title: IL-34 deficiency impairs FOXP3+ Treg function and increases susceptibility to autoimmunity

abstract: Immune homeostasis requires fully functional Tregs with a stable phenotype to control autoimmunity. Although IL-34 is a cytokine first described as mainly involved in monocyte cell survival and differentiation, we recently described its expression by CD8+ Tregs in a rat model of transplantation tolerance and by activated FOXP3+ CD4+ and CD8+ Tregs in human healthy individuals. However, its role in autoimmunity and potential in human diseases remain to be determined. Here we report that the absence of expression of IL-34 in Il34-/- rodents leads to an unstable phenotype, with production of multiple auto-antibodies, exacerbated under inflammatory conditions with increased susceptibility to DSS- and TNBS-colitis in deficient animals. Moreover, we strikingly revealed the inability of Il34-/- CD4+ Tregs to protect Il2rg-/- rats from a wasting disease induced by transfer of pathogenic cells, in contrast to Il34+/+ CD4+ Tregs. We also showed that IL-34 treatment delayed EAE in mice as well as GVHD and human skin allograft rejection in immune humanized immunodeficient NSG mice. Finally, we show that presence of IL-34 in the serum is associated with a longer rejection-free period in kidney transplanted patients. Altogether, our data emphasize on the crucial necessity of IL-34 for immune homeostasis and for CD4+ Tregs suppressive function. Our data also shows the therapeutic potential of IL-34 in human transplantation and auto-immunity.


title: Chlamydomonas RABL4/IFT27 Mediates Phototaxis via Promoting BBSome-dependent Ciliary Export of Phospholipase D

abstract: Phospholipase D (PLD) interacts with the BBSome for loading onto retrograde intraflagellar transport (IFT) trains to exit cilia in Chlamydomonas reinhardtii. PLD ciliary retention and depletion of Rab-like 4 (RABL4) GTPase IFT27 cause the same non-phototactic phenotype but not impair IFT and ciliation. Here, we show that the IFT-B1 subunit IFT27 binds its partner IFT25 to form the heterodimeric IFT25/27 in an IFT27 nucleotide state-independent manner. IFT25/27, IFT-A, and IFT-B are irrelevant for maintaining the stability of one another. GTP-loading onto IFT27 enhances the IFT25/27 affinity for IFT-B1 in cytoplasm, while GDP-loaded IFT27 does not prevent IFT25/27 from entering and cycling through cilia by integrating into IFT-B1. Upon at the ciliary tip, IFT25/27 cycles on and off IFT-B1 and this process is irrelevant with the nucleotide state of IFT27. During BBSome remodeling at the ciliary tip, IFT25/27 promotes BBSome reassembly independent of IFT27 nucleotide state, making post-remodeled BBSomes available for PLD to interact with. Therefore, IFT25/27 facilitates BBSome-dependent PLD export from cilia via controlling availability of intact BBSomes at the ciliary tip, providing a regulatory mechanism for IFT27 to mediate phototaxis in C. reinhardtii.

category: cell biology


title: Nociception-dependent CCL21 induce dorsal root ganglia axonal growth via CCR7-ERK activation

abstract: While chemokines were originally described for their ability to induce cell migration, many studies show how chemokines also take part in a variety of other cell functions, acting as adaptable messengers in the communication between a diversity of cell types. In the nervous system, chemokines participate both in physiological and pathological processes, and while their expression is often described on glial and immune cells, growing evidence describe the expression of chemokines and their receptors in neurons, highlighting, their potential in auto- and paracrine signalling. In this study we analysed the role of nociception in the neuronal chemokinome, and their role in axonal growth. We found that stimulating TRPV1+ nociceptors induces a transient increase in CCL21. Interestingly we found that, this CCL21 increases neurite growth of large diameter proprioceptors in vitro. Consistent with this, we show that proprioceptors express the CCL21 receptor CCR7, and a CCR7 neutralizing antibody dose-dependently attenuates CCL21-induced neurite outgrowth. Mechanistically, we found that CCL21 binds locally to its receptor CCR7 at the growth cone, activating the downstream MEK-ERK pathway, that in turn activates N-WASP, triggering actin filament ramification in the growth cone, resulting in increased axonal growth.


title: A Yap-dependent transcriptional program directs cell migration for embryo axis assembly

abstract: The condensation of the embryo primary axis is a fundamental landmark in the establishment of the vertebrate body plan. Although the complex morphogenetic movements directing cell convergence towards the midline have been described extensively, little is known on how gastrulating cells interpret mechanical cues. Yap proteins are among the best characterized transcriptional mechanotransducers, yet their role in gastrulation has remained elusive. Here we show that the double knockout of yap and its paralog yap1b in medaka results in an axis assembly failure. Quantitative live imaging reveals that mutant cells display reduced displacement and migratory persistence. By characterizing the Yap-dependent transcriptional program, we identified genes involved in cytoskeletal organization and cell-ECM adhesion, rather than in germ layer specification, as direct Yap targets. Dynamic analysis of Tead sensors and downstream targets reveals Yap is acting in migratory cells, and not as a midline beacon, to direct gastrulating precursors trajectories by promoting cortical actin recruitment and focal adhesions assembly. We propose that Yap is engaged in a mechano-regulatory loop that is essential to maintain the directed cell migration sustaining embryo axis formation.

category: developmental biology


title: Distinct mechanisms of germ cell factor regulation for an inductive germ cell fate

abstract:
Specification of primordial germ cells (PGCs), the lineage which gives rise to eggs and sperm, is essential for sexually reproducing organisms. The mechanism by which animals specify their PGCs generally falls into two categories: inherited or inductive. The inductive mechanism, used by mammals, relies on cell signaling interactions to direct a subset of embryonic cells to a germ cell fate. Previous work suggested that sea star embryos, which develop in simple culture and are markedly transparent, also use inductive mechanisms to specify their germline. The germ cell factors Nanos and Vasa become restricted during early development into a localized region of cells within the posterior enterocoel (PE), the presumptive germline. Nodal signaling was observed to negatively regulate Vasa and Nanos mRNAs outside of the PE and restrict the germline to the PE. Here we employed single cell RNA sequencing to identify the transcriptional program of germ cells and their changes during development. We never see Nodal pathway members within Nanos/Vasa positive cells in the region known to give rise to the PE, and instead see members of the Wnt-signaling pathway and the FoxY family of transcription factors. We learned that Wnt and Delta/Notch signaling enhances expression of both Nanos and Vasa, whereas a test of cell interactions reveals that Nanos and Vasa are regulated distinctly. This work provides insights into the sequence of events that leads to PGC specification and enables deeper mechanistic studies in a tractable in vivo model.

HighlightsO_LISingle-cell RNA-sequencing of sea star embryos demonstrates temporal differences in cell fate commitment among echinoderms.
C_LIO_LISea urchin and sea star embryos appear to ascribe their germ line by two extreme different mechanisms but share similar pathways in regulation of the germline genes.
C_LIO_LIExpression of the germline factors, Vasa and Nanos, is regulated by distinct mechanisms in the sea star.
C_LIO_LIGermline induction in the sea star uses similar signaling mechanisms as mammals.
C_LI


title: Brain aging is faithfully modelled in organotypic brain slices and accelerated by prions

abstract: Mammalian models are essential for brain aging research. However, the long lifespan and limited amenability to genetic and pharmacological perturbations have hindered the use of mammals for dissecting aging-regulatory molecular networks and discovering new anti-aging interventions. To circumvent these limitations, we developed an ex vivo model system that faithfully mimics the aging process of the mammalian brain using cultured mouse brain slices. Genome-wide gene expression analyses showed that brain slices spontaneously upregulated senescence-associated genes over time and reproduced many of the transcriptional characteristics of aged brains. Treatment with rapamycin, a classical anti-aging compound, largely abolished the time-dependent transcriptional changes in brain slices. Using this model system, we discovered that prions drastically accelerated the development of age-related molecular signatures and the pace of brain aging. We confirmed this finding in mouse models and human victims of Creutzfeldt-Jakob disease. These data establish a novel, eminently tractable mammalian model of brain aging, and uncover a surprising acceleration of brain aging in prion diseases.

category: neuroscience


title: Interference of Neuronal TrkB Signaling by the Cannabis-Derived Flavonoids Cannflavins A and B

abstract: AO_SCPLOWBSTRACTC_SCPLOWCannflavins A and B are two flavonoids that accumulate in the Cannabis sativa plant. These specialized metabolites are uniquely prenylated and highly lipophilic, which, a priori, may permit their interaction with membrane-bound enzymes and receptors. Although previous studies found that cannflavins can produce anti-inflammatory responses by inhibiting the biosynthesis of pro-inflammatory mediators, the full extent of their cellular influence remains to be understood. Here, we studied these flavonoids in relation to the Tropomyosin receptor kinase B (TrkB), a receptor tyrosine kinase that is activated by the growth factor brain-derived neurotrophic factor (BDNF). Using mouse primary cortical neurons, we first collected evidence that cannflavins prevent the accumulation of Activity-regulated cytoskeleton-associated (Arc) protein upon TrkB stimulation by exogenous BDNF in these cells. Consistent with this effect, we also observed a reduced activation of TrkB and downstream signaling effectors that mediate Arc mRNA transcription when BDNF was co-applied with the cannflavins. Of note, we also performed a high-throughput screen that demonstrated a lack of agonist action of cannflavins towards 320 different G protein-coupled receptors, a result that specifically limit the possibility of a TrkB transinactivation scenario via G protein signaling to explain our results with dissociated neurons. Finally, we used Neuro2a cells overexpressing TrkB to show that cannflavins can block the growth of neurites and increased survival rate produced by the higher abundance of the receptor in this model. Taken together, our study offers a new path to understand the reported effects of cannflavins and other closely related compounds in different cellular contexts.

category: pharmacology and toxicology


title: Reading instruction causes changes in category-selective visual cortex

abstract: Education sculpts specialized neural circuits for skills like reading that are critical to success in modern society but were not anticipated by the selective pressures of evolution. Does the emergence of brain regions that selectively process novel visual stimuli like words occur at the expense of cortical representations of other stimulus categories like faces and objects? To answer this question we designed a randomized controlled trial where pre-literate children (five years of age) were randomly assigned to intervention programs that either taught reading skills (Letter Intervention) or oral language comprehension skills (Language Intervention). Magnetoencephalography (MEG) data collected before and after the interventions revealed that being assigned to the Letter versus Language Intervention induced different patterns of changes in category selective responses in high-level visual cortex. We found moderate support for the notion that words compete with other objects for cortical territory as children become attuned to this new class of visual stimuli. How these changes play out over a longer timescale is still unclear but, based on these data, we can surmise that high-level visual cortex undergoes rapid changes as children enter school and begin establishing new skills like literacy.


title: Traveling waves in the monkey frontoparietal network predict recent reward memory

abstract: Brain function depends on neural communication, but the mechanisms of this communication are not well understood. Recent studies suggest that one form of neural communication is through traveling waves (TWs)--patterns of neural oscillations that propagate within and between areas. We show that TWs appear robustly in microarray recordings in monkey frontal and parietal cortex and encode memory for recent rewards. While making saccades to obtain probabilistic rewards, monkeys were sensitive to the (statistically irrelevant) prior reward, which is consistent with behavioral biases produced by reward history. TWs in frontal and parietal areas were stronger in trials following a prior reward versus a lack of reward and, in the frontal lobe, correlated with the monkeys sensitivity to the prior reward. The findings suggest that neural communication across fronto-parietal areas, reflected by TWs, maintains default reward memories, while communication within the frontal lobe mediates the read out of the memories for prospective expectations.


title: Hindbrain catecholaminergic inputs to the paraventricular thalamus scale feeding and metabolic efficiency in stress-related contexts

abstract:
The regulation of food intake and energy balance relies on the dynamic integration of exteroceptive and interoceptive signals monitoring nutritional, metabolic, cognitive and emotional states. The paraventricular thalamus (PVT) is a central hub that, by integrating sensory, metabolic and emotional states, may contribute to the regulation of feeding and homeostatic/allostatic processes. However, the underlying PVT circuits remain still elusive. Here, we aimed at unraveling the role of catecholaminergic (CA) inputs to the PVT in scaling feeding and metabolic efficiency. First, using region-specific retrograde disruption of CA projections, we show that PVT CA inputs mainly arise from the hindbrain, notably the locus coeruleus (LC) and the nucleus tractus solitarius (NTS). Second, taking advantage of integrative calorimetric measurements of metabolic efficiency, we reveal that CA inputs to the PVT scale adaptive feeding and metabolic responses in environmental, behavioral, physiological and metabolic stress-like contexts. Third, we show that hindbrainTH[->]PVT inputs contribute in modulating the activity of PVT as well as lateral (LH) and dorsomedial (DMH) hypothalamic neurons.

In conclusion, this study, by assessing the key role of CA inputs to the PVT in scaling homeostatic/allostatic regulations of feeding patterns, reveals the integrative and converging hindbrainTH[->]PVT paths that contribute to whole-body metabolic adaptations in stress-like contexts.

Key pointsO_LIThe paraventricular thalamus (PVT) is known to receive projections from the hindbrain. Here, we confirm and further extend current knowledge on the existence of hindbrainTH[->]PVT catecholaminergic (CA) inputs, notably from the locus coeruleus (LC) and the nucleus tractus solitarius (NTS), with the NTS representing the main source.
C_LIO_LIDisruption of hindbrainTH[->]PVT inputs contribute to the modulation of PVT-neurons activity.
C_LIO_LIHindbrainTH[->]PVT inputs scale feeding strategies in environmental, behavioral, physiological and metabolic stress-like contexts.
C_LIO_LIHindbrainTH[->]PVT inputs participate in regulating metabolic efficiency and nutrient partitioning in stress-like contexts.
C_LIO_LIHindbrainTH[->]PVT, directly and/or indirectly, contribute in modulating the downstream activity of lateral (LH) and dorsomedial (DMH) hypothalamic neurons.
C_LI


title: Preferential activation of proprioceptive and cutaneous sensory fibers compared to motor fibers during cervical transcutaneous spinal cord stimulation: A computational study

abstract:
ObjectiveCervical transcutaneous spinal cord stimulation (tSCS) is a promising technology that can support motor function recovery of upper-limbs after spinal cord injury. Its efficacy may depend on the ability to recruit sensory afferents and convey excitatory inputs onto motoneurons. Therefore, understanding its physiological mechanisms is critical to accelerate its development towards clinical applications. In this study, we used an anatomically realistic computational model of the cervical spine to compare -motor, A-sensory, and A{beta}-sensory fiber activation thresholds and activation sites.

ApproachWe developed a tridimensional geometry of the cervical body and tSCS electrodes with a cathode centred at the C7 spinous process and an anode placed over the anterior neck. The geometrical model was used to estimate the electric potential distributions along motor and sensory fiber trajectories at the C7 spinal level using a finite element method. We implemented dedicated motor and sensory fiber models to simulate the -motor and A-sensory fibers using 12, 16, and 20 m diameter fibers, and A{beta}-sensory fibers using 6, 9, and 12 m diameter fibers. We estimated nerve fiber activation thresholds and sites for a 2 ms monophasic stimulating pulse and compared them across the fiber groups.

Main resultsOur results showed lower activation thresholds of A- and A{beta}-sensory fibers compared with -motor fibers, suggesting preferential sensory fiber activation. We also found no differences between activation thresholds of A-sensory and large A{beta}-sensory fibers, implying they were co-activated. The activation sites were located at the dorsal and ventral root levels.

SignificanceUsing a realistic computational model, we demonstrated preferential activation of dorsal root A- and A{beta}-sensory fibers compared with ventral root -motor fibers during cervical tSCS. These findings suggest high proprioceptive and cutaneous contributions to neural activations during cervical tSCS, which inform the underlying mechanisms of upper-limb functional motor recovery.


title: Defensive and offensive behaviors in social interaction settings in a Kleefstra syndrome mouse model

abstract: Kleefstra syndrome in humans is characterized by general delay in development, intellectual disability and autistic features. The mouse model of this disease (Ehmt1+/-) expresses anxiety, autistic-like traits, and aberrant social interactions with non-cagemates. To investigate how Ehmt1+/- mice behave with unfamiliar conspecifics, we allowed adult, male animals to freely interact for 10 minutes in a neutral, novel environment within a host-visitor setting. In 17 out of 74 trials there were defensive and offensive behaviors. Our key finding was that Ehmt1+/- mice displayed defensive postures, attacking and biting; in contrast, wild-type (WT) interacting with other WT did not enact such behaviors. Further, if there was a fight between an Ehmt1+/- and a WT mouse, it was always the Ehmt1+/- who initiated these behaviors.


title: An improved MS2-MCP imaging system with minimal perturbation of mRNA stability

abstract: The MS2-MCP imaging system is widely used to study the mRNA spatial distribution in living cells. Here, we report that the MS2-MCP system may destabilize the tagged mRNA by targeting it to the nonsense-mediated mRNA decay pathway. We introduce an improved version, which has minimal perturbation of the mRNA stability.


title: Can the Kuznetsov Model Replicate and Predict Cancer Growth in Humans?

abstract: Several mathematical models to predict tumor growth over time have been developed in the last decades. A central aspect of such models is the interaction of tumor cells with immune effector cells. The Kuznetsov model (Kuznetsov et al. (1994), Bulletin of Mathematical Biology, vol. 56, no. 2, pp. 295-321) is the most prominent of these models and has been used as a basis for many other related models and theoretical studies. However, none of these models have been validated with large-scale real-world data of human patients treated with cancer immunotherapy. In addition, parameter estimation of these models remains a major bottleneck on the way to model-based and data-driven medical treatment. In this study, we quantitatively fit Kuznetsovs model to a large dataset of 1472 patients, of which 210 patients have more than six data points, by estimating the model parameters of each patient individually. We also conduct a global practical identifiability analysis for the estimated parameters. We thus demonstrate that several combinations of parameter values could lead to accurate data fitting. This opens the potential for global parameter estimation of the model, in which the values of all parameters are fixed for all patients. Furthermore, by omitting the last two or three data points, we show that the model can be extrapolated and predict future tumor dynamics. This paves the way for a more clinically relevant application of mathematical tumor modeling, in which the treatment strategy could be adjusted in advance according to the models future predictions.

category: bioinformatics


title: CRFalign: A Sequence-structure alignment of proteins based on a combinationof HMM-HMM comparison and conditional random fields

abstract: We built a method of sequence-structure alignment (called CRFalign) which improves upon a base alignment model based on HMM-HMM comparison by employing pairwise conditional random fields (pCRF) in combination with nonlinear scoring functions of structural and sequence features. The total scoring function consists of a base scoring part based on HMM-HMM profile comparison plus additional nonlinear scoring part which is implemented by a set of gradient boosted regression trees. In addition to sequence profile features, various structural features are employed including secondary structures, solvent accessibilities, environment-dependent properties that give rise to position-dependent as well as environment-dependent match scores and gap penalties. Training is performed on reference alignments at superfamily levels or twilight zone chosen from the SABmark benchmark set. We found that our alignment method produce relative improvement in terms of average alignment accuracies, especially for the alignment of remote homologous proteins. We found that our alignment method produced (by using Modeller) better modeling results especially in the relatively hard targets compared with other methods. CRFalign was successfully applied to the stages of fold recognition and multiple sequence alignment in CASP11 and CASP12 competition on protein structure predictions.


title: Transfer Learning for Cognitive Reserve Quantification

abstract:
Cognitive reserve (CR) has been introduced to explain individual differences in susceptibility to cognitive or functional impairment in the presence of age or pathology. We developed a deep learning model to quantify the CR as residual variance in memory performance using the structural MRI data from a lifespan healthy cohort. The generalizability of the sMRI-based deep learning model was tested in two independent healthy and Alzheimers cohorts using transfer learning framework.

Structural MRIs were collected from three cohorts: 495 healthy adults (initially aged 20-80) from RANN, 620 healthy participants (age 36-100) from lifespan Human Connectome Project Aging (HCPA), and 941 subjects from Alzheimers Disease Neuroimaging Initiative (ADNI). Region of interest (ROI)-specific cortical thickness and volume measures were extracted using the Desikan-Killiany Atlas. Cognitive reserve was quantified by residuals which subtract the predicted memory from the true memory. Cascade neural network (CNN) models were used to train RANN dataset for memory prediction. Transfer learning was applied to transfer the T1 imaging-based model from source domain (using RANN) to the target domain (HCPA or ADNI).

The CNN model trained on the RANN dataset exhibited strong linear correlation between true and predicted memory based on the chosen T1 cortical thickness and volume predictors. In addition, the model generated from healthy lifespan data (RANN) was able to generalize to an independent healthy lifespan data (HCPA) and older demented participants (ADNI) across different scanner types. The estimated CR was correlated with CR proxies such education and IQ across all three datasets.

The current findings suggest that the transfer learning approach is an effective way to generalize the residual-based CR estimation. It is applicable to various diseases and may flexibly incorporate different imaging modalities such as fMRI and PET, making it a promising tool for scientific and clinical purposes.

HighlightO_LIQuantification of cognitive reserve using brain measures for pre-symptomatic Alzheimers patients can be estimated by leveraging lifespan data.
C_LIO_LIMulti-center, multi-scanner, multi-sequence can affect the performance of the quantification.
C_LIO_LILeveraging lifespan data from a single site can improve the performance.
C_LIO_LITransfer learning allows the pre-trained network to successfully reconstruct the dataset acquired from different domains or age groups.
C_LI


title: RaPID-Query for Fast Identity by Descent Search and Genealogical Analysis

abstract: The size of genetic databases has grown large enough such that, genetic genealogical search, a process of inferring familial relatedness by identifying DNA matches, has become a viable approach to help individuals finding missing family members or law enforcement agencies locating suspects. However, a fast and accurate method is needed to search an out-of-database individual against millions of individuals in such databases. Most existing approaches only offer all-vs-all within panel match. Some prototype algorithms offer 1-vs-all query from out-of-panel individual, but they do not tolerate errors. A new method, random projection-based identical-by-descent (IBD) detection (RaPID) query, referred as RaPID-Query, is introduced to make fast genealogical search possible. RaPID-Query method identifies IBD segments between a query haplotype and a panel of haplotypes. By integrating matches over multiple PBWT indexes, RaPID-Query method is able to locate IBD segments quickly with a given cutoff length while allowing mismatched sites in IBD segments. A single query against all UK biobank autosomal chromosomes can be completed within 2.76 seconds CPU time on average, with the minimum 7 cM IBD segment length and minimum 700 markers. Using the same criteria, RaPID-Query can achieve 0.099 false negative rate and 0.017 false positive rate at the same time on a chromosome 20 sequencing panel having 92,296 sites, which is comparable to the state-of-the-art IBD detection method Hap-IBD. For the relatedness degree separation experiments, RaPID-Query is able to distinguish up to fourth degree of the familial relatedness for a given individual pair, and the area under the receiver operating characteristic curve values are at least 97.28%. It is anticipated that RaPID-Query will make genealogical search convenient and effective, potentially with the integration of complex inference models.


title: mcPBWT: Space-efficient Multi-column PBWT Scanning Algorithm for Composite Haplotype Matching

abstract: Positional Burrows-Wheeler Transform (PBWT) is a data structure that supports efficient algorithms for finding matching segments in a panel of haplotypes. It is of interest to study the composite patterns of multiple matching segments or blocks arranged contiguously along a same haplotype as they can indicate recombination crossover events, gene-conversion tracts, or, some-times, errors of phasing algorithms. However, current PBWT algorithms do not support search of such composite patterns efficiently. Here, we present our algorithm, mcPBWT (multi-column PBWT), that uses multiple synchronized runs of PBWT at different variant sites providing a "look-ahead" information of matches at those variant sites. Such "look-ahead" information allows us to analyze multiple contiguous matching pairs in a single pass. We present two specific cases of mcPBWT, namely double-PBWT and triple-PBWT which utilize two and three columns of PBWT respectively. double-PBWT finds two matching pairs combinations representative of crossover event or phasing error while triple-PBWT finds three matching pairs combinations representative of gene-conversion tract.


title: DisPhaseDB, an integrative database of diseases related variations in liquid-liquid phase separation proteins

abstract:
MotivationProteins involved in liquid-liquid phase separation (LLPS) and membraneless organelles (MLOs) are recognized to be decisive for many biological processes and also responsible for several diseases. The recent explosion of research in the area still lacks tools for the analysis and data integration among different repositories. Currently, there is not a comprehensive and dedicated database that collects all disease-related variations in combination with the protein location, biological role in the MLO and all the metadata available for each protein and disease. Disease related protein variants and additional features are dispersed and the user has to navigate many databases, with different focus, formats and often not user friendly.

ResultsWe present DisPhaseDB, a database dedicated to disease related variants of LLPS proteins and/or are involved in MLOs. It integrates 10 databases, contains 5.741 proteins, 1.660.059 variants and 4.051 disease terms. It also offers intuitive navigation and an informative display. It constitutes a pivotal starting point for further analysis, encouraging the development of new computational tools.

Availability and ImplementationThe database is freely available at http://disphasedb.leloir.org.ar.

Contactjiserte@leloir.org.ar and cmb@leloir.org.ar

Graphical abstract

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title: Data-driven dynamical modelling of a pathogen-infected plant gene regulatory network: a comparative analysis

abstract: Recent advances in synthetic biology have enabled the design of genetic feedback control circuits that could be implemented to build resilient plants against pathogen attacks. To facilitate the proper design of these genetic feedback control circuits, an accurate model that is able to capture the vital dynamical behaviour of the pathogen-infected plant is required. In this study, using a data-driven modelling approach, we develop and compare four dynamical models (i.e. linear, Michaelis-Menten, standard S-System and extended S-System) of a pathogen-infected plant gene regulatory network (GRN). These models are then assessed across several criteria, i.e. ease of identifying the type of gene regulation, the predictive capability, Akaike Information Criterion (AIC) and the robustness to parameter uncertainty to determine its viability of modelling the pathogen-infected plant GRN. Using our defined ranking score, our analyses show that while the extended S-System model ranks highest in the overall comparison, the performance of the linear model is more consistent throughout the comparison, making it the preferred model for this pathogen-infected plant GRN.

category: systems biology


title: Interferon regulates stem cell output via post-transcriptional repression of Sox2 independent of its antiviral function in the brain

abstract: Stem cells show intrinsic interferon signaling, which protects them from viral infections. In the brain, type I interferon signaling increases in stem cells as they age and reduces their ability to become activated for repair. Whether interferons impact stem cell activity only in aging, how, and if it is coupled to the intrinsic immune function remains elusive. Our study shows that interferon-{beta} arrested stem cells in G0 of the cell cycle and transiently increased their TOR signaling before switching it off. This bimodal regulation of TOR is needed to repress translation of Sox2. Single-cell transcriptomics reveals that in the absence of interferon receptors, interferon signaling levels change and its increase with age disappears. Mathematical simulations indicate that interferons are beneficial in the young and harmful in the old brain. Our study identifies interferons as genuine regulators of stem cell homeostasis and potential therapeutic target to repair the aging brain.


title: Interferon adjusts stem cell output in the young and old brain to meet demand independently of its antiviral function


title: Multiscale model of primary motor cortex circuits reproduces in vivo cell type-specific dynamics associated with behavior

abstract: Understanding cortical function requires studying multiple scales: molecular, cellular, circuit and behavior. We developed a biophysically detailed multiscale model of mouse primary motor cortex (M1) with over 10,000 neurons and 30 million synapses. Neuron types, densities, spatial distributions, morphologies, biophysics, connectivity and dendritic synapse locations were tightly constrained by experimental data. The model includes long-range inputs from 7 thalamic and cortical regions, as well as noradrenergic inputs from locus coeruleus. Connectivity depended on cell class and cortical depth at sublaminar resolution. The model accurately predicted in vivo layer- and cell type-specific responses (firing rates and LFP) associated with behavioral states (quiet wakefulness and movement) and experimental manipulations (noradrenaline receptor blocking and thalamus inactivation). It also enabled evaluation of multiple mechanistic hypotheses underlying the observed activity. This quantitative theoretical framework can be used to integrate and interpret M1 experimental data and sheds light on the cell type-specific multiscale dynamics associated with a range of experimental conditions and behaviors.


title: Multiscale model of primary motor cortex circuits predicts in vivo cell type-specific, behavioral state-dependent dynamics


title: Tau seeding without tauopathy

abstract: Neurodegenerative tauopathies such as Alzheimers disease (AD) are caused by brain accumulation of tau assemblies. Evidence suggests tau functions as a prion, and cells and animals efficiently propagate unique tau assemblies. This suggests a dedicated cellular replication machinery, with normal physiologic function for tau seeds. Consequently, we hypothesized that healthy control brains would have seeding activity. We recently developed a novel monoclonal antibody (MD3.1) specific for tau seeds. We used this antibody to immunopurify tau from the parietal and cerebellar cortices of 19 healthy subjects ranging 19-65 years. We detected seeding in the parietal cortex, but not in the cerebellum, or in wild-type or human tau knockin mice, suggesting that cellular/genetic context dictates development of seed-competent tau. Seeding did not correlate with subject age or brain tau levels. Dot blot analyses revealed no AT8 immunoreactivity above background levels in parietal and cerebellar extracts and <1/100 of that present in AD. Based on binding to a panel of antibodies, the conformational characteristics of control seeds differed from AD, suggesting a unique underlying assembly, or structural ensemble. Taus ability to adopt self-replicating conformations under non-pathogenic conditions may reflect normal function that goes awry in disease states.


title: Multiplex imaging of human induced pluripotent stem cell-derived neurons with CO-Detection by indEXing (CODEX) technology

abstract:
BackgroundHuman induced pluripotent stem cell (iPSC) models have been hailed as a breakthrough for understanding disease and developing new therapeutics. The major advantage of iPSC-derived neurons is that they carry the genetic background of the donor, and as such could be more predictive for clinical translation. However, the development of these cell models is time-consuming and expensive and it is thus critical to maximize biomarker readout from every model that is developed. One option is to use a highly multiplexed biomarker imaging assay, like CO-Detection by indEXing (CODEX), which allows detection of 50+ targets in situ at single-cell resolution.

New MethodThis paper describes the development of CODEX in neuronal cell cultures derived from human iPSCs.

ResultsWe differentiated human iPSCs into mixed neuronal and glial cultures on glass coverslips. We then developed and optimized a panel of 21 antibodies to phenotype iPSC-derived neuronal subtypes of cortical, dopaminergic, and striatal neurons, as well as astrocytes, and pre-and postsynaptic proteins.

Comparison with existing methodsCompared to standard immunocytochemistry, CODEX oligoconjugated fluorophores circumvent antibody host interactions and allow for highly customized multiplexing.

ConclusionWe show that CODEX can be applied to iPSC neuronal cultures and developed fixation and staining protocols for the neurons to sustain the multiple wash-stain cycles of the technology. Furthermore, we demonstrate both cellular and subcellular resolution imaging of multiplexed biomarkers in the same samples. CODEX is a powerful technique that complements other single-cell omics technologies for in-depth phenotype analysis.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/479039v1_ufig1.gif" ALT="Figure 1">
View larger version (54K):
org.highwire.dtl.DTLVardef@68d49dorg.highwire.dtl.DTLVardef@5ea424org.highwire.dtl.DTLVardef@16a763forg.highwire.dtl.DTLVardef@7950fc_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOC_FLOATNO CODEX(R) Multiplex Imaging in human iPSC neurons

[A-D] Schematic drawings of the tools and steps used for Co-Detection by indEXing (CODEX) imaging. [A] Target-specific antibodies are conjugated to unique DNA oligonucleotide barcodes. Fluorescent reporter (excitation wavelength at 488 nm, 550 nm, or 647 nm/Cy5) carrying the complementary DNA (to the barcode) enables barcode-specific binding of the reporter to the antibody and detection by fluorescence microscopy. [B] Neuronal cell cultures are prepared for the CODEX staining and imaging by several fixation steps with different PFA concentrations followed by incubation with 100% acetone. Residual acetone is removed by drying the sample. After rehydration with PBS, autofluorescence is quenched by exposure to broad-spectrum LED light. Following a pre-staining fixation step, the sample is incubated with a mix of all conjugated primary antibodies. Excessive, unbound antibodies are removed by a washing step, leaving only the bound antibodies followed by a final post-staining fixation. [C] The CODEX Instrument Manager performs the multicycle run and controls the microscope software for automated addition of reporters, imaging, and washing of the samples (pre-stained with primary antibodies) to remove reporters from each cycle. After imaging, bound reporters are removed without damaging the tissue using a solvent, and the next set of reporters (conjugated to different barcodes) are added. [D] CODEX(R) Processor processes raw files and performs stitching, deconvolution, background subtraction, and cell segmentation. The processed images can be viewed and analyzed with the CODEX(R) Multiplex Analysis Viewer (MAV) plugin using Fiji software.

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title: Data-driven multiscale model of macaque auditory thalamocortical circuits reproduces in vivo dynamics

abstract: We developed a biophysically-detailed model of the macaque auditory thalamocortical circuits, including primary auditory cortex (A1), medial geniculate body (MGB) and thalamic reticular nuclei (TRN), using the NEURON simulator and NetPyNE multiscale modeling tool. We simulated A1 as a cortical column with a depth of 2000 m and 200 m diameter, containing over 12k neurons and 30M synapses. Neuron densities, laminar locations, classes, morphology and biophysics, and connectivity at the long-range, local and dendritic scale were derived from published experimental data. The A1 model included 6 cortical layers and multiple populations of neurons consisting of 4 excitatory and 4 inhibitory types, and was reciprocally connected to the thalamus (MGB and TRN), mimicking anatomical connectivity. MGB included core and matrix thalamocortical neurons with layer-specific projection patterns to A1, and thalamic interneurons projecting locally. Auditory stimulus-related inputs to the MGB were simulated using phenomenological models of the cochlear/auditory nerve and the inferior colliculus. The model generated cell type and layer-specific firing rates consistent with experimentally observed ranges, and accurately simulated the corresponding local field potentials (LFPs), current source density (CSD), and electroencephalogram (EEG) signals. Laminar CSD patterns during spontaneous activity, and in response to speech input, were similar to those recorded experimentally. Physiological oscillations emerged spontaneously across frequency bands without external rhythmic inputs and were comparable to those recorded in vivo. We used the model to unravel the contributions from distinct cell type and layer-specific neuronal populations to oscillation events detected in CSD, and explored how these relate to the population firing patterns. Overall, the computational model provides a quantitative theoretical framework to integrate and interpret a wide range of experimental data in auditory circuits. It also constitutes a powerful tool to evaluate hypotheses and make predictions about the cellular and network mechanisms underlying common experimental measurements, including MUA, LFP and EEG signals.


title: Recurrent circuits amplify corticofugal signals and drive feed-forward inhibition in the inferior colliculus

abstract: The inferior colliculus (IC) is a midbrain hub critical for perceiving complex, time-varying sounds such as speech. In addition to processing ascending inputs from most auditory brainstem nuclei, the IC receives descending inputs from auditory cortex that control IC neuron feature selectivity, plasticity, and certain forms of perceptual learning. Although these corticofugal synapses primarily release the excitatory transmitter glutamate, many physiology studies show that auditory cortical activity has a net inhibitory effect on IC neuron spiking. Perplexingly, anatomy studies indicate that corticofugal axons primarily target glutamatergic IC neurons while only sparsely innervating IC GABA neurons, thereby suggesting that corticofugal inhibition of the IC occurs largely independently of feedforward activation of local GABA neurons. We shed light on this paradox using in vitro electrophysiology and optogenetics in acute IC slices from fluorescent reporter mice. We find that corticofugal synaptic strength is indeed stronger onto IC glutamate compared to GABA neurons. Nevertheless, two mechanisms enable reliable corticofugal activation of local GABA neurons. 1) Many IC GABA neurons fire tonically at rest, such that sparse and weak excitation suffices to significantly increase their spike rates. 2) Corticofugal activity triggers barrages of large amplitude, polysynaptic EPSPs in IC GABA neurons, owing to a dense intra-collicular connectivity from local axon collaterals of IC glutamate neurons. Consequently, repetitive activity in corticofugal fibers drives spikes in IC GABA neurons and generates substantial recurrent inhibition. Thus, descending signals trigger intra-collicular inhibition despite apparent constraints of monosynaptic connectivity between auditory cortex and IC GABA neurons.


title: Large-scale biophysically detailed model of somatosensory thalamocortical circuits in NetPyNE

abstract: The primary somatosensory cortex (S1) of mammals is critically important in the perception of touch and related sensorimotor behaviors. In 2015 the Blue Brain Project developed a groundbreaking rat S1 microcircuit simulation with over 31,000 neurons with 207 morpho-electrical neuron types, and 37 million synapses, incorporating anatomical and physiological information from a wide range of experimental studies. We have implemented this highly-detailed and complex model S1 model in NetPyNE, using the data available in the Neocortical Microcircuit Collaboration Portal. NetPyNE provides a Python high-level interface to NEURON and allows defining complicated multiscale models using an intuitive declarative standardized language. It also facilitates running parallel simulations, automates the optimization and exploration of parameters using supercomputers, and provides a wide range of built-in analysis functions. This will make the S1 model more accessible and simpler to scale, modify and extend in order to explore research questions or interconnect to other existing models. Despite some implementation differences, the NetPyNE model closely reproduced the original cell morphologies, electrophysiological responses, spatial distribution for all 207 cell types; and the connectivity properties of the 1941 pathways, including synaptic dynamics and short-term plasticity (STP). The NetPyNE S1 simulations produced reasonable physiological rates and activity patterns across all populations, and, when STP was included, produced a 1 Hz oscillation comparable to that of the original model. We also extended the model by adding thalamic circuits, including 6 distinct thalamic populations with intrathalamic, thalamocortical and corticothalamic connectivity derived from experimental data. Overall, our work provides a widely accessible, data-driven and biophysically-detailed model of the somatosensory thalamocortical circuits that can be utilized as a community tool for researchers to study neural dynamics, function and disease.


title: Abnormal molecular signatures of inflammation, energy metabolism and vesicle biology in human Huntington disease peripheral tissues

abstract:
BackgroundA major challenge in neurodegenerative diseases concerns identifying biological disease signatures that track with disease progression or respond to an intervention. Several clinical trials in Huntington disease (HD), an inherited, progressive neurodegenerative disease, are currently ongoing. Therefore, we examined whether peripheral tissues can serve as a source of readily accessible biological signatures at the RNA and protein level in HD patients.

ResultsWe generated large, high-quality human datasets from skeletal muscle, skin and adipose tissue to probe molecular changes in human premanifest and early manifest HD patients - those most likely involved in clinical trials. In-depth single nucleotide polymorphism data across the HTT gene will facilitate the use of the generated primary- and iPSC cell lines in allele-specific targeting approaches. The analysis of the transcriptomics and proteomics data shows robust, stage-dependent dysregulation. Gene ontology analysis confirmed the involvement of inflammation and energy metabolism in peripheral HD pathogenesis. Furthermore, we observed changes in the homeostasis of extracellular vesicles, where we found consistent changes of genes and proteins involved in this process.

ConclusionsOur  omics data document the involvement of inflammation, energy metabolism and extracellular vesicle homeostasis. This demonstrates the potential to identify biological signatures from peripheral tissues in HD suitable as biomarkers in clinical trials. Together with the primary cell lines established from peripheral tissues and a large panel of iPSC lines that can serve as human models of HD, the generated data are a valuable and unique resource to advance the current understanding of molecular mechanisms driving HD pathogenesis.


title: Experimental Suppression of TMS-EEG Sensory Potentials

abstract:
BackgroundThe sensory experience of transcranial magnetic stimulation (TMS) evokes cortical responses measured in EEG that confound interpretation of TMS-evoked potentials (TEPs). Methods for sensory masking have been proposed to minimize sensory contributions to the TEP, but the most effective combination for suprathreshold TMS to dorsolateral prefrontal cortex (dlPFC) is unknown.

ObjectiveWe applied sensory suppression techniques and quantified electrophysiology and perception from suprathreshold dlPFC TMS to identify the best combination to minimize the sensory TEP.

MethodsIn 21 healthy adults, we applied single pulse TMS at 120% resting motor threshold (rMT) to the left dlPFC and compared EEG vertex N100-P200 and perception. Conditions included three protocols: No masking (no auditory masking, no foam, jittered inter-stimulus interval (ISI)), Standard masking (auditory noise, foam, jittered ISI), and our ATTENUATE protocol (auditory noise, foam, over-the-ear protection, unjittered ISI).

ResultsATTENUATE reduced vertex N100-P200 by 56%, "click" loudness perception by 50%, and scalp sensation by 36%. We show that sensory prediction, induced with predictable ISI, has a suppressive effect on vertex N100-P200, and that combining standard suppression protocols with sensory prediction provides the best N100-P200 suppression. ATTENUATE was more effective than Standard masking, which only reduced vertex N100-P200 by 22%, loudness by 27%, and scalp sensation by 24%.

ConclusionsWe introduce a sensory suppression protocol superior to Standard masking and demonstrate that using an unjittered ISI can contribute to minimizing sensory confounds. ATTENUATE provides superior sensory suppression to increase TEP signal-to-noise and contributes to a growing understanding of TMS-EEG sensory neuroscience.

HighlightsO_LIATTENUATE is a novel sensory suppression protocol for suprathreshold dlPFC TMS
C_LIO_LIATTENUATE is superior to standard masking for minimizing sensory confounds
C_LIO_LIATTENUATE reduced vertex N100-P200 by 56% with no effect on the early TEP
C_LIO_LIATTENUATE reduced "click" loudness rating by 50% and scalp sensation by 36%
C_LIO_LIIndividual modifications are not sufficient to reduce vertex N100-P200 or perception
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title: Interference with lipoprotein maturation sensitizes methicillin-resistant Staphylococcus aureus to human group IIA secreted phospholipase A2 and daptomycin.

abstract: Methicillin-resistant Staphylococcus aureus (MRSA) has been classified as a high priority pathogen by the World Health Organization underlining the high demand for new therapeutics to treat infections. Human group IIA secreted phospholipase A2 (hGIIA) is among the most potent bactericidal proteins against Gram-positive bacteria, including S. aureus. To determine hGIIA-resistance mechanisms of MRSA we screened the Nebraska Transposon Mutant Library using a sublethal concentration of recombinant hGIIA. We identified and confirmed the role of lspA, encoding the lipoprotein signal peptidase LspA, as a new hGIIA resistance gene in both in vitro assays and an infection model in hGIIA-transgenic mice. Increased susceptibility of the lspA mutant was associated with faster and increased cell wall penetration of hGIIA. Moreover, lspA deletion also increased susceptibility to daptomycin, a last-resort antibiotic to treat MRSA infections. Exposure of MRSA wild-type to the LspA-specific inhibitors globomycin and myxovirescin A1 induced a lspA mutant phenotype with regard to hGIIA and daptomycin killing. Analysis of >26,000 S. aureus genomes showed that LspA is highly sequence-conserved, suggesting that LspA inhibition could be applied universally. The role of LspA in hGIIA resistance was not restricted to MRSA since Streptococcus mutans and Enterococcus faecalis were also more hGIIA-susceptible after lspA deletion or LspA inhibition, respectively. Overall, our data suggest that pharmacological blocking of LspA may disarm Gram-positive pathogens, including MRSA, to enhance clearance by innate host defense molecules and clinically-applied antibiotics.

category: microbiology


title: Janus Kinase 3 phosphorylation and the JAK/STAT pathway are positively modulated by follicle-stimulating hormone (FSH) in bovine granulosa cells.

abstract: JAK3 is a member of the JAK family of tyrosine kinase proteins involved in cytokine receptor-mediated intracellular signal transduction through the JAK/STAT signaling pathway. JAK3 was shown as differentially expressed in granulosa cells (GC) of bovine pre-ovulatory follicles suggesting that JAK3 could modulate GC function and activation/inhibition of downstream targets. We used JANEX-1, a JAK3 inhibitor, and FSH treatments and analyzed proliferation markers, steroidogenic enzymes and phosphorylation of target proteins including STAT3, CDKN1B/p27Kip1 and MAPK8IP3/JIP3. GC were treated with or without FSH in the presence or not of JANEX-1. Expression of steroidogenic enzyme CYP11A1, but not CYP19A1, was upregulated in GC treated with FSH and both were significantly decreased when JAK3 was inhibited. Proliferation markers CCND2 and PCNA were reduced in JANEX-1-treated GC and upregulated by FSH. Western blots analyses showed that JANEX-1 treatment reduced pSTAT3 amounts while JAK3 overexpression increased pSTAT3. Similarly, FSH treatment increased pSTAT3 even in JANEX-1-treated GC. UHPLC-MS/MS analyses revealed phosphorylation of specific amino acid residues within JAK3 as well as CDKN1B and MAPK8IP3 suggesting possible activation or inhibition post-FSH or JANEX-1 treatments. We show that FSH activates JAK3 in GC, which could phosphorylate target proteins and likely modulate other signaling pathways involving CDKN1B and MAPK8IP3.

category: molecular biology


title: Body mass and latitude as global predictors of vertebrate populations exposure to multiple threats

abstract: The interactive effects of multiple threats are one of the main causes of biodiversity loss, yet our understanding of what predisposes species to be impacted by multiple threats remains limited. Here we analyse a global dataset of over 7000 marine, freshwater, and terrestrial vertebrate populations, alongside trait, threat and geographical data, to identify the factors influencing the number of threats a species is subjected to at the population level. Out of a suite of predictors tested, we find that body mass and latitude both are broadly available for vertebrate species, and influence the number of threats a population is subjected to. Larger bodied species and those nearer the equator are typically affected by a higher number of threats. However, whilst this pattern broadly holds across ecosystems for most taxa, amphibians and reptiles show opposing trends. We suggest that latitude and body mass should be considered as key predictors to identify which vertebrate populations are likely to be impacted by multiple threats. These general predictors can help to better understand the impacts of the Anthropocene on global vertebrate biodiversity and design effective conservation policies.

category: ecology


title: Idiosyncratic fitness costs of ampicillin-resistant mutants derived from a long-term experiment with Escherichia coli

abstract: Antibiotic resistance is a growing concern that has prompted a renewed focus on drug discovery, stewardship, and evolutionary studies of the patterns and processes that underlie this phenomenon. A resistant strains competitive fitness relative to its sensitive counterparts in the absence of drug can impact its spread and persistence in both clinical and community settings. In a prior study, we examined the fitness of tetracycline-resistant clones that evolved from five different Escherichia coli genotypes, which had diverged during a long-term evolution experiment. In this study, we build on that work to examine whether ampicillin-resistant mutants are also less fit in the absence of the drug than their sensitive parents, and whether the cost of resistance is constant or variable among independently derived lines. Like the tetracycline-resistant lines, the ampicillin-resistant mutants were often less fit than their sensitive parents, with significant variation in the fitness costs among the mutants. This variation was not associated with the level of resistance conferred by the mutations, nor did it vary across the different parental backgrounds. In our earlier study, some of the variation in fitness costs associated with tetracycline resistance was explained by the effects of different mutations affecting the same cellular pathway and even the same gene. In contrast, the variance among the ampicillin-resistant mutants was associated with different sets of target genes. About half of the resistant clones suffered large fitness deficits, and their mutations impacted major outer-membrane proteins or subunits of RNA polymerases. The other mutants experienced little or no fitness costs and with, one exception, they had mutations affecting other genes and functions. Our findings underscore the importance of comparative studies on the evolution of antibiotic resistance, and they highlight the nuanced processes that shape these phenotypes.

category: evolutionary biology


title: Interdisciplinary analyses of Bronze Age communities from Western Hungary reveal complex population histories

abstract:
In this study we report 20 ancient shotgun genomes from present-day Western Hungary (3530 - 1620 cal BCE), mainly from previously understudied Baden, Somogyvar-Vinkovci, Kisapostag, and Encrusted Pottery archaeological cultures. Besides analysing archaeological, anthropological and genetic data, 14C and strontium isotope measurements complemented reconstructing the dynamics of the communities discovered at the site Balatonkeresztur. Our results indicate the appearance of an outstandingly high Mesolithic hunter-gatherer ancestry in the largest proportion (up to ~46%) among Kisapostag associated individuals, despite this component being thought to be highly diluted by the Early Bronze Age. We show that hunter-gatherer ancestry was likely derived from a previously unrecognised source in Eastern Europe that contributed mostly to prehistoric populations in Central Europe and the Baltic region. We revealed a patrilocal residence system and local female exogamy for this Kisapostag population that was also the genetic basis of the succeeding community of the Encrusted Pottery culture, represented by a mass grave that likely resulted from an epidemic. We also created a bioinformatic pipeline dedicated for archaeogenetic data processing. By developing and applying analytical methods for analysing genetic variants we found carriers of aneuploidy and inheritable genetic diseases. Furthermore, based on genetic and anthropological data, we present here the first female facial reconstruction from the Bronze Age Carpathian Basin.

SignificanceHere we present a genomic time transect study from the Carpathian Basin (3530 - 1620 cal BCE), that sheds light on local and interregional population processes. We not only discovered long-distance mobility to provide detailed analysis of yet understudied Bronze Age communities, but we also recovered a previously hidden remnant hunter-gatherer genetic ancestry and its contribution to various populations in Eastern and Central Europe. We integrated 14C and strontium isotope measurements to the interdisciplinary interpretation of a site with 19 individuals analysed, where patrilocal social organisation and several health-related genetic traits were detected. Furthermore, we developed new methods and method standards for computational analyses of archaic DNA, implemented to our newly developed and freely available bioinformatic pipeline.

category: genomics


title: Uncovering the hologenomic basis of an extraordinary plant invasion

abstract:
While invasive species are a key driver of the global biodiversity crisis, the drivers of invasiveness remain debated. To investigate the genomic basis of invasiveness in plants, we use the invasive weed Ambrosia artemisiifolia, introduced to Europe in the late 19th century, resequencing 655 ragweed genomes, including 308 herbarium specimens collected up to 190 years ago. In introduced European populations, we report selection signatures in defense genes and lower prevalence of particular plant pathogens in the invasive range. Together with temporal changes in population structure associated with introgression from closely related Ambrosia species, escape from microbial enemies likely favoured the plants remarkable success as an invasive species.

One-Sentence SummaryThe invasive success of European ragweed was facilitated by release from enemy microbes and inter-species hybridization.


title: Inhibition of a critical malaria host-pathogen interaction by a computationally designed inhibitor targeting Plasmodium vivax DBP

abstract:
Malaria is a substantial global health burden with 229 million cases in 2019 and 450,000 deaths annually. Plasmodium vivax is the most widespread malaria-causing parasite putting 2.5 billion people at risk of infection. P. vivax has a dormant liver stage and therefore can exist for long periods undetected. Its blood-stage can cause severe reactions and hospitalization. Few treatment and detection options are available for this pathogen. To address this need, we developed nanomolar inhibitor that could serve as a therapeutic and a diagnostic. A unique characteristic of P. vivax is that it depends on the Duffy antigen/Receptor for chemokines (DARC) on the surface of host red blood cells for invasion. P. vivax employs the Duffy binding protein (DBP) to bind to DARC. We first de novo designed a three helical bundle scaffolding database which was screened via protease digestions for stability. Protease-resistant scaffolds highlighted thresholds for stability, which we utilized for selecting DARC mimetics that we subsequentially designed through grafting and redesign of these scaffolds. The optimized design small helical protein disrupts the DBP:DARC interaction. The inhibitor blocks the receptor binding site on DBP and thus forms a strong foundation for a therapeutic that will inhibit reticulocyte infection and prevent the pathogenesis of P. vivax malaria.

TeaserDe novo designed proteins present a new alternative for the development of therapeutics. They can be small, highly stable and easily manufactured. Here we designed a potential new therapeutic to inhibit entry of Plasmodium vivax into red blood cells by interfering its interactions of surface displayed DBP molecules with the host receptor DARC.

category: bioengineering


title: Multiplexed Detection of Molecular Interactions with DNA Origami Engineered Cells in 3-D Collagen Matrices

abstract: The interactions of cells with signaling molecules present in their local microenvironment maintain cell proliferation, differentiation, and spatial organization, and mediate progression of diseases such as metabolic disorders and cancer. Real-time monitoring of the interactions between cells and their extracellular ligands in a 3-D microenvironment can inform detection and understanding of cell processes and development of effective therapeutic agents. DNA-origami technology allows for the design and fabrication of biocompatible and 3-D functional nanodevices via molecular self-assembly for various applications including molecular sensing. Here, we report a robust method to monitor live cell interactions with molecules in their surrounding environment in a 3-D tissue model using a microfluidic device. We used a DNA origami Cell Sensing Platform (CSP) to detect two specific nucleic acid sequences on the membrane of B cells and dendritic cells. We further demonstrated real-time detection of biomolecules with the DNA sensing platform on the surface of dendritic cells in a 3-D microfluidic tissue model. Our results establish the integration of live cells with membranes engineered with DNA nanodevices into microfluidic chips as a highly capable biosensor approach to investigate subcellular interactions in physiologically relevant 3-D environments under controlled biomolecular transport.


title: Optocoder: computational decoding of spatially indexed bead arrays

abstract:
MotivationAdvancing technologies that quantify gene expression in space are transforming contemporary biology research. A class of spatial transcriptomics methods uses barcoded bead arrays that are optically decoded via microscopy and are later matched to sequenced data from the respective libraries. To obtain a detailed representation of the tissue in space, robust and efficient computational pipelines are required to process microscopy images and accurately basecall the bead barcodes.

ResultsOptocoder is a computational framework that processes microscopy images to decode bead barcodes in space. It efficiently aligns images, detects beads, and corrects for confounding factors of the fluorescence signal, such as crosstalk and phasing. Furthermore, Optocoder employs supervised machine learning to strongly increase the number of matches between optically decoded and sequenced barcodes. We benchmark Optocoder using data from an in-house spatial transcriptomics platform, as well as from Slide-Seq(V2), and we show that it efficiently processes all datasets without modification.

AvailabilityOptocoder is publicly available, open-source and provided as a stand-alone Python package on GitHub: https://github.com/rajewsky-lab/optocoder


title: Genomic neighbor typing for bacterial outbreaksurveillance

abstract: Genomic neighbor typing enables heuristic inference of bacterial lineages and phenotypes from nanopore sequencing data. However, small reference databases may not be sufficiently representative of the diversity of lineages and genotypes present in a collection of isolates. In this study, we explore the use of genomic neighbor typing for surveillance of community-associated Staphylococcus aureus outbreaks in Papua New Guinea (PNG) and Far North Queensland, Australia (FNQ). We developed Sketchy, an implementation of genomic neighbor typing that queries exhaustive whole genome reference databases using MinHash. Evaluations were conducted using nanopore read simulations and six species-wide reference sketches (4832 - 47616 genomes), as well as two S. aureus outbreak data sets sequenced at low depth using a sequential multiplex library protocol on the MinION (n = 160, with matching Illumina data). Heuristic inference of lineages and antimicrobial resistance profiles allowed us to conduct multiplex genotyping in situ at the Papua New Guinea Institute of Medical Research in Goroka, on low-throughput Flongle adapters and using multiple successive libraries on the same MinION flow cell (n = 24 - 48). Comparison to phylogenetically informed genomic neighbor typing with RASE on the dominant outbreak sequence type suggests slightly better performance at predicting lineage-scale genotypes using large sketch sizes, but inferior performance in resolving clade-specific genotypes (methicillin resistance). Sketchy can be used for large-scale bacterial outbreak surveillance and in challenging sequencing scenarios, but improvements to clade-specific genotype inference are needed for diagnostic applications. Sketchy is available open-source at: https://github.com/esteinig/sketchy


title: CDMAP/CDVIS: Context-dependent Mutation Analysis Package and Visualization Software

abstract: The Context-dependent Mutation Analysis Package and Visualization Software (CDMAP/CDVIS) is an automated, modular toolkit used for analysis and visualization of context-dependent mutation patterns (site-specific variation in mutation rate from neighboring-nucleotide effects). The CDMAP algorithm calculates context-dependent mutation rates in chromosomes or replichores, and can generate high-resolution figures to analyze variation in mutation rate across spatiotemporal scales. Output from CDMAP can be integrated into CDVIS, an interactive database for visualizing mutation patterns across multiple taxa simultaneously.


title: Towards Effective and Generalizable Fine-tuning for Pre-trained Molecular Graph Models

abstract: Graph Neural Networks (GNNs) and Transformer have emerged as dominant tools for AI-driven drug discovery. Many state-of-the-art methods first pre-train GNNs or the hybrid of GNNs and Transformer on a large molecular database and then fine-tune on downstream tasks. However, different from other domains such as computer vision (CV) or natural language processing (NLP), getting labels for molecular data of downstream tasks often requires resource-intensive wet-lab experiments. Besides, the pre-trained models are often of extremely high complexity with huge parameters. These often cause the fine-tuned model to over-fit the training data of downstream tasks and significantly deteriorate the performance. To alleviate these critical yet under-explored issues, we propose two straightforward yet effective strategies to attain better generalization performance: 1. MolAug, which enriches the molecular datasets of down-stream tasks with chemical homologies and enantiomers; 2. WordReg, which controls the complexity of the pre-trained models with a smoothness-inducing regularization built on dropout. Extensive experiments demonstrate that our proposed strategies achieve notable and consistent improvements over vanilla fine-tuning and yield multiple state-of-the-art results. Also, these strategies are model-agnostic and readily pluggable into fine-tuning of various pre-trained molecular graph models. We will release the code and the fine-tuned models.


title: Early removal of the infrapatellar fat pad pad beneficially alters the pathogenesis of moderate stage idiopathic knee osteoarthritis in the Dunkin Hartley guinea pig

abstract:
BackgroundThe infrapatellar fat pad (IFP) is the largest adipose deposit in the knee; however, its contributions to the homeostasis of this organ remain unknown. To determine the influence of this depot on joint health, this study determined the progression of osteoarthritis (OA) following excision of the IFP in a rodent model of naturally-occurring disease.

MethodsMale Dunkin-Hartley guinea pigs (n=10) received surgical removal of the IFP in one knee at 3 months of age; contralateral knees received sham surgery as matched internal controls. Treadmill-based gait analysis was performed prior to IFP removal and monthly thereafter. Animals were harvested at 7 months of age. Both knees were processed for microcomputed tomography (microCT), histopathology, transcript expression analyses, and immunohistochemistry (IHC).

ResultsFibrous connective tissue (FCT) developed in place of the native fat pad. Gait demonstrated no significant differences between IFP removal and contralateral limbs. MicroCT OA scores were improved in knees with the FCT. Histopathology confirmed maintenance of articular cartilage structure, proteoglycan content, and chondrocyte cellularity in FCT-containing knees. Transcript analyses revealed decreased expression of adipose-related molecules and inflammatory mediators in FCTs compared to IFPs. This was corroborated via IHC for select inflammatory mediators.

Discussion/ConclusionFormation of the FCT resulted in reduced OA-associated changes in both bone and cartilage. A decrease in inflammatory mediators at transcript and protein levels may be associated with these improvements. The IFP may therefore play a role in the pathogenesis of knee OA in this strain, with removal prior to disease onset appearing to have short-term benefits.

category: pathology


title: Cell-type specific transcriptional networks in root xylem adjacent cell layers

abstract: Transport of water, ions and signals from roots to leaves via the xylem vessels is essential for plant life and needs to be tightly regulated. The final composition of the transpiration stream before passage into the shoots is controlled by the xylem-adjacent cell layers, namely xylem parenchyma and pericycle, in the upper part of the root. To unravel regulatory networks in this strategically important location, we generated Arabidopsis lines expressing a nuclear tag under the control of the HKT1 promoter. HKT1 retrieves sodium from the xylem to prevent toxic levels in the shoot, and this function depends on its specific expression in upper root xylem-adjacent tissues. Based on FACS RNA-sequencing and INTACT ChIP-sequencing, we identified the gene repertoire that is preferentially expressed in the tagged cell types and discovered transcription factors experiencing cell-type specific loss of H3K27me3 demethylation. For one of these, ZAT6, we show that H3K27me3-demethylase REF6 is required for de-repression. Analysis of zat6 mutants revealed that ZAT6 activates a suite of cell-type specific downstream genes and restricts Na+ accumulation in the shoots. The combined Files open novel opportunities for  bottom-up causal dissection of cell-type specific regulatory networks that control root-to-shoot communication under environmental challenge.

category: plant biology


title: Sensitisation of colonic nociceptors by TNFα is dependent on TNFR1 expression and p38 MAPK activity

abstract:
Visceral pain is a leading cause of morbidity in gastrointestinal diseases, which is exacerbated by the gut related side-effects of many analgesics. New treatments are needed and further understanding of the mediators and mechanisms underpinning visceral nociception in disease states is required to facilitate this. The pro-inflammatory cytokine TNF is linked to pain in patients with inflammatory bowel disease or irritable bowel syndrome and has been shown to sensitise colonic sensory neurons. Somatic, TNF-triggered thermal and mechanical hypersensitivity is mediated by TRPV1 signalling and p38 MAPK activity respectively, downstream of TNFR1 receptor activation. We therefore hypothesised that TNFR1-evoked p38 MAPK activity may also be responsible for TNF sensitisation of colonic afferent responses to the TRPV1 agonist capsaicin, and noxious distension of the bowel. Using Ca2+ imaging of dorsal root ganglia sensory neurons, we observed TNF-mediated increases in intracellular [Ca2+] and sensitisation of capsaicin responses. The sensitising effects of TNF were dependent on TNFR1 expression and attenuated by TRPV1 and p38 MAPK inhibition. Consistent with these findings, ex vivo colonic afferent fibre recordings demonstrated enhanced response to noxious ramp distention of the bowel and bath application of capsaicin following TNF pre-treatment. Responses were reversed by p38 MAPK inhibition and absent in tissue from TNFR1 knockout mice. Our findings demonstrate a contribution of TNFR1, p38 MAPK and TRPV1 to TNF-induced sensitisation of colonic afferents, highlighting the potential utility of these drug targets for the treatment of visceral pain in GI disease.

Key Points SummaryO_LITNF sensitises sensory neurons and colonic afferents to the TRPV1 agonist capsaicin.
C_LIO_LITNF mediated sensitisation of sensory neurons and colonic nociceptors is dependent on TNFR1 expression.
C_LIO_LITNF sensitisation of sensory neurons and colonic afferents to capsaicin and noxious ramp distension is abolished by inhibition of p38 MAPK.
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title: Speech understanding oppositely affectsacoustic and linguistic neural tracking in aspeech rate manipulation paradigm.

abstract:
When listening to continuous speech, the human brain can track features of the presented speech signal. It has been shown that neural tracking of acoustic features is a prerequisite for speech understanding and can predict speech understanding in controlled circumstances. However, the brain also tracks linguistic features of speech, which may be more directly related to speech understanding. We investigated acoustic and linguistic speech processing as a function of varying speech understanding by manipulating the speech rate. In this paradigm, acoustic and linguistic speech processing are affected simultaneously but in opposite directions: When the speech rate increases, more acoustic information per second is present. In contrast, the tracking of linguistic information becomes more challenging when speech is less intelligible at higher speech rates. We measured the EEG of 18 participants (4 male) who listened to speech at various speech rates. As expected and confirmed by the behavioral results, speech understanding decreased with increasing speech rate. Accordingly, linguistic neural tracking decreased with increasing speech rate, but acoustic neural tracking increased. This indicates that neural tracking of linguistic representations can capture the gradual effect of decreasing speech understanding. In addition, increased acoustic neural tracking does not necessarily imply better speech understanding. This suggests that, although more challenging to measure due to the low signal-to-noise ratio, linguistic neural tracking may be a more direct predictor of speech understanding.

Significance statementAn increasingly popular method to investigate neural speech processing is to measure neural tracking. Although much research has been done on how the brain tracks acoustic speech features, linguistic speech features have received less attention. In this study, we disentangled acoustic and linguistic characteristics of neural speech tracking via manipulating the speech rate. A proper way of objectively measuring auditory and language processing paves the way towards clinical applications: An objective measure of speech understanding would allow for behavioral-free evaluation of speech understanding, which allows to evaluate hearing loss and adjust hearing aids based on brain responses. This objective measure would benefit populations from whom obtaining behavioral measures may be complex, such as young children or people with cognitive impairments.


title: Abnormal brain state distribution and network connectivity in a SYNGAP1 rat model

abstract: Mutations in the SYNGAP1 gene are one of the common predictors of neurodevelopmental disorders, commonly resulting in individuals developing autism, intellectual disability, epilepsy, and sleep deficits. EEG recordings in neurodevelopmental disorders show potential to identify clinically translatable biomarkers to both diagnose and track the progress of novel therapeutic strategies, as well as providing insight into underlying pathological mechanisms. In a rat model of SYNGAP1 haploinsufficiency in which the exons encoding the calcium/lipid binding and GTPase activating protein (GAP) domains have been deleted (Syngap+/{Delta}-GAP) we analysed the duration and occurrence of wake, non rapid eye movement (NREM) and rapid eye movement (REM) brain states during 6 hour multi-electrode EEG recordings. We find that although Syngap+/{Delta}-GAP animals spend an equivalent percent time in wake and sleep states, they have an abnormal brain state distribution as the number of wake and NREM bouts are reduced and there is an increase in the average duration of both wake and NREM epochs. We perform connectivity analysis by calculating the average imaginary coherence between electrode pairs at varying distance thresholds during these states. In group averages from pairs of electrodes at short distances from each other, a clear reduction in connectivity during NREM is present between 11.5 Hz and 29.5 Hz, a frequency range that overlaps with sleep spindles, oscillatory phenomena thought to be important for normal brain function and memory consolidation. Sleep spindles occurrence, amplitude, power and spread across multiple electrodes were not reduced in Syngap+/{Delta}-GAP rats, with only a small decrease in duration detected. Nonetheless, by analysing the dynamic imaginary coherence during sleep spindles, we found a reduction in high connectivity instances between short-distance electrode pairs. Finally, by comparing the dynamic imaginary coherence during sleep spindles between individual electrode pairs, we identified a group of channels over the right somatosensory, association and visual cortices that have a significant reduction in connectivity during sleep spindles in mutant animals. These data suggest that Syngap+/{Delta}-GAP rats have altered brain state dynamics and EEG connectivity, which may have clinical relevance for SYNGAP haploinsufficiency in humans.


title: Dopamine transporter and synaptic vesicle sorting defects initiate auxilin-linked Parkinson's disease

abstract: Auxilin participates in the uncoating of clathrin-coated vesicles (CCVs), thereby facilitating synaptic vesicle (SV) regeneration at presynaptic sites. Auxilin (DNAJC6/PARK19) loss-of- function mutations cause early-onset Parkinsons disease (PD). Here, we utilized auxilin-knockout (KO) mice to elucidate the mechanisms through which auxilin deficiency and clathrin-uncoating deficits lead to PD. We demonstrate that auxilin KO mice display the cardinal features of PD, including progressive motor deficits, -synuclein pathology, nigral dopaminergic loss, and neuroinflammation. Through unbiased proteomic and neurochemical analyses, we demonstrate that dopamine homeostasis is disrupted in auxilin KO brains, including via slower dopamine reuptake kinetics in vivo, an effect associated with dopamine transporter misrouting into axonal membrane deformities in the dorsal striatum. We also show that elevated macroautophagy and defective SV protein sorting contribute to ineffective dopamine sequestration and homeostasis, ultimately leading to neurodegeneration. This study advances our knowledge of how presynaptic endocytosis deficits lead to dopaminergic vulnerability and pathogenesis of PD.


title: Human Cerebral Spheroids Undergo Activity Dependent Changes In Cellular Composition And MicroRNA Expression

abstract: Activity-induced neurogenesis has been extensively studied in rodents but the lack of ante mortem accessibility to human brain at the cellular and molecular levels limits studies of the process in humans. Using cerebral spheroids derived from human induced pluripotent stem cells (iPSCs), we investigated the effects of increased neuronal activity on neurogenesis. Our studies demonstrate that increasing neuronal activity with 4-aminopyridine in 3-month-old cerebral spheroids is associated with increases in the numbers of new neurons and decreases in the population of new glial cells. We also observed a significant decrease in the expression of miR-135a, which has previously been shown to be decreased in exercise-induced neurogenesis. Predicted targets of miR-135a include key participants in the SMAD2/3 and BDNF pathways. Together, our results suggest that iPSC-derived cerebral spheroids are an attractive model to study some aspects of activity-induced neurogenesis.


title: Ventral pallidum GABA neurons bidirectionally control opioid relapse across rat behavioral models

abstract:
Opioid addiction is a chronic, relapsing disorder. Whether addicted individuals are forced to abstain or they decide themselves to quit using drugs, relapse rates are high--especially upon encountering contexts and stimuli associated with prior opioid use. Rodents similarly show context- and cue-induced reinstatement of drug seeking following abstinence, and intriguingly, the neural circuits underlying these relapse-like behaviors differ when abstinence is involuntarily imposed, versus when animals decide themselves to cease taking drug. Here, we employ two complementary rat behavioral models of relapse-like behavior for the highly reinforcing opioid drug remifentanil, and asked whether GABAergic neurons in the ventral pallidum (VPGABA) control opioid seeking under these behavioral conditions. Specifically, we asked how chemogenetically stimulating VPGABA neurons with clozapine-N-oxide (CNO) influences the ability of contextual or discrete remifentanil-paired cues to reinstate drug seeking following either voluntary (punishment-induced; GroupPunish), or experimenter-imposed (extinction training; GroupExt) abstinence. In GroupPunish rats, we also chemogenetically inhibited VPGABA neurons, and examined spontaneous VP activity (Fos) during cued-reinstatement. In both GroupPunish and GroupExt rats, stimulating Gq-signaling in VPGABA neurons augmented remifentanil reinstatement in a cue- and context-dependent manner. Conversely, engaging inhibitory Gi-signaling in VPGABA neurons in GroupPunish suppressed cue-induced reinstatement, and additionally cue-triggered seeking was correlated with Fos in rostral, but not caudal VP. In contrast, neither stimulating nor inhibiting VPGABA neurons influenced unpunished remifentanil self-administration. We conclude that VPGABA neurons bidirectionally control opioid seeking regardless of the specific relapse model employed, highlighting their fundamental role in opioid relapse-like behavior across behavioral models, and potentially across species.

HighlightsO_LIWe acutely inhibit or stimulate VP GABA neurons during opioid seeking
C_LIO_LIVP GABA neurons mediate relapse-like behavior across behavioral models
C_LIO_LIBehavioral context impacts DREADD stimulation of behavior, not VP activity
C_LIO_LIRostral, not caudal VP Fos correlates with opioid reinstatement
C_LI


title: The AAA+ ATPase RavA and its binding partner ViaA modulate E. coli aminoglycoside sensitivity through interaction with the inner membrane

abstract: Enteric bacteria have to adapt to environmental stresses in the human gastrointestinal tract such as acid and nutrient stress, oxygen limitation and exposure to antibiotics. Membrane lipid composition has recently emerged as a key factor for stress adaptation. The E. coli ravA-viaA operon is essential for aminoglycoside bactericidal activity under anaerobiosis but its mechanism of action is unclear. Here we characterise the VWA domain-protein ViaA and its interaction with the AAA+ ATPase RavA, and find that both proteins localise at the inner cell membrane. We demonstrate that RavA and ViaA target specific phospholipids and subsequently identify their lipid-binding sites. We further show that mutations abolishing interaction with lipids restore induced changes in cell membrane morphology and lipid composition. Finally we reveal that these mutations render E. coli gentamicin-resistant under fumarate respiration conditions. Our work thus uncovers a ravA-viaA-based pathway which is mobilised in response to antibiotics under anaerobiosis and has a major impact on cell membrane regulation.


title: Non-essential tRNA and rRNA modifications impact the bacterial response to sub-MIC antibiotic stress

abstract: Antimicrobial resistance (AMR) develops as a major problem in infectious diseases treatment. While antibiotic resistance mechanisms are usually studied using lethal antibiotic doses, lower doses allowing bacterial growth are now considered as factors influencing the development and selection of resistance. Based on high throughput transposon insertion sequencing (TN-seq) in V. cholerae, we have undertaken the phenotypic characterization of 23 transfer RNA (tRNA) and ribosomal RNA (rRNA) modifications deletion mutants, for which growth is globally not affected in the absence of stress. We uncover a specific involvement of different RNA modification genes in the response to aminoglycosides (tobramycin (TOB), gentamicin (GEN)), fluoroquinolones (ciprofloxacin (CIP)), {beta}-lactams (carbenicillin (CRB)), chloramphenicol (CM) and trimethoprim (TRM). Our results identify t/rRNA modification genes, not previously associated to any antibiotic resistance phenotype, as important factors affecting the bacterial response to sub-MIC antibiotics from different families. This suggests differential translation and codon decoding as critical factors involved in the bacterial response to stress.


title: Exploring Genomic Conservation in Actinobacteriophages with Small Genomes

abstract: Actinobacteriophages of a wide range of genome sizes continue to be isolated and characterized, but only a handful of these have atypically small genomes, defined in this work as genome sizes under 20,000 bp. These "small phages" are relatively rare and have received minimal study thus far. Of the actinobacteriophages published in PhagesDB.org, small phages have been isolated on Arthrobacter, Gordonia, Rhodococcus, and Microbacterium hosts. A previous study by Pope et al. showed that Gordonia small phages have similar gene products and amino acid sequences. Here, we set out to further examine relationships between small Gordonia phages as well as small phages that infect other hosts. Of the 3222 sequenced phages listed on PhagesDB, we identified 109 distinctly small phages with genome sizes under 20,000 bp. The majority of the small phages were isolated on Arthrobacter or Microbacterium hosts. Using comparative genomics, we searched for patterns of similarity among 34 cluster-representative small phages. Dot plot comparisons showed that there was more amino acid conservation than nucleotide identity amongst small phages. Gene content similarity (GCS) analysis revealed that the temperate Gordonia phages in Cluster CW share significant GCS values (over 35%) with the lytic Arthrobacter phages in Cluster AN, suggesting that some small phages have a considerable degree of genomic similarity with each other. SplitsTree analyses of shared phams (genes with substantial amino acid identity) supported the complexity of clustering criteria in small phages, given shuffling of genes across phages of different clusters and close relationships despite varied cluster membership. We observed this continuum of phage diversity through Rhodococcus phage RRH1s closer similarity to phages in Gordonia subcluster CW1 than CW1 is to Gordonia subcluster CW3. Finally, we were able to confirm the presence of conserved phams across not only small Gordonia phages but also within small phages from different clusters and hosts. Studying these genomic trends hidden in small phages allows us to better understand and appreciate the overall diversity of phages.


title: Heteractis magnifica Sea Anemone Population Dynamics at Village Reef, Perhentian Kecil, Malaysia: Monitoring of Growth, Formations, and Hosting Status.

abstract: The coastal waters of Malaysia have been known to allow proliferation of sea anemone assemblages, which are resident species of tropical coral reefs. Along the Perhentian Islands of Terengganu, no efforts have been made thus far to investigate the presence and population dynamics of sea anemone assemblages locally. In this study, Heteractis magnifica assemblages at Village Reef at Perhentian Kecil were monitored during May, July, and August of 2020, thus providing a first assessment of their abundance. Sea anemone formation size, individual size, habitat location, and hosting status of anemonefish were assessed. Results demonstrate significantly larger counts of individuals within aggregated formations in the patch reef as compared to the fringe reef, without the presence of larger individual sizes. In addition, Heteractis magnifica specimens that were actively hosting anemonefish had significantly larger cover, larger individual sizes, and demonstrated higher individual counts within their aggregated formations compared to non-hosting specimens. There was no significant overall effect of time on sea anemone growth throughout the monitoring period, nor were there any significant changes in abundance levels regarding formation make-up throughout the monitoring phase. However, time related effects where present upon data inspection per assessment period. The restricted time frame of the monitoring period could play a role in explaining the absence of overall time related effects. A prolonged monitoring will help to further understand Heteractis magnifica population dynamics at this location, which can increase knowledge capacity for reef management and conservation strategies.


title: metaPR2: a database of eukaryotic 18S rRNA metabarcodes with an emphasis on protists.

abstract: In recent years, metabarcoding has become the method of choice for investigating the composition and assembly of microbial eukaryotic communities, and an increasing number of environmental datasets are being published. Although unprocessed sequence files are often publicly available, processed data, i.e. sequences clustered as operational taxonomic units (OTUs) or amplicon sequence variants (ASVs) are rarely at hand in a comparable format. This hampers comparative studies between different environments and datasets, for example examining the biogeographical patterns of specific groups/species, as well analysing the micro-genetic diversity within these groups. Here, we present a newly-assembled database of processed 18S rRNA metabarcodes that are annotated with the PR2 reference sequence database. This database, called metaPR2, contains 41 datasets corresponding to more than 4,000 samples and 73,000 ASVs. The database is accessible through both a web-based interface (https://shiny.metapr2.org) and as an R package, and should prove very useful to all researchers working on protist diversity in a variety of systems.


title: Delimiting Continuity: Comparison of Target Enrichment and ddRAD for Delineating Admixing Parapatric Melitaea Butterflies

abstract: Parapatrically distributed taxa pose a challenge for species delimitation due to the presence of gene flow and inherent arbitrariness of exactly defining the species boundaries in such systems. We tackled the problem of species delimitation in a parapatric species pair of Melitaea butterflies using two popular genomic methods - double digest restriction-site associated DNA sequencing (ddRAD) and target enrichment. The former is mainly applied at shallow phylogenetic scales and the latter at both deep and shallow scales. Although both of these methods have been adequately utilised for species delimitation purposes, there are only a handful of studies that have compared these two genomic approaches in the same study system. We applied phylogenetic, population genetic and species delimitation methods and compared the results obtained from the two approaches. Using a recently developed target enrichment probe kit, we were able to capture 1,743 loci with a low amount of missing data and compared these with already available ddRAD data from a previous study on the same set of specimens. We recovered consistent phylogenetic relationships across the datasets, both demonstrating the presence of a genetically distinct Balkan lineage and paraphyly of Melitaea athalia with respect to Melitaea celadussa. The same relationships were also found in a species tree analysis of the target enrichment dataset using ASTRAL. Population genetic STRUCTURE analyses supported the presence of two species when using ddRAD data, but three species when using target enrichment, while the Bayes factor delimitation analysis found both two and three species scenarios equally decisive in both datasets. From the geographic distribution of genomic admixture, we confirm the patterns observed by a previous study that used ddRAD data. As the results obtained from both methods were largely congruent, we discuss some practical considerations and benefits of target enrichment over RAD sequencing. We conclude that the choice of method of genomic data collection does not influence the results of phylogenetic analyses at alpha taxonomic level, given a sufficient number of loci. Finally, we recommend a solution for delineating species in parapatric scenarios by proposing that parapatric taxa be consistently classified as subspecies or complete species, but not both, to promote taxonomic stability.


title: Evolutionary quantitative proteomics of reproductive interactions in Drosophila

abstract: Reproductive traits evolve rapidly between species. Understanding the causes and consequences of this rapid divergence requires characterization of male and female reproductive proteins and how their interaction mediates fertilisation success. Species in the Drosophila virilis clade exhibit interspecific postmating prezygotic reproductive isolation, making them ideal for studies on diversification of reproductive proteins and their role in speciation. Here we present a novel method to simultaneously identify and quantify transferred male ejaculate proteins and the female reproductive proteome using multiplexed Tandem-Mass-Tag (TMT) isobaric labelling of the lower female reproductive tract before and immediately after mating in three species of the virilis group. We identified over 200 putative male ejaculate proteins, many of which show differential abundance between species. We also identified over 2000 proteins that provide the first description of the female reproductive tract proteome outside of the D. melanogaster group. The female reproductive tract proteome shows significant divergence between species, especially female-specific serine-type endopeptidases, which showed differential abundance between species and elevated rates of molecular evolution that is similar to that of male seminal fluid proteins. We also found that only ~1/3 of male ejaculate proteins are likely produced in the accessory glands and code for a signal peptide sequence. Finally, we assessed the utility of species-specific compared to single species query databases for protein identification and quantification from multiple species samples. Our results show that using species-specific query databases dramatically improves protein identification and quantification.


title: Genetic and genomic analysis of oxygen consumption in mice

abstract: We estimated genetic parameters of oxygen consumption (OC), OC per metabolic body weight (OCMBW), and body weight at three through eight weeks of age in divergently selected mice populations, with an animal model considering maternal genetic, common litter environmental, and cytoplasmic inheritance effects. Cytoplasmic inheritance was considered based on maternal lineage information. For OC, estimated direct heritability was moderate (0.32) and estimated maternal heritability and proportion of the variance of cytoplasmic inheritance effects to the phenotypic variance were very low (both <0.03), implying that causal genes for OC could be located on autosomes. To assess this hypothesis, we attempted to identify possible candidate causal genes by performing pool-seq using pooled DNA samples from mice in high and low OC lines and selective signature detection. We made a list of possible candidate causal genes for OC, including those relating to electron transport chain and ATP-binging proteins (Ndufa12, Sdhc, Atp10b, etc.), Prr16 encoding Largen protein, Cry1 encoding a key component of the circadian core oscillator, and so on. The results could contribute to elucidate the genetic mechanism of OC, an indicator for maintenance energy requirement and therefore feed efficiency.

category: genetics