagery differs from overt action primarily through the use of executive resources to monitor and elaborate a motor image during execution. This can result in a lack of correspondence between motor imagery and its overt action counterpart. The present study examined the importance of executive resources in motor imagery using TMS to impair the function of the dorsolateral prefrontal cortex, while measuring the time to complete imagined versus overt actions. In two experiments, TMS over the dorsolateral prefrontal cortex slowed motor imagery but did not affect overt actions. TMS over the same region also interfered with performance of a mental calculation task, though it did not reliably affect less demanding cognitive tasks also thought to rely on executive functions. Taken together, these results were consistent with the Motor-Cognitive model but not with the idea of functional equivalence. The implications of these results for the theoretical understanding of motor imagery, and potential applications of the Motor-Cognitive model to the use of motor imagery in training and rehabilitation, are discussed.


title: A molecular mechanism for high salt taste in Drosophila

abstract: Dietary salt detection and consumption are crucial to maintaining fluid and ionic homeostasis. To optimize salt intake, animals employ salt-dependent activation of multiple taste pathways. Generally, sodium activates attractive taste cells, but attraction is overridden at high salt concentrations by cation non-selective activation of aversive taste cells. In flies, high salt avoidance is driven by both  bitter taste neurons and a class of glutamatergic  high salt neurons expressing pickpocket23 (ppk23). Although the cellular basis of salt taste has been described, many of the molecular mechanisms remain elusive. Here, we show that ionotropic receptor 7c (IR7c) is expressed in glutamatergic high salt neurons, where it functions with co-receptors IR76b and IR25a to detect high salt. Misexpression of IR7c in sweet neurons, which endogenously express IR76b and IR25a, confers responsiveness to non-sodium salts, indicating that IR7c is sufficient to convert a sodium-selective receptor to a cation non-selective receptor. Furthermore, the resultant transformation of taste neuron tuning switches potassium chloride from an aversive to an attractive tastant. This research provides insight into the molecular basis of monovalent and divalent salt taste coding and the full repertoire of IRs needed to form a functional salt receptor.


title: Conserved neuropeptidergic regulation of intestinal integrity in invertebrate models of aging

abstract: Age-related decline in intestinal barrier function impacts survival across species, but the underlying cell intrinsic and extrinsic factors remain unclear. Here, we demonstrate a role for neuropeptides in regulating aging-associated increases in intestinal leakiness. Adult-specific knockdown of insulin-like peptides daf-28 or ins-7 in C. elegans neurons or dilp3 in D. melanogaster neurons improves intestinal integrity and lifespan of these animals, respectively. Neuropeptide knockdowns activate intestinal DAF-16/FOXO transcription factor that likely increases expression of epithelial barrier components. Furthermore, age-associated changes in neuronal DAF-28 peptide secretion mirrors C. elegans reproductive span and overexpression of this peptide suppresses the improved intestinal health in long-lived germline-less animals. Collectively, we show that intestinal integrity is subject to neuronal regulation, and this neuropeptidergic axis may be modulated by the animals germline.


title: The decision to develop into αβ or γδ T cells is pre-programmed in distinct subpopulations of DN1 thymocytes

abstract: T cells are divided into the {beta} and {gamma}{delta} lineages. It is currently thought that these lineages differentiate in the thymus from uncommitted progenitor thymocytes. However, in this study we show that the decision to differentiate into one or either lineage is in fact already fixed in these apparent uncommitted progenitors. Using single-cell RNA sequencing, we reassemble de novo a model of early T cell development based on the transcriptional profiles of individual CD4-CD8- double negative and {gamma}{delta} thymocytes. We show that the earliest thymocyte stage, known as CD4-CD8- double negative 1 (DN1), is actually comprised of a mixture of transcriptionally distinct subpopulations. Although not yet expressing definitive markers of {beta} and {gamma}{delta} lineages, such as the lineage-defining T cell receptors, specific DN subpopulations exhibit restricted developmental potential for either {beta} or {gamma}{delta} lineage. Furthermore, specific {gamma}{delta}-primed DN1 subpopulations preferentially develop into IL-17 or IFN{gamma}-producing {gamma}{delta} T cells. Thus, T cell lineage decisions are hardwired from the earliest stages of T cell development.

category: immunology


title: Machine learning meets classical computer vision for accurate cell identification

abstract: High-parameter multiplex immunostaining techniques have revolutionized our ability to image healthy and diseased tissues with unprecedented depth; however, accurate cell identification and segmentation remain significant downstream challenges. Identifying individual cells with high precision is a requisite to reliably and reproducibly interpret acquired data. Here we introduce CIRCLE, a cell identification pipeline that combines classical and modern machine learning-based computer vision algorithms to address the shortcomings of current cell segmentation tools for 2D images. CIRCLE is a fully automated hybrid cell detection model, eliminating subjective investigator bias and enabling high-throughput image analysis. CIRCLE accurately distinguishes cells across diverse tissues microenvironments, resolves low-resolution structures, and can be applied to any 2D image that contains nuclei. Importantly, we quantitatively demonstrate that CIRCLE outperforms current state-of-the-art image segmentation tools using multiple accuracy measures. As high-throughput multiplex imaging grows closer toward standard practice for histology, integration of CIRCLE into analysis protocols will deliver unparalleled segmentation quality.

category: cell biology


title: Crosstalk between β-catenin and WT1 signalling activity in acute myeloid leukemia

abstract:
Wnt signalling is an evolutionary conserved signal transduction pathway heavily implicated in normal development and disease. The central mediator of this pathway, {beta}-catenin, is frequently overexpressed, mislocalised and overactive in acute myeloid leukaemia (AML) where it mediates the establishment, maintenance and drug resistance of leukaemia stem cells. Critical to the stability, localisation and activity of {beta}-catenin are the protein-protein interactions it forms, yet these are poorly defined in AML. We recently performed the first {beta}-catenin interactome study in blood cells of any kind and identified a plethora of novel interacting partners. This study shows for the first time that {beta}-catenin interacts with Wilms tumour protein (WT1), a protein frequently overexpressed and mutated in AML, in both myeloid cell lines and also primary AML samples. We demonstrate crosstalk between the signalling activity of these two proteins in myeloid cells, and show that modulation of either protein can affect expression of the other. Finally, we demonstrate that WT1 mutations frequently observed in AML can increase stabilise {beta}-catenin and augment Wnt signalling output. This study has uncovered new context-dependent molecular interactions for {beta}-catenin which could inform future therapeutic strategies to target this dysregulated molecule in AML.

Article summaryO_LI{beta}-Catenin is frequently dysregulated in acute myeloid leukemia (AML) and protein interactions govern its stability, localization and activity, but these are poorly defined in AML.
C_LIO_LIThis study shows for the first time that {beta}-catenin and Wilms tumour protein (WT1) interact and influence each others expression level and signalling activity in AML cells, which could inform future therapeutic strategies.
C_LI

category: cancer biology


title: Molecular and mineral responses of corals grown under artificial Calcite Sea conditions

abstract: The formation of skeletal structures composed of different calcium carbonate polymorphs (aragonite and calcite) appears to be regulated both biologically and environmentally. Among environmental factors influencing aragonite and calcite precipitation, changes in sea water conditions - primarily in the molar ratio of magnesium and calcium during so-called "Calcite" (mMg:mCa below 2) or "Aragonite" seas (mMg:mCa above 2) - have had profound impacts on the distribution and performance of marine calcifiers throughout the Earths history. Nonetheless, the fossil record shows that some species appear to have counteracted such changes and kept their skeleton polymorph unaltered. Here, the aragonitic octocoral Heliopora coerulea and the aragonitic scleractinian Montipora digitata were exposed to Calcite Sea-like mMg:mCa with various levels of changes in magnesium and calcium concentration, and both mineralogical (i.e.,CaCO3 polymorph) and gene expression changes were monitored. The two species exhibited different responses: no calcite deposition could be detected in H. coerulea, while M. digitata presented considerable amounts of calcite under mMg:mCa of 1.5. Nonetheless, aragonite was the main skeleton constituent in both species and under both treatment conditions. Despite a strong variability between experimental replicates, expression for a set of putative calcification-related genes, including known components of scleractinians skeleton organic matrix, was found to consistently change when mMg:mCa was lowered. These results are consistent with the hypothesis of an involvement of the skeleton organic matrix in counteracting decreases in seawater mMg:mCa. On the other hand, no clear changes in expression for calcium and magnesium transporters were detected. Nonetheless, different calcium channels were found downregulated in H. coerulea when exposed to mMg:mCa of 2.5, pointing to an active calcium uptake regulation by the corals under altered mMg:mCa to maintain aragonite deposition.

category: paleontology


title: Variation of sexual dimorphism of wing shape in the family Dolichopodidae (Diptera)

abstract: Although sexual dimorphism manifestations are widespread in the family Dolichopodidae, a detailed characterization of their phylogenetic significance is lacking. Purpose: in order to study the distribution patterns of wing sexual dimorphism, we analysed 57 species from 17 genera of 9 subfamilies. Methods: A comparative analysis of the evidence, obtained by geometric morphometry and molecular data, allowed us to assess the phylogenetic signal in the sexual dimorphism of the wing. Results: The results of the study confirm the presence of diverse patterns of sexual variability in the wings of this family. More often, females have larger wings with blunted apexes, whereas males are characterized by a more pointed apex. In some cases, the larger size of females wings is associated with an increase in body size, while in other cases, differences in shape and size can be explained by differences in behavioural and life patterns. Although there exists a general pattern of sexual dimorphism, its features differ even in closely related species. Conclusion: the absence of a significant phylogenetic signal in seven out of nine studied wing points indicates that the sexual dimorphism in form evolved, at least partially, in each of the studied species.


title: Deciphering the developmental patterns of white matter tracts in a diffusion MRI based fetal brain atlas

abstract:
White matter of the fetal brain undergoes rapid development to form the early structural connections. Diffusion MRI has shown to be a useful tool to depict the fetal brain white matter in utero, and many studies have observed increasing fractional anisotropy and decreasing mean diffusivity in the fetal brain during the second-to-third trimester, whereas others reported non-monotonic changes. Unbiased diffusion MRI atlases of the fetal brain are important for characterizing developmental trajectories of white matter and providing normative references for in-utero diagnosis of prenatal abnormalities. To date, the sole fetal brain diffusion MRI atlas was collected from a Caucasian/mixed population, and was constructed based on the diffusion tensor model with limited spatial resolution.

In this work, we proposed a fiber orientation distribution (FOD) based pipeline for the generation of fetal brain diffusion MRI atlases, which showed better registration accuracy than diffusion tensor-based pipeline. Based on the FOD pipeline, we constructed the first Chinese fetal brain diffusion MRI atlas using 89 normal fetal diffusion MRI scans at GA between 24 and 38 weeks. Complex non-monotonic trends of tensor- and FOD-derived microstructural parameters in eight white matter tracts were observed, which jointly pointed to different phases of microstructural development. Specifically, we speculated that the turning point of the diffusivity trajectory may correspond to the starting point of pre-myelination, based on which, the developmental order of white matter tracts can be mapped and the order was in agreement with order of myelination from histological studies. The normative atlas also provided a reference for detection of abnormal white matter development, such as that in congenital heart disease. Therefore, the high-order fetal brain diffusion MRI atlas established in this study depicted the spatiotemporal pattern of early white matter development, and findings may help decipher the distinct microstructural events in utero.


title: Deciphering the developmental order and microstructural patterns of early white matter pathways in a diffusion MRI based fetal brain atlas


title: A Bayesian approach to single-particle electron cryo-tomography in RELION-4.0

abstract: We present a new approach for macromolecular structure determination from multiple particles in electron cryo-tomography (cryo-ET) data sets. Whereas existing subtomogram averaging approaches are based on 3D data models, we propose to optimise a regularised likelihood target that approximates a function of the 2D experimental images. In addition, analogous to Bayesian polishing and CTF refinement in single-particle analysis, we describe approaches that exploit the increased signal-to-noise ratio in the averaged structure to optimise tilt series alignments, beam-induced motions of the particles throughout the tilt series acquisition, defoci of the individual particles, as well as higher-order optical aberrations of the microscope. Implementation of our approaches in the open-source software package RELION aims to facilitate their general use, in particular for those researchers who are already familiar with its single-particle analysis tools. We illustrate for two cases that our approaches allow structure determination from cryo-ET data to resolutions sufficient for de novo atomic modelling.

category: biophysics


title: Why link diverse citizen science surveys? Widespread arboreal habits of a terrestrial amphibian revealed by mammalian tree surveys in Britain

abstract: Terrestrial anurans, with their typically short limbs, heavy-set bodies and absent claws or toe pads are incongruous tree climbers, but even occasional arboreal locomotion could offer substantial advantages for evading predators or accessing new shelter or food resources. Despite recent interest, arboreal behaviour remains rarely and unsystematically described for terrestrial amphibians in Europe, likely due to fundamental differences in survey methods and therefore a lack of field data. However, other taxa surveys specifically target trees and tree cavities. We undertook collaborations and large-scale data searches with citizen science projects surveying for arboreal mammals in Britain to investigate potential tree climbing by amphibians at a national scale. Remarkably, we found widespread arboreal usage by amphibians in England and Wales, with occupancy of hazel dormouse (Muscardinus avellenarius) nest boxes, tree cavities investigated as potential bat roosts and even a bird nest by common toads (Bufo bufo), but few additional records of frogs or newts. Toads are potentially attracted to tree cavities and arboreal nests because they provide safe and damp microenvironments which can support an abundance of invertebrate prey but the importance of such tree microhabitats for toad conservation remains unknown. Possible interactions with arboreal mammals are also unclear, but such mammals and even some birds may benefit from the occasional presence of toads if they feed on the mites and other arthropods that frequently infest their nests. We encourage expanding and linking of unrelated monitoring surveys and citizen science initiatives as valuable tools for investigating ecological traits and interactions.

category: ecology


title: Detection of nervous necrosis virus RGNNV genotype in pearl gentian grouper (Epinephelus lanceolatus male x E. fuscoguttatus female) fry imported to Thailand

abstract:
Nervous necrosis virus (NNV) is a deadly virus that affects more than 120 fish species worldwide, but there is little information about it in Thailand. In August 2019, a population of pearl gentian grouper fry imported to Thailand experienced mass mortality. The diseased fish exhibited darkening, floating on the water surface,  sleepy behaviour, and erratic swimming. We received a set of samples for disease diagnosis. PCR analysis revealed that these samples were positive for NNV but negative for Megalocytivirus ISKNV and Ranavirus. Sequencing the viruss genome and phylogenetic analysis revealed that it is a member of the red-spotted grouper nervous necrosis virus (RGNNV) genotype. In situ hybridization using an NNV-specific probe revealed localization of the virus in the vacuolation lesions of the central nervous system (brain and spinal cord) and the retina of infected fish. The virus was successfully isolated from tissue homogenate from diseased fish using the E-11 cell line. This study identified NNV (RGNNV genotype) as a virus associated with massive die-offs in imported pearl grouper fry in Thailand. Considering the infectious nature of the virus and its broad host range, appropriate biosecurity measures are needed to prevent any loss to the marine aquaculture industry.

HighlightsO_LIThis study reports detection of NNV in grouper larvae imported to Thailand
C_LIO_LIThe virus was assigned to the RGNNV genotype, which is the most extensively distributed genotype
C_LIO_LIHistopathology revealed a pathognomonic lesion of NNV
C_LIO_LIISH using an NNV-specific probe revealed localization of NNV in the CNS and retina
C_LIO_LIThe virus was successfully propagated from diseased fish using the E-11 cell line
C_LI

category: pathology


title: Widening the landscape of transcriptional regulation of algal photoprotection

abstract: Availability of light and CO2, substrates of microalgae photosynthesis, is frequently far from optimal. Microalgae activate photoprotection under strong light, to prevent oxidative damage, and the CO2 Concentrating Mechanism (CCM) under low CO2, to raise intracellular CO2 levels. The two processes are interconnected; yet, the underlying transcriptional regulators remain largely unknown. By employing a large transcriptomics data compendium of Chlamydomonas reinhardtiis responses to different light and carbon supply we reconstructed a consensus genome-scale gene regulatory network from complementary inference approaches and used it to elucidate the transcriptional regulation of photoprotection. We showed that the CCM regulator LCR1 also controls photoprotection, and that QER7, a Squamosa Binding Protein, suppresses photoprotection- and CCM-gene expression under the control of the blue light photoreceptor Phototropin. Along with demonstrating the existence of regulatory hubs that channel light- and CO2-mediated signals into a common response, our study provides a unique resource to dissect gene expression regulation in this microalga.

category: plant biology


title: Telomerase deficiency in humans is associated with systemic age-related changes in energy metabolism

abstract:
Underlying mechanisms of plasma metabolite signatures of human ageing and age-related diseases are not clear but telomere attrition and dysfunction are central to both. Dyskeratosis Congenita (DC) is associated with mutations in the telomerase enzyme complex (TERT, TERC, DKC1) and progressive telomere attrition. We show extracellular citrate is repressed by canonical telomerase function in vitro and associated with DC leukocyte telomere attrition in vivo; leading to the hypothesis that altered citrate metabolism detects telomere dysfunction. However, citrate and senescence factors only weakly distinguished DC patients from controls, whereas other tricarboxylic acid cycle metabolites, lactate and especially pyruvate distinguished them with high significance, consistent with further metabolism of citrate and lactate in the liver and kidneys. Citrate uptake in certain organs modulates age-related disease in mice and our data has similarities with age-related disease signatures in humans. Our results have implications for the early diagnosis of telomere dysfunction and anti-senescence therapeutics.

HighlightsO_LIExtracellular citrate is regulated by telomere function in vitro and in vivo.
C_LIO_LIDyskeratosis Congenita (DC) is a human disease characterized by systemic telomere attrition, which showed an age-related plasma energetic profile, distinct from age-related disease and that of centenarians.
C_LIO_LIThe DC profile strikingly out-performed senescence factors in discriminating DC from controls, and pyruvate associated with a low lactate:pyruvate ratio is potentially a useful and cheap minimally invasive diagnostic aid for DC and telomere dysfunction.
C_LIO_LIMechanistically DC systemic metabolism is indicative of a shift to reduced pyruvate dehydrogenase activity, glycolysis and/or increased citrate and lactate production followed by further metabolism in the kidneys and liver.
C_LI


title: Loading-Induced Bone Formation is Mediated by Wnt1 Induction in Osteoblast-Lineage Cells

abstract: Mechanical loading on the skeleton stimulates bone formation. Although the exact mechanism underlying this process remains unknown, a growing body of evidence indicates that the Wnt signaling pathway is necessary for the skeletal response to loading. Recently, we showed that Wnts produced by osteoblast lineage cells mediate the osteo-anabolic response to tibial loading in adult mice. Here, we report that Wnt1 specifically plays a crucial role in mediating the mechano-adaptive response to loading. Independent of loading, short-term loss of Wnt1 in the Osx-lineage resulted in decreased cortical bone area in the tibias of 5-month old mice. In females, strain-matched loading enhanced periosteal bone formation in Wnt1F/F controls, but not in Wnt1F/F; OsxCreERT2 knockouts. In males, strain-matched loading increased periosteal bone formation in both control and knockout mice; however, the periosteal relative bone formation rate was 65% lower in Wnt1 knockouts versus controls. Together, these findings show that Wnt1 supports adult bone homeostasis and mediates the bone anabolic response to mechanical loading.


title: Interrogating Metabolic Interactions Between Skeletal Muscle and Liver Circadian Clocks In Vivo

abstract: Expressed throughout the body, the circadian clock system achieves daily metabolic homeostasis at every level of physiology, with clock disruption associated with metabolic disease (1, 2). Molecular clocks present in the brain, liver, adipose, pancreas and skeletal muscle each contribute to glucose homeostasis (3). However, it is unclear; 1) which organ clocks provide the most essential contributions, and 2) if these contributions depend on inter-organ communication. We recently showed that the liver clock alone is insufficient for most aspects of daily liver glucose handling and requires connections with other clocks (4). Considering the pathways that link glucose metabolism between liver and skeletal muscle, we sought to test whether a clock connection along this axis is important. Using our previous published methodology for tissue-specific rescue of Bmal1 in vivo (4, 5), we now show that in the absence of feeding-fasting cycles, liver and muscle clocks are not sufficient for systemic glucose metabolism, nor do they form a functional connection influencing local glucose handling or daily transcriptional rhythms in each tissue. However, the introduction of a daily feeding-fasting rhythm enables a synergistic state between liver and muscle clocks that leads to restoration of systemic glucose tolerance. These findings reveal limited autonomous capabilities of liver and muscle clocks and highlight the need for inter-organ clock communication for glucose homeostasis which involves at least two peripheral metabolic organs.


title: RNA binding is essential for NONO condensates to modulate pre-mRNA processing in neuroblastoma

abstract: High-risk neuroblastoma patients have poor survival rates and require better therapeutic options. High expression of a multifunctional DNA and RNA binding protein, NONO, in neuroblastoma is associated with poor patient outcome, however there is little understanding of the mechanism of NONO-dependent oncogenic gene regulatory activity in neuroblastoma. Here, we used cell imaging, biophysical and molecular analysis to reveal complex NONO-dependent regulation of gene expression, finding that NONO forms RNA- and DNA-tethered phase-separated condensates throughout the nucleus. CLIP analyses show that NONO mainly binds to the 5 end of pre-mRNAs and modulates pre-mRNA processing, dependent on its RNA binding activity. NONO preferentially regulates super enhancer-associated genes, including HAND2 and GATA2. In the absence of functional NONO-RNA condensates, inefficient pre-mRNA processing at these loci leads to decreased expression of HAND2 and GATA2. Thus, future development of agents that target RNA binding activity of NONO may have therapeutic potential in this cancer context.


title: Maturation of cortical endoplasmic reticulum clusters in the mouse oocyte: changes at fertilization.

abstract:
Oocytes from many invertebrate and vertebrate species exhibit unique endoplasmic reticulum specializations (cortical ER clusters) thought be essential for egg activation. In examination of cortical ER clusters, we observed they were tethered to previously unreported fenestrae within the cortical actin layer. Further, studies demonstrated sperm preferentially bind to plasma membrane overlying the fenestrae, establishing close proximity to underlying ER clusters. Moreover, following sperm-oocyte fusion, cortical ER clusters undergo a previously unrecognized global maturational change in volume, shape, and calreticulin content that persists through sperm incorporation, before dispersing at the pronuclear stage. These changes did not occur in oocytes from females mated with Izumo1 -/- males demonstrating that gamete fusion plays an important role in ER cluster maturation. In addition to these global changes seen at sites distant to the sperm, highly localized ER modifications were noted at the sperm binding site as cortical ER clusters surround the sperm head during incorporation, then form a diffuse cloud surrounding the decondensing sperm nucleus. This study provides the first evidence that cortical ER clusters interact with the fertilizing sperm, indirectly through a previous unknown lattice work of actin fenestrae, then directly during sperm incorporation. These observations raise the possibility that oocyte ER cluster-sperm interactions provide a competitive advantage to the oocyte, which may not occur during assisted reproductive technologies such as intracytoplasmic sperm injection.

Summary StatementSperm-oocyte interactions stimulate global changes in cortical endoplasmic reticulum cluster structure as well as localized responses at the sperm binding site.

category: developmental biology


title: LipidMS 3.0: an R-package and a web-based tool for LC-MS/MS data processing and lipid annotation

abstract:
SummaryLipidMS was initially envisioned to use fragmentation rules and data-independent acquisition (DIA) for lipid annotation. However, data-dependent acquisition (DDA) remains the most widespread acquisition mode for untargeted LC-MS/MS-based lipidomics. Here we present LipidMS 3.0, an R package that not only adds DDA and new lipid classes to its pipeline, but also the required functionalities to cover the whole data analysis workflow from pre-processing (i.e., peak-peaking, alignment and grouping) to lipid annotation. We applied the new workflow in the analysis of a serum dataset acquired in MS, DDA and DIA modes. Our results show that LipidMS 3.0 data pre-processing outperforms XCMS and complements those lipids annotated using MS-DIAL, one of the most widely used tools in lipidomics. To extend and facilitate LipidMS 3.0 usage among less experienced R-programming users the workflow has been also implemented as a web-based application.

Availability and ImplementationLipidMS R-package is freely available at https://CRAN.R-project.org/package=LipidMS and as a website at http://www.lipidms.com.


title: Memory Consolidation with Orthogonal Gradients for avoiding Catastrophic Forgetting

abstract:
The memory consolidation process enables the accumulation of recent and remote memories in the long-term memory store. In general, the deep network models of memory suffer from forgetting old information while learning new information, called catastrophic forgetting/interference. The human brain overcomes this problem quite effectively, a problem that continues to challenge current deep neural network models.

We propose a regularization-based model to solve the problem of catastrophic forgetting. According to the proposed training mechanism, the network parameters are constrained to vary in a direction orthogonal to the average of the error gradients corresponding to the previous tasks. We also ensure that the constraint used in parameter updating satisfies the locality principle.

The proposed models performance is compared with Elastic Weight Consolidation on standard datasets such as permuted MNIST and split MNIST on classification tasks using fully connected networks, and Convolution-based networks. The model performance is also compared to an autoencoder on split MNIST dataset, and to complex core50 dataset on two types of classification tasks with EWC.

The proposed model gives a new view on plasticity at the neuronal level. In the proposed model, the parameter updating is controlled by the neuronal level plasticity rather than synapse level plasticity as in other standard models. The biological plausibility of the proposed model is discussed by linking the extra parameters to synaptic tagging, which represents the state of the synapse involved in Long Term Potentiation (LTP).


title: Data based slurry treatment decision tree to minimize antibiotic resistance and pathogen transfer while maximizing nutrient recycling

abstract:
Direct application of pig slurry to agricultural land, as a means of nutrient recycling, introduces pathogens, antibiotic resistant bacteria, or genes, to the environment. With global environmental sustainability policies mandating a reduction in synthetic fertilisation and a commitment to a circular economy it is imperative to find effective on-farm treatments of slurry that maximises its fertilisation value and minimises risk to health and the environment. We assessed and compared the effect of storage, composting, and anaerobic digestion on pig slurry microbiome, resistome and nutrient content. Shotgun metagenomic sequencing and HT-qPCR arrays were implemented to understand the dynamics across the treatments. Our results identified that each of the treatment methods had advantages and disadvantages, depending on the parameter measured. The data suggests that storage and composting are optimal for the removal of human pathogens and anaerobic digestion for the reduction in AMR genes and mobile genetic elements. The nitrogen content is increased in storage and AD and reduced in composting. Thus, depending on the requirement for increased or reduced nitrogen the optimum treatment varies. Combining the results indicates that composting provides the greatest gain by reducing risk to human health and the environment. Network analysis revealed reducing Proteobacteria and Bacteroidetes while increasing Firmicutes will reduce the AMR content. KEGG analysis identified no significant change in the pathways across all treatments. This novel study provides a data driven decision tree to determine the optimal treatment for best practice to minimise pathogen, AMR and excess or increasing nutrient transfer from slurry to environment.

Graphical abstract

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category: microbiology


title: Increased birth rank of homosexual males: disentangling the older brother effect and sexual antagonism hypothesis

abstract: Male homosexual orientation remains a Darwinian paradox, as there is no consensus on its evolutionary determinants. One intriguing feature of homosexual men is their higher male birth rank compared to heterosexual men. This can be explained by two non-exclusive mechanisms: an antagonistic effect (AE), implying that more fertile women have a higher chance of having a homosexual son, or an older brother effect (OBE), where each additional older brother increases the chances for a male embryo to develop a homosexual orientation due to an immunoreactivity process. However, there is no consensus on whether both OBE and AE are present in human populations, or if only one of these mechanisms is at play and its effect mimicking the signature of the other mechanism. An additional older sister effect (OSE) has also recently been proposed. To clarify this situation, we developed theoretical and statistical tools to study OBE and AE independently or in combination, taking into account all known sampling biases. These tools were applied on new individual data, and on various available published data (two individual datasets, all relevant aggregated data, and twin data). Support for OBE was apparent in aggregated data, with the OBE increasing linearly with fertility. The OBE was also supported in two individual datasets. An OSE seems to result from a sampling bias in presence of OBE, and is likely to be artefactual. AE was not supported, either in individual datasets, including the analysis of the extended maternal family, nor in twin data. The evolutionary implications of these findings are discussed.


title: Cellular deconstruction of inflamed synovium defines diverse inflammatory phenotypes in rheumatoid arthritis

abstract: Rheumatoid arthritis (RA) is a prototypical autoimmune disease that causes destructive tissue inflammation in joints and elsewhere. Clinical challenges in RA include the empirical selection of drugs to treat patients, inadequate responders with incomplete disease remission, and lack of a cure. We profiled the full spectrum of cells in inflamed synovium from patients with RA with the goal of deconstructing the cell states and pathways characterizing pathogenic heterogeneity in RA. Our multicenter consortium effort used multi-modal CITE-seq, RNA-seq, and histology of synovial tissue from 79 donors to build a >314,000 single-cell RA synovial cell atlas with 77 cell states from T, B/plasma, natural killer, myeloid, stromal, and endothelial cells. We stratified tissue samples into six distinct cell type abundance phenotypes (CTAPs) individually enriched for specific cell states. These CTAPs demonstrate the striking diversity of RA synovial inflammation, ranging from marked enrichment of T and B cells (CTAP-TB) to a congregation of specific myeloid, fibroblast, and endothelial cells largely lacking lymphocytes (CTAP-EFM). Disease-relevant cytokines, histology, and serology metrics are associated with certain CTAPs. This comprehensive RA synovial atlas and molecular, tissue-based CTAP stratification reveal new insights into RA pathology and heterogeneity, which could lead to novel targeted-treatment approaches in RA.

category: genomics


title: The role of liver fat in cardiometabolic diseases is highlighted by genome-wide association study of MRI-derived measures of body composition

abstract:
Background & AimsObesity and associated morbidities, metabolic associated liver disease (MAFLD) included, constitute some of the largest public health threats worldwide. Body composition and related risk factors are known to be heritable and identification of their genetic determinants may aid in the development of better prevention and treatment strategies. Recently, large-scale whole-body MRI data has become available, providing more specific measures of body composition than anthropometrics such as body mass index. Here, we aimed to elucidate the genetic architecture of body composition, by conducting the first genome-wide association study (GWAS) of these MRI-derived measures.

MethodsWe ran both univariate and multivariate GWAS on fourteen MRI-derived measurements of adipose and muscle tissue distribution, derived from scans from 34,036 White European UK Biobank participants (mean age of 64.5 years, 51.5% female).

ResultsThrough multivariate analysis, we discovered 108 loci with distributed effects across the body composition measures and 256 significant genes primarily involved in immune system functioning. Liver fat stood out, with a highly discoverable and oligogenic architecture and the strongest genetic associations. Comparison with 21 common cardiometabolic traits revealed both shared and specific genetic influences, with higher mean heritability for the MRI measures (h2=.25 vs. .16, p=1.4x10-6). We found substantial genetic correlations between the body composition measures and a range of cardiometabolic diseases, with the strongest correlation between liver fat and type 2 diabetes (rg=.48, p=1.6x10-22).

ConclusionsThese findings show that MRI-derived body composition measures complement conventional body anthropometrics and other biomarkers of cardiometabolic health, highlighting the central role of liver fat, and improving our knowledge of the genetic architecture of body composition and related diseases.


title: Optimization of Neuroprosthetic Vision via End-to-end Deep Reinforcement Learning

abstract: Visual neuroprostheses are a promising approach to restore basic sight in visually impaired people. A major challenge is to condense the sensory information contained in a complex environment into meaningful stimulation patterns at low spatial and temporal resolution. Previous approaches considered task-agnostic feature extractors such as edge detectors or semantic segmentation, which are likely suboptimal for specific tasks in complex dynamic environments. As an alternative approach, we propose to optimize stimulation patterns by end-to-end training of a feature extractor using deep reinforcement learning agents in virtual environments. We present a task-oriented evaluation framework to compare different stimulus generation mechanisms, such as static edge-based and adaptive end-to-end approaches like the one introduced here. Our experiments in Atari games show that stimulation patterns obtained via task-dependent end-to-end optimized reinforcement learning result in equivalent or improved performance compared to fixed feature extractors on high difficulty levels. These findings signify the relevance of adaptive reinforcement learning for neuroprosthetic vision in complex environments.


title: Automated, Reproducible Investigation of gene set Differential Enrichment via the AUTO-go framework

abstract:
Reproducibility in Life Sciences is challenged in the analysis of large multi-omics datasets. One of the final steps of said processes is Gene Set enrichment, where web tools represent a valuable resource but not a reliable surrogate for standardized, high-quality visualizations. The AUTO-go framework proposes standardization of the Gene Functional Enrichment process along with an R framework able to produce high-quality visualization in an automated manner, improving the reproducibility of the whole analytical process. We present three use cases in Cancer Transcriptomics and Epigenomics datasets as a proof-of-concept to visualize Multiple Differential Expression and Single Sample Gene Set Enrichment Analysis.

Author SummaryBioinformatics and Data Science are routinely challenged to distill intelligible results from huge amounts of data. These results, in turn, are conveyed through plots and visualizations that should be easily reproducible for scientific soundness and ethical reasons. A specific area in which these analyses are of critical importance is Genomics, where Genes functions need to be enriched when comparing pathological states or treatments. Here we present a software framework that aims at standardizing said differential analyses and visualizations when dealing with genomics data. Finally, we show how it can be employed to shear light on publicly available datasets, even in small casuistry of Rare Cancers.


title: Photizo: an open-source library for cross-sample analysis of FTIR spectroscopy data

abstract:
With continually improved instrumentation, Fourier transform infrared (FTIR) microspectroscopy can now be used to capture thousands of high-resolution spectra for chemical characterisation of a sample. The spatially resolved nature of this method lends itself well to histological characterisation of complex biological specimens. However, commercial software currently available can make joint analysis of multiple samples challenging and, for large datasets, computationally infeasible. In order to overcome these limitations, we have developed Photizo - an open-source Python library for spectral analysis which includes functions for pre-processing, visualisation and downstream analysis, including principal component analysis, clustering, macromolecular quantification and biochemical mapping. This library can be used for analysis of spectroscopy data without a spatial component, as well as spatially-resolved data, such as data obtained via infrared (IR) microspectroscopy in scanning mode and IR imaging by focal plane array (FPA) detector.

Contactcdendrou@well.ox.ac.uk


title: The Development of 2-stage Microfermentation Protocols for High Throughput Cell Factory Evaluations

abstract: Cell based factories can be engineered to produce a wide variety of products. Advances in DNA synthesis and genome editing have greatly simplified the design and construction of these factories. It has never been easier to generate hundreds or even thousands of cell factory strain variants for evaluation. These advances have amplified the need for standardized, higher throughput means of evaluating these designs. Toward this goal, we have previously reported the development of engineered E. coli strains and associated 2-stage production processes to simplify and standardize strain engineering, evaluation and scale up. This approach relies on decoupling growth (stage 1), from production, which occurs in stationary phase (stage 2). Phosphate depletion is used as the trigger to stop growth as well as induce heterologous expression. Here, we describe in detail the development of optimal protocols used for the evaluation of engineered E. coli strains in 2-stage microfermentations. These protocols are readily adaptable to the evaluation of strains producing a wide variety of protein as well as small molecule products. Additionally, the development approach described is adaptable to additional cellular hosts, as well as other 2-stage processes with various additional triggers.

category: synthetic biology


title: Fast and functionally specific cortical thickness changes induced by visual stimulation

abstract: Structural characteristics of the human brain serve as important markers of brain development, aging, disease progression and neural plasticity. They are considered stable properties, changing slowly over time. Multiple recent studies reported that structural brain changes measured with MRI may occur much faster than previously thought, happening on a time scale as fast as several minutes. The mechanisms behind such fast changes remain unclear, with hemodynamics as one possible explanation. Here we investigated the functional specificity of cortical thickness changes induced by a flickering checkerboard stimulus. We also compared the thickness effects to those measured with BOLD fMRI. We found that checkerboard stimulation led to a significant thickness increase, which was functionally specific to V1 and spatially confined to the retinotopic representation of the checkerboard stimulus. Although functional specificity and the effect size of these changes were comparable to those of the BOLD signal in V1, thickness effects were substantially weaker in V3. Furthermore, a comparison of predicted and measured thickness changes for different stimulus timings suggested a slow increase of thickness over time, speaking against a hemodynamic explanation. Altogether, our findings suggest that visual stimulation can induce structural gray matter enlargement measurable with MRI.

category: neuroscience


title: Hippocampal GFAP-positive astrocyte responses to amyloid and tau pathologies

abstract:
IntroductionIn Alzheimers disease clinical research, glial fibrillary acidic protein (GFAP) released in the cerebrospinal fluid and blood is widely measured and perceived as a biomarker of reactive astrogliosis. However, it was recently demonstrated that plasma GFAP levels are associated with amyloid-{beta} (A{beta}) but not tau pathology. The molecular underpinnings behind this specificity are unexplored. Here we investigated biomarker and transcriptomic associations of GFAP-positive astrocytes with A{beta} and tau pathologies in humans and mouse models.

MethodsWe studied 90 individuals with plasma GFAP, A{beta}- and Tau-PET to investigate the association between biomarkers. Then, transcriptomic analysis in hippocampal GFAP-positive astrocytes isolated from mouse models presenting A{beta} (PS2APP) or tau (P301S) pathologies was applied to explore differentially expressed genes (DEGs), Gene Ontology processes, and protein-protein interaction networks associated with each phenotype.

FindingsIn humans, we found that plasma GFAP associates with A{beta} but not tau pathology. Supporting these results, mouse transcriptomics showed scarce overlap of DEGs between the A{beta} and tau mouse models, revealing the unique nature of GFAP-positive astrocytic responses to A{beta} or tau pathology. While A{beta} GFAP-positive astrocytes were overrepresented with genes associated with proteostasis and exocytosis-related processes, tau hippocampal GFAP-positive astrocytes presented greater abnormalities in functions related to DNA/RNA processing and cytoskeleton dynamics.

InterpretationOur results offer insights into A{beta}- and tau-driven specific signatures in GFAP-positive astrocytes. Characterizing how different underlying pathologies distinctly influence astrocyte responses is critical for the biological interpretation of astrocyte-related biomarker and suggests the need to develop context-specific astrocyte targets to study AD.

FundingThis study was supported by Instituto Serrapilheira, Alzheimers Association, CAPES, CNPq and FAPERGS.

Evidence before the studyThe glial fibrillary acidic protein (GFAP) is commonly used as a proxy of reactive astrogliosis in Alzheimers disease (AD) clinical studies. Recent evidence demonstrated that increases in GFAP levels are associated with a specific aspect of AD pathology, namely amyloid-{beta} (A{beta}). However, the biological interpretation behind it was not explored in depth molecularly. Uncovering the phenotypes acquired by GFAP-positive astrocytes under A{beta} or tau influence might directly impact clinical research by improving the biological interpretation of clinical glial biomarkers used in AD.

Added value of this studyThis study revealed molecular signatures driven by AD hallmarks in hippocampal GFAP-positive astrocytes, including 1) a scarce overlap of differentially expressed genes (DEG) driven by A{beta} and tau; 2) abnormalities in proteostasis and exocytosis-related processes in A{beta}-driven reactive astrocytes and 3) impaired DNA/RNA processing and cytoskeleton dynamics in tau-driven astrocytes. Additionally, this study revealed several astrocyte-related DEGs and biological pathways specific to each aspect of AD core pathology.

Implications of all the available evidenceThese results shed light on the existence of GFAP-positive astrocyte signatures driven by A{beta}- and tau pathologies in AD. Characterizing how upstream triggers influence GFAP-positive astrocyte molecular signals may contribute to the biological interpretation of GFAP as a biomarker of AD. Additionally, the signature genes and related biological processes identified in this study might guide other studies to identify context-specific astrocyte biomarkers for AD. Finally, it paves the way to unraveling novel mechanisms and therapeutic targets capable of potentiating the effects of emerging anti[Formula] and anti-tau therapies.


title: Efficient Neutralization of SARS-CoV-2 Omicron and Other VOCs by a Broad Spectrum Antibody 8G3

abstract: Numerous mutations in the spike protein of SARS-CoV-2 B.1.1.529 Omicron variant pose a crisis for antibody-based immunotherapies. The efficacy of emergency use authorized (EUA) antibodies that developed in early SARS-CoV-2 pandemic seems to be in flounder. We tested the Omicron neutralization efficacy of an early B cell antibody repertoire as well as several EUA antibodies in pseudovirus and authentic virus systems. More than half of the antibodies in the repertoire that showed good activity against WA1/2020 previously had completely lost neutralizing activity against Omicron, while antibody 8G3 displayed non-regressive activity. EUA antibodies Etesevimab, Casirivimab, Imdevimab and Bamlanivimab were entirely desensitized by Omicron. Only Sotrovimab targeting the non-ACE2 overlap epitope showed a dramatic decrease activity. Antibody 8G3 efficiently neutralized Omicron in pseudovirus and authentic virus systems. The in vivo results showed that Omicron virus was less virulent than the WA1/2020 strain, but still caused deterioration of health and even death in mice. Treatment with 8G3 quickly cleared virus load of mice. Antibody 8G3 also showed excellent activity against other variants of concern (VOCs), especially more efficient against authentic Delta plus virus. Collectively, our results suggest that neutralizing antibodies with breadth remains broad neutralizing activity in tackling SARS-CoV-2 infection despite the universal evasion from EUA antibodies by Omicron variant.


title: A strategy to optimize the peptide-based inhibitors against different mutants of the spike protein of SARS-CoV-2

abstract: SARS-CoV-2 virus has caused high-priority health concerns at a global level. Vaccines have stalled the proliferation of viruses to some extent. Yet, the emergence of newer, potentially more infectious, and dangerous mutants such as delta and omicron are among the major challenges in finding a more permanent solution for this pandemic. The effectiveness of antivirals Molnupiravir and Paxlovid, authorized for emergency use by the FDA, are yet to be assessed at larger populations. Patients with a high risk of disease progression or hospitalization have received treatment with a combination of antibodies (antibody-cocktail). Most of the mutations leading to the new lineage of SARS-CoV-2 are found in the spike protein of this virus that plays a key role in facilitating host entry. The current study has investigated how to modify a promising peptide-based inhibitor of spike protein, LCB3, against common mutations in the target protein so that it retains its efficacy against the spike protein. LCB3 being a prototype for protein-based inhibitors is an ideal testing system to learn about protein-based inhibitors. Two common mutations N501Y and K417N are considered in this work. Using a structure-based approach that considers free energy decomposition of residues, distance, and the interactions between amino acids, we propose the substitutions of amino acid residues of LCB3 inhibitors. Our binding free energy calculations suggest a possible improvement in the binding affinity of existing inhibitor LCB3 to the mutant forms of the S-protein using simple substitutions at specific positions of the inhibitor. This approach, being general, can be used in different inhibitors and other mutations and help in fighting against SARS-CoV-2.


title: An immunoPET probe to SARS-CoV-2 reveals early infection of the male genital tract in rhesus macaques

abstract:
The systemic nature of SARS-CoV-2 infection is highly recognized, but poorly characterized. A non-invasive and unbiased method is needed to clarify whole body spatiotemporal dynamics of SARS-CoV-2 infection after transmission. We recently developed a probe based on the anti-SARS-CoV-2 spike antibody CR3022 to study SARS-CoV-2 pathogenesis in vivo. Herein, we describe its use in immunoPET to investigate SARS-CoV-2 infection of three rhesus macaques. Using PET/CT imaging of macaques at different times post-SARS-CoV-2 inoculation, we track the 64Cu-labelled CR3022-F(ab)2 probe targeting the spike protein of SARS-CoV-2 to study the dynamics of infection within the respiratory tract and uncover novel sites of infection. Using this method, we uncovered differences in lung pathology between infection with the WA1 isolate and the delta variant, which were readily corroborated through computed tomography scans. The 64Cu-CR3022-probe also demonstrated dynamic changes occurring between 1- and 2-weeks post-infection. Remarkably, a robust signal was seen in the male genital tract (MGT) of all three animals studied. Infection of the MGT was validated by immunofluorescence imaging of infected cells in the testicular and penile tissue and severe pathology was observed in the testes of one animal at 2-weeks post-infection. The results presented here underscore the utility of using immunoPET to study the dynamics of SARS-CoV-2 infection to understand its pathogenicity and discover new anatomical sites of viral replication. We provide direct evidence for SARS-CoV-2 infection of the MGT in rhesus macaques revealing the possible pathologic outcomes of viral replication at these sites.

Graphic AbstractPET/CT detected SARS-CoV-2 infection of 4 different tissues in the male genital tract illuminates the cause of COVID-19 clinical sequalae of male sexual health and fertility

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title: Different efficacies of neutralizing antibodies and antiviral drugs on SARS-CoV-2 Omicron subvariants, BA.1 and BA.2

abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.2 has spread in many countries, replacing the earlier Omicron subvariant BA.1 and other variants. Here, using a cell culture infection assay, we quantified the intrinsic sensitivity of BA.2 and BA.1 compared with other variants of concern, Alpha, Gamma, and Delta, to five approved-neutralizing antibodies and antiviral drugs. Our assay revealed the diverse sensitivities of these variants to antibodies, including the loss of response of both BA.1 and BA.2 to casirivimab and of BA.1 to imdevimab. In contrast, EIDD-1931 and nirmatrelvir showed a more conserved activities to these variants. The viral response profile combined with mathematical analysis estimated differences in antiviral effects among variants in the clinical concentrations. These analyses provide essential evidence that gives insight into variant emergences impact on choosing optimal drug treatment.


title: Kinesin-8-specific loop-2 controls the dual activities of the motor domain according to tubulin protofilament shape

abstract: Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains unclear. We addressed this by obtaining cryo-EM structures (2.6-3.9 [A]) of Candida albicans Kip3 in different catalytic states on the microtubule lattice and on a curved microtubule end mimic, as well as a microtubule-unbound CaKip3-ADP crystal structure (2.0 [A]). Together with biochemical analyses of CaKip3 and kinesin-1 mutants, we define a model that explains the kinesin-8 mechanism. The microtubule depolymerization activity originates in conformational changes of the kinesin-8 motor core that are amplified by its dynamic loop-2. On curved microtubule ends, loop-1 assists depolymerization by inserting into preceding motor domains, forming head-to-tail arrays of kinesin-8s that complement loop-2 contacts with curved tubulin. On straight tubulin protofilaments in the microtubule lattice, extended loop-2-tubulin contacts inhibit conformational changes in the motor core, but in the ADP-Pi state these contacts are relaxed, allowing neck-linker docking for motility. These tubulin shape-induced alternations between pro-microtubule-depolymerization and pro-motility kinesin states, regulated by loop-2, are the key to the dual activity of kinesin-8 motors.

category: biochemistry


title: Inhibition of advanced glycation end product formation and serum protein infiltration in bioprosthetic heart valve leaflets: Investigations of anti-glycation agents.

abstract: Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde pretreated heterograft tissue, such as bovine pericardium (BP) are the most commonly used heart valve replacements. However, BHV durability is limited by structural valve degeneration (SVD) resulting from both calcification and advanced glycation end product (AGE) deposition together with serum protein infiltration. In the present study we investigated the hypothesis that anti-AGE agents, Aminoguanidine (AG), Pyridoxamine (PYR), and N-Acetylcysteine (NAC) could mitigate AGE-serum protein mechanisms in model studies, both in vitro and in vivo, using rat subdermal implants of BP. In vitro studies demonstrated that each of these agents could significantly inhibit AGE formation in BP. However, in rat 28 days BP subdermal implants, only PYR demonstrated both significant inhibition of AGE and serum albumin accumulation per immunostaining. BHV calcification was not mitigated by PYR. It is concluded that AGE-serum protein pathophysiology contributing to SVD can be ameliorated by PYR.


title: Design, Synthesis and Evaluation of Inhibitors of the SARS-CoV-2 nsp3 Macrodomain

abstract:
A series of amino acid based 7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV-2 nsp3 macrodomain (Mac1), an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis. Structure activity studies identified compound 15c as a low-micromolar inhibitor of Mac1 in two ADP-ribose binding assays. This compound also demonstrated inhibition in an enzymatic assay of Mac1 and displayed a thermal shift comparable to ADPr in the melting temperature of Mac1 supporting binding to the target protein. A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network with Asp22 and the amide backbone NH of Ile23 in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe157 and Asp156, part of the oxyanion subsite of Mac1. Compound 15c also demonstrated notable selectivity for coronavirus macrodomains when tested against a panel of ADP-ribose binding proteins. Together, this study identified several low MW, low M Mac1 inhibitors to use as small molecule chemical probes for this potential anti-viral target and offers starting points for further optimization.

Graphical Abstract

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title: Enabling spatiotemporal regulation within biomaterials using DNA reaction- diffusion waveguides

abstract: In multicellular organisms, cells and tissues coordinate biochemical signal propagation across length scales spanning microns to meters. Endowing synthetic materials with similar capacities for coordinated signal propagation could allow these systems to adaptively regulate themselves across space and over time. Here we combine ideas from cell signaling and electronic circuitry to design a biochemical waveguide that transmits information in the form of a concentration of a DNA species on a directed path. The waveguide can be seamlessly integrated into a soft material because there is virtually no difference between the chemical or physical properties of the waveguide and the material it is embedded within. We propose the design of DNA strand displacement reactions to construct the system and, using reaction-diffusion models, identify kinetic and diffusive parameters that enable super-diffusive transport of DNA species via autocatalysis. Finally, to support experimental waveguide implementation, we show how a sink reaction could mitigate the spurious amplification of an autocatalyst within the waveguide, allowing for controlled waveguide triggering. Chemical waveguides could facilitate the design of synthetic biomaterials with distributed sensing machinery integrated throughout their structure and enable coordinated self-regulating programs triggered by changing environmental conditions.


title: Achieving pan-microbiome biological insights via the dbBact knowledge base

abstract: 16S rRNA amplicon sequencing provides a relatively cheap culture-independent method for studying the microbial world. Thousands of studies have examined microbial populations in various habitats, yet a global pan-microbiome perspective integrating these studies is still missing. Here we introduce dbBact, an open wiki-like bacterial knowledge base that combines information from hundreds of studies across diverse habitats, creating a collaborative central repository amenable to subsequent analysis. 16S rRNA amplicon sequence variants (ASVs) are manually extracted from each study and assigned multiple ontology-based terms. dbBact currently contains about 850 studies, covering more than 1,300,000 associations between 315,000 ASVs and 3,500 ontology terms. We demonstrate how dbBact can lead to better understanding of underlying bacterial communities and the formulation of novel biological hypotheses. dbBact can be accessed using a website (http://dbbact.org), plugins for qiime2 and Calour, and programmatically, using a REST-API.


title: Mycobacterial Serine/Threonine phosphatase PstP is phospho-regulated and localized to mediate control of cell wall metabolism

abstract: The mycobacterial cell wall is profoundly regulated in response to environmental stresses, and this regulation contributes to antibiotic tolerance. The reversible phosphorylation of different cell wall regulatory proteins is a major mechanism of cell wall regulation. Eleven Serine/Threonine protein kinases (STPKs) phosphorylate many critical cell wall-related proteins in mycobacteria. PstP is the sole serine/ threonine phosphatase, but few proteins have been verified as PstP substrates. PstP is itself phosphorylated but the role of its phosphorylation in regulating its activity has been unclear. In this study we aim to discover novel substrates of PstP in Mycobacterium tuberculosis (Mtb). We show in vitro that PstP dephosphorylates two peptidoglycan regulators of Mtb, FhaA and Wag31. We also show that a phospho-mimetic mutation of T137 on PstP negatively regulates its catalytic activity against the cell wall regulatory substrates FhaA, Wag31, CwlM, PknB and PknA, and that the corresponding mutation in Mycobacterium smegmatis (Msmeg) causes mis-regulation of peptidoglycan in vivo. We show that PstP is localized to the septum, which likely restricts its access to certain substrates. These findings on the regulation of PstP provide insight into the control of cell wall metabolism in mycobacteria.


title: RNA fluorescence in situ hybridization (FISH) to visualize microbial colonization and infection in the Caenorhabditis elegans intestines

abstract:
The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools.

This protocol introduces RNA fluorescence in situ hybridization (FISH) as a tool used for the identification, visualization, and quantification of the microbes within the intestines of C. elegans. FISH probes that label the highly abundant small subunit ribosomal RNA can produce a bright signal for bacteria and microsporidian cells, and similar probes can be used to label viral RNA. FISH probes can be ordered from a commercial source as single-stranded DNA end-labeled with fluorophores. One limitation is that FISH may not provide robust signal against low copy targets, although signal can be boosted by using multiple probes (so-called  single-molecule FISH). FISH staining involves collecting colonized or infected animals, washing to eliminate external contamination, followed by fixation in either paraformaldehyde or acetone. After fixation, FISH probes are incubated with samples to allow for the hybridization of probes to the desired target. To remove excess background, the animals are washed again, and then examined on microscope slides or using automated approaches.

Overall, this protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

SUMMARYGut microbiome bacteria and intestinal intracellular pathogens, like the Orsay virus and microsporidia, are often found associated with wild Caenorhabditis nematodes. This protocol presents RNA FISH as a method for the detection, quantification, and identification of colonizing or infectious microbes within the context of intact C. elegans nematodes.


title: A nepenthesin insert allosterically controls catalysis in the malaria parasite protease plasmepsin V

abstract: Plasmepsin V (PM V) is a pepsin-like aspartic protease essential for growth of the malaria parasite Plasmodium falciparum. Previous work has shown PM V to be an ER-resident protease that processes parasite proteins destined for export into the host cell. Depletion or inhibition of the enzyme is lethal during asexual replication within red blood cells, as well as during the formation of sexual stage gametocytes. The structure of the P. vivax PM V has been characterized by x-ray crystallography, revealing a canonical pepsin fold punctuated by structural features uncommon to secretory aspartic proteases. Here we use parasite genetics to probe these structural features by attempting to rescue lethal PM V depletion with various mutant enzymes. We find an unusual nepenthesin 1-type insert to be essential for parasite growth and PM V activity. Mutagenesis of the nepenthesin insert suggests that both its amino acid sequence and one of the two disulfide bonds that undergird its structure are required for the nepenthesin inserts role in PM V function. Molecular dynamics simulations paired with Markov state modelling suggest that the nepenthesin insert allosterically controls PM V catalysis through multiple mechanisms.


title: Macrolide therapy in Pseudomonas aeruginosa infections causes uL4 ribosomal protein mutations leading to high-level resistance

abstract:
BackgroundCystic fibrosis (CF) patients have reduced mucociliary clearance resulting in recurring and chronic bacterial lung infections. Pseudomonas aeruginosa is one of the most common pathogens to colonize the airways of CF patients and can persist in the lungs for decades. CF patients infected with P. aeruginosa are treated with macrolides to inhibit quorum sensing, mucoidity and has additional immunomodulatory effects. However, according to the EUCAST committee, P. aeruginosa is not susceptible to macrolides leaving resistance mechanisms largely overlooked.

MethodsUsing a modified susceptibility testing protocol, P. aeruginosa isolates harbouring a mutated uL4 ribosomal protein were tested for resistance against macrolide antibiotics. Quorum sensing related properties, alterations in proteome composition and ribosome subunits distribution were further analysed to characterize the effect of the uL4 mutations on the physiology of the bacteria.

FindingsSeveral uL4 mutations were identified in isolates from P. aeruginosa collections from various sources and geographical locations. Most of them mapped to the conserved loop region of uL4 and resulted in increased survival upon macrolide exposure. uL4 mutations did not negatively impact the physiology of the bacteria and greater concentrations of antibiotic were needed to inhibit the growth, reduce swimming motility, and induce redox sensitivity. Proteome analysis revealed that pathways involved in ribosome adaptation displayed altered expression levels possibly to compensate for the uL4 mutations, which changed the subunit distribution of the ribosome.

InterpretationMacrolides used against P. aeruginosa cause selection of macrolide resistant mutants which is a widespread - but uncharacterized - phenomenon. Using a modified susceptibility test, revealed that macrolides are indeed effective bacteriostatic antibiotics against P. aeruginosa and that this effect - along with macrolide-induced quorum sensing modulation - are drastically reduced in uL4 mutants. Macrolide antibiotics should, therefore, be considered as active antimicrobial agents against P. aeruginosa and resistance development should be contemplated especially when patients are treated with prolonged courses of macrolides.

FundingCystic Fibrosis Foundation, Independent Research Fund Denmark, Novo Nordisk Foundation

Research in contextO_ST_ABSEvidence before this studyC_ST_ABSMacrolide antibiotics are readily prescribed as anti-inflammatory therapy against Pseudomonas aeruginosa infections in cystic fibrosis patients. This treatment strategy has largely overlooked the direct antimicrobial effect of this drug class on this pathogen, which is considered non-susceptible according to the European Committee on Antimicrobial Susceptibility Testing. This is because standardized antimicrobial susceptibility testing is sub-optimal for the quantification of macrolide minimum inhibitory concentrations due to the interference of the growth medium. Mutations in ribosomal RNA and increased efflux has been shown to reduce the effect of macrolides on P. aeruginosa, however, the involvement of ribosomal proteins has not been investigated in P. aeruginosa. Work done in other bacterial species such as Escherichia coli, Streptococcus pneumoniae, Legionella pneumophila and Neisseria gonorrhoeae has identified the ribosomal proteins uL4 and uL22 as targets for evolved macrolide resistance in vitro and in vivo. Especially the extended loop region of uL4 has been identified as important for macrolide susceptibility. We found no other studies published addressing the emergence of macrolide resistance through uL4 or uL22 mutations in P. aeruginosa.

Added value of this studyWe present evidence that macrolide antibiotics used as anti-inflammatory agents and bacterial modulators against P. aeruginosa infection in cystic fibrosis (CF) patients cause the emergence of resistant strains through mutations in the ribosomal protein uL4. The importance of this finding is underlined by the identification of uL4 mutations in multiple strains not only within our strain collection comprised of CF associated isolates but throughout all available sequences of clinical P. aeruginosa isolates spanning several continents and different infection types. uL4 mutations resulted in significantly reduced susceptibility towards macrolide antibiotics with respect to bacteriostatic and quorum sensing modulation effects. The generation time in presence of macrolide antibiotics was unaffected in strains harbouring uL4 mutations, while it was reduced in wild type strains indicating a fitness advantage of the mutation.

Implications of all the available evidenceThe uL4 mutants identified in this study were significantly more resistant towards macrolide antibiotics than strains harbouring a wild type uL4 hereby revealing that not only are macrolides effective as antimicrobial agents against P. aeruginosa but also that the imposed selective pressure causes resistant mutants to arise through mutations in the ribosomal uL4 protein. These mutations are also present but uncharacterized in other collections of clinical P. aeruginosa isolates from patient groups who typically receive long courses of macrolide treatment. The effect of the uL4 and possibly other hitherto unknown mutations on macrolide susceptibility can be determined via modification of the standard susceptibility testing protocol. Along with evidence describing other types of macrolide resistance mechanisms in P. aeruginosa such as rRNA modification and increased efflux activity, our results demonstrate that macrolide resistance development is widespread in P. aeruginosa and macrolide antibiotics should be considered as antimicrobial agents against P. aeruginosa with the same precautions being taken to avoid resistance development as for any other antimicrobial agent.


title: Complex extracellular biology drives surface competition in Bacillus subtilis

abstract: In nature, many bacteria grow on surfaces, where they form cell collectives that compete for space. Within these collectives, cells often secrete molecules that benefit surface spreading by, for example, reducing surface tension or promoting filamentous growth. Although we have a detailed understanding of how these molecules are produced, much remains unknown about their role in surface competition. Here, we examine sliding motility in Bacillus subtilis and compare how secreted molecules, essential for sliding, affect cooperation and competition on the surface. We specifically examine (i) the lipopeptide surfactin, (ii) the hydrophobin protein BslA and (iii) exopolysaccharides (EPS). We find that these molecules have a remarkably different effect on competition: whereas surfactin acts like a common good, which is costly to produce and benefits cells throughout the surface, BslA and EPS are cost-free and act locally. Accordingly, surfactin deficient mutants can exploit the wild-type strain in competition for space, while BslA and EPS mutants cannot. Using a mathematical model, we show that three factors are important in predicting the outcome of surface competition: the costs of molecule synthesis, the private benefits of molecule production, and the diffusion rate. Our results underscore the intricate extracellular biology that can drive bacterial surface competition.


title: Synthetic oxepanoprolinamide iboxamycin is highly active against human pathogen Listeria monocytogenes

abstract: Listeriosis is a dangerous food-borne bacterial disease caused by the Gram-positive Bacillota (Firmicute) bacterium Listeria monocytogenes. In this report, we show that the synthetic lincosamide iboxamycin is highly active against L. monocytogenes and can overcome the intrinsic lincosamide resistance mediated by VgaL/Lmo0919, a member of ABCF ATPase resistance determinants that act by directly removing the antibiotic from the ribosome. While iboxamycin is not bactericidal against L. monocytogenes, it displays a pronounced postantibiotic effect, which is a valuable pharmacokinetic feature. Experiments in L. monocytogenes infection models are necessary to further assess the potential of iboxamycin as a novel drug for treatment of listeriosis. We demonstrate that VmlR ARE ABCF of Bacillota bacterium Bacillus subtilis grants significant (33-fold increase in MIC) protection from iboxamycin, while LsaA ABCF of Enterococcus faecalis grants an 8-fold protective effect. Furthermore, the VmlR-mediated iboxamycin resistance is cooperative with that mediated by the Cfr 23S rRNA methyltransferase resistance determinant, resulting in up to a 512-fold increase in MIC. Therefore, emergence and spread of ABCF ARE variants capable of defeating next-generation lincosamides in the clinic is possible and should be closely monitored.