title: A Geometric Framework for Understanding Dynamic Information Integration in Context-Dependent Computation

abstract: Prefrontal cortex plays a prominent role in performing flexible cognitive functions and working memory, yet the underlying computational principle remains poorly understood. Here we trained a rate-based recurrent neural network (RNN) to explore how the context rules are encoded, maintained across seconds-long mnemonic delay, and subsequently used in a context-dependent decision-making task. The trained networks emerged key experimentally observed features in the prefrontal cortex (PFC) of rodent and monkey experiments, such as mixed-selectivity, sparse representations, neuronal sequential activity and rotation dynamics. To uncover the high-dimensional neural dynamical system, we further proposed a geometric framework to quantify and visualize population coding and sensory integration in a temporally-defined manner. We employed dynamic epoch-wise principal component analysis (PCA) to define multiple task-specific subspaces and task-related axes, and computed the angles between task-related axes and these subspaces. In low-dimensional neural representations, the trained RNN first encoded the context cues in a cue-specific subspace, and then maintained the cue information with a stable low-activity state persisting during the delay epoch, and further formed line attractors for sensor integration through low-dimensional neural trajectories to guide decision making. We demonstrated via intensive computer simulations that the geometric manifolds encoding the context information were robust to varying degrees of weight perturbation in both space and time. Overall, our analysis framework provides clear geometric interpretations and quantification of information coding, maintenance and integration, yielding new insight into the computational mechanisms of context-dependent computation.


title: tDCS modulates effective connectivity during motor command following; a potential therapeutic target for disorders of consciousness

abstract: Transcranial direct current stimulation (tDCS) is attracting increasing interest as a potential therapeutic route for unresponsive patients with prolonged disorders of consciousness (PDOC). However, research to date has had mixed results. Here, we propose a new direction by directly addressing the mechanisms underlying lack of responsiveness in PDOC, and using these to define our targets and the success of our intervention in the healthy brain first. We report 2 experiments that assess whether tDCS to the primary motor cortex (M1-tDCS; Experiment 1) and the cerebellum (cb-tDCS; Experiment 2) administered at rest modulate thalamo-cortical coupling in a subsequent command following task typically used to clinically assess awareness. Both experiments use sham- and polarity-controlled, randomised, double-blind, crossover designs. In Experiment 1, 22 participants received anodal, cathodal, and sham M1-tDCS sessions while in the MRI scanner. A further 22 participants received the same protocol with cb-tDCS in Experiment 2. We use Dynamic Causal Modelling of fMRI to characterise the effects of tDCS on brain activity and dynamics during simple thumb movements in response to command. We found that M1-tDCS increased thalamic excitation and that Cathodal cb-tDCS increased excitatory coupling from thalamus to M1. All these changes were polarity specific. Combined, our experiments demonstrate that tDCS can successfully modulate long range thalamo-cortical dynamics during command following via targeting of cortical regions. This suggests that M1- and cb-tDCS may allow PDOC patients to overcome the motor deficits at the root of their reduced responsiveness, improving their rehabilitation options and quality of life as a result.

category: neuroscience


title: Decrease of Functional Connectivity within the Default Mode Network by a Brief Training of Focused Attention on the Breath in Novices

abstract: Experienced meditators reduce the activity of the default mode network (DMN), a brain system preferentially active when people are not engaged in specific tasks. However, the neural modulation of the DMN in novices remain largely unexplored. By using electroencephalography, we investigated the DMN functional connectivity in two groups of novices: the meditation group practiced six consecutive days of focused attention on the breath; the control group practiced only on the first and last days. After the brief training, results showed a decrease in the DMN functional connectivity between the ventromedial prefrontal cortex and the posterior cingulate cortex in theta and alpha bands during the focused attention condition, in the meditation group compared to the control group. The change in DMN functional connectivity was significantly correlated with the increase in state-level mindfulness scores. These data elucidate DMN modifications already arising at the initial stages of mindfulness meditation training in novices.


title: Targeting Gys1 with AAV-SaCas9 decreases pathogenic polyglucosan bodies and neuroinflammation in Adult Polyglucosan Body and Lafora disease mouse models

abstract: Many adult and most childhood neurological diseases have a genetic basis. CRISPR/Cas9 biotechnology holds great promise in neurological therapy, pending the clearance of major delivery, efficiency and specificity hurdles. We apply CRISPR/Cas9 genome editing in its simplest modality, namely inducing gene sequence disruption, to one adult and one pediatric disease. Adult polyglucosan body disease is a neurodegenerative disease resembling amyotrophic lateral sclerosis. Lafora disease is a severe late childhood onset progressive myoclonus epilepsy. The pathogenic insult in both is formation in the brain of glycogen with overlong branches, which precipitates and accumulates into polyglucosan bodies that drive neuroinflammation and neurodegeneration. We packaged Staphylococcus aureus Cas9 and a guide RNA targeting the glycogen synthase gene Gys1 responsible for brain glycogen branch elongation in AAV9 virus, which we delivered by neonatal intracerebroventricular injection to one mouse model of adult polyglucosan body disease and two mouse models of Lafora disease. This resulted, in all three models, in editing of approximately 17% of Gys1 alleles and a similar extent of reduction of Gys1 mRNA across the brain. The latter led to approximately 50% reductions of GYS1 protein, of abnormal glycogen accumulation and of polyglucosan bodies, as well as corrections of neuroinflammatory markers in all three models. Our work represents proof of principle for virally-delivered CRISPR/Cas9 neurotherapeutics in an adult-onset (adult polyglucosan body) and a childhood-onset (Lafora) neurological diseases.


title: Relationships between biomechanical parameters, neurological recovery, and neuropathology following concussion in swine

abstract: Mild traumatic brain injury (mTBI) affects millions of individuals annually primarily through falls, traffic collisions, or blunt trauma and can generate symptoms that persist for years. Closed-head rotational injury is the most common form of mTBI and is defined by a rapid change in acceleration within an intact skull. Injury kinematics - the mechanical descriptors of injury-inducing motion - explain movement of the head, energy transfer to the brain, and, therefore, determine injury severity. However, the relationship between closed-head rotational injury kinematics - such as angular velocity, angular acceleration, and injury duration - and outcome after mTBI is currently unknown. To address this gap in knowledge, we analyzed archived surgical records of 24 swine experiencing a diffuse closed-head rotational acceleration mTBI against 12 sham animals. Kinematics were contrasted against acute recovery outcomes, specifically apnea, extubation time, standing time, and recovery duration. Compared to controls, animals with mTBI were far more likely to have apnea (p<0.001) along with shorter time to extubation (p=0.023), and longer time from extubation to recovery (p=0.006). Using regression analyses with variable selection, we generated simplified linear models relating kinematics to apnea (R2=0.27), standing time (R2=0.39) and recovery duration (R2=0.42). Neuropathology was correlated with multiple kinematics, with maximum acceleration exhibiting the strongest correlation (R2=0.66). Together, these data suggest the interplay between multiple injury kinematics, including minimum velocity and middle to minimum acceleration time, best explain acute recovery parameters and neuropathology after mTBI in swine. Future experiments that independently manipulate individual kinematics could be instrumental in developing translational diagnostics for clinical mTBI.


title: Arboreal aperitifs: Space use and activity of a major songbird nest predator in a tropical Thailand forest

abstract: Predator-prey interactions are fundamental drivers of population dynamics, yet rarely are both predator and prey species simultaneously studied. Despite being significant, widespread avian nest predators, research on the ecology of Southeast Asian snakes in relation to birds remains scarce. The green cat snake (Boiga cyanea) is a primary nest predator, responsible for {approx}24% of forest songbird depredation in Northeast Thailand. We explored both diurnal and nocturnal movements of 14 (5 male, 9 female) adult B. cyanea with radio-telemetry for an average of 68 {+/-} 16 days per individual, between 21 October 2017 and 8 June 2019 in the dry evergreen forest of the Sakaerat Biosphere Reserve (SBR). We quantified area of space use (ha) and activity through motion variance (m2) during the study period using dynamic Brownian bridge movement models, and linked our findings to a simultaneously-run avian nest monitoring study, initiated in 2013 within the same forest fragment. On average, movements, space use and activity differed between males and females, and between the avian nesting and non-nesting seasons. Males moved 51.37 m/day farther than females. They used areas 15.09 ha larger than females, and their activity was 3.91 m2 higher than that of females. In general, individuals moved 50.30 m/day farther during the nesting season than the non-nesting season. The snakes used areas 9.84 ha larger during the nesting season than the non-nesting season, and their activity during the nesting season was 3.24 m2 higher than that during the non-nesting season. All individuals were exclusively nocturnal, moving throughout the night, and often descending from higher diurnal refugia (>2 m) to forage closer to the ground after sunset. Boiga cyanea activity followed a similar trend to that of the recorded nest depredations at SBR. Our study links snake activity to nest depredations in SBR. Our openly-available data may yield further insight when combined with other major avian nest predator species like the congeneric invasive brown tree snake (Boiga irregularis) on the island of Guam.

category: ecology


title: Lack of parent-of-origin effects in Nasonia jewel wasp: a replication and extension study

abstract: In diploid cells, the paternal and maternal alleles are, on average, equally expressed. There are exceptions from this: a small number of genes express the maternal or paternal allele copy exclusively. This phenomenon, known as genomic imprinting, is common among eutherian mammals and some plant species; however, genomic imprinting in species with haplodiploid sex determination is not well characterized. Previous work reported no parent-of-origin effects in the hybrids of closely related haplodiploid Nasonia vitripennis and Nasonia giraulti jewel wasps, suggesting a lack of epigenetic reprogramming during embryogenesis in these species. Here, we replicate the gene expression dataset and observations using different individuals and sequencing technology, as well as reproduce these findings using the previously published RNA sequence data following our data analysis strategy. The major difference from the previous dataset is that they used an introgression strain as one of the parents and we found several loci that resisted introgression in that strain. Our results from both datasets demonstrate a species-of-origin effect, rather than a parent-of-origin effect. We present a reproducible workflow that others may use for replicating the results. Overall, we reproduced the original report of no parent-of-origin effects in the haplodiploid Nasonia using the original data with our new processing and analysis pipeline and replicated these results with our newly generated data.

category: genomics


title: KCTD19 associates with ZFP541 and HDAC1 and is required for meiotic exit in male mice

abstract:
Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. Immunoprecipitation-mass spectrometry identified zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1) as binding partners of KCTD19, indicating that KCTD19 is involved in chromatin modification. Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 were essential for meiotic exit in male mice.

Author summaryMeiosis is a fundamental process that consisting of one round of genomic DNA replication and two rounds of chromosome segregation producing four haploid cells. To properly distribute their genetic material, cells need to undergo complex chromosome events such as a physical linkage of homologous chromosomes (termed synapsis) and meiotic recombination. The molecules involved in these events have not been fully characterized yet, especially in mammals. Using a CRISPR/Cas9-screening system, we identified the potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis in male mice. Further, we identified zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1) as binding partners of KCTD19. By observing meiosis of Zfp541 knockout germ cells, we found that Zfp541 was also essential for meiotic completion. These results show that the KCTD19/ZFP541 complex plays a critical role and is indispensable for male meiosis and fertility.

category: developmental biology


title: Dynamics of primary productivity in relation to submerged vegetation of a shallow, eutrophic lagoon: a field and mesocosm study

abstract: Aquatic ecosystems nowadays are under constant pressure, either from recent or historical events. In most systems with increased nutrient supply, submerged macrophytes got replaced by another stable state, dominated by phytoplankton as main primary producer. Yet, reducing the nutrient supply did not yield the aimed goal of restored habitats for submerged macrophytes in systems worldwide. The question arises, why submerged macrophytes do not re-colonize, and if they are actually competitive. Therefore, primary production assays were conducted in ex-situ bentho-pelagic mesocosms and compared to the actual ecosystem, a turbid brackish lagoon of the southern Baltic Sea. Mesocosm were either manipulated to be colonized by macrophytes, or stayed phytoplankton dominated. Oxygen evolution was monitored over a period of five months in 5 min (mesocosms) to 10 min (ecosystem) intervals. Surface and depth-integrated production was calculated to analyse seasonal and areal resolved production patterns. It was found that macrophyte mesocosms were more stable, when considering only surface O2 production. However, calculating depth-integrated production resulted in net-heterotrophy in both shallow mesocosms approaches and the actual ecosystem. This heterotrophy is likely mediated by sediment respiration and POC accumulation in mesocosms, and a low share of productive to respiring water column in the actual ecosystem. Therefore, it seems unlikely that macrophytes will re-settle, as constant net-heterotrophy may allow for high-nutrient turnover at sediment-water interfaces and within the water column, favouring phytoplankton. Changes within the ecosystem cannot be expected without further restoration measures within and in the systems proximity.


title: Life histories as mosaics: plastic and genetic components differ among traits that underpin life-history strategies

abstract:
Life-history variation reflects phenotypic variation across suites of traits. Differences among life-history strategies result from genetic differentiation, phenotypic plasticity, and genotype-by-environment interactions. If the relative strength and direction of these components differed among traits underlying a strategy, life histories might not evolve as a cohesive unit.

We tested this hypothesis on the high- and low-predation ecotypes of Trinidadian guppies, defined by distinct life-history strategies. Using common garden experiments, we assessed how strongly 36 traits were determined by ancestral habitat (i.e., ecotype) or food availability, a key environmental difference between ecotypes. Our dataset was large (1178 individuals) and included six putatively independent origins of the derived ecotype.

Traits could be confidently assigned to four groups, defined by highly significant effects of only food (13 traits), only habitat (6), both (6), or neither (11), revealing substantial variation among traits in levels of genetic and environmental control. Ecotype-food (i.e., genotype-by-environment) interactions were negligible. The directions of plastic and genetic effects were usually aligned.

This suggests that life histories are mosaics with unequal rates of phenotypic and evolutionary change. Broadly speaking of "life-history evolution" masks a complex interplay of genes and environment on the multiple traits that underpin life-history strategies.

category: evolutionary biology


title: Promiscuous specialists: Host specificity patterns among generalist louse flies

abstract: Ectoparasites such as louse flies (Diptera: Hippoboscidae) have tendency for host specialization, which is driven by adaptation to host biology as well as competition avoidance between parasites of the same host. However, some louse fly species, especially in genera attacking birds, show wide range of suitable hosts. In the presented study, we have surveyed the current status of bird attacking louse flies in Finland to provide comprehensive host association data to analyse the ecological requirements of the generalist species. A thorough sampling of 9342 birds, representing 134 species, recovered 576 specimens of louse flies, belonging to six species: Crataerina hirundinis, C. pallida, Ornithomya avicularia, O. chloropus, O. fringillina and Ornithophila metallica. Despite some overlapping hosts, the three Ornithomya species showed a notable pattern in their host preference, which was influenced not only by the host size but also by the habitat and host breeding strategy. We also provide DNA barcodes for most Finnish species of Hippoboscidae, which can be used as a resource for species identification as well as metabarcoding studies in the future.

category: zoology


title: Neural markers for Musical Creativity in Jazz Improvisation and Classical Interpretation

abstract:
Two main types of musical creativity in the western canon are improvisation and interpretation. With improvisation, the fundamental structure of the melody, chords, rhythm and tempo of a piece can be modified, while with interpretation, the focus is on the emotional dynamics. Here we characterise electrical brain activity from professional jazz and classical pianists, whilst they were engaged in these different creative tasks with musical excerpts from both genres. Multivariate EEG was recorded during two phases of each task, mental planning and actual performance. Subsequently neuronal activity patterns were source localised with standardised low resolution electromagnetic tomography (sLORETA). For each musical performance, we obtained both subjective (self-rated) and objective (blind, expert-rated) measures of musical creativity. Across both tasks and genre backgrounds, within the first and middle 4 second segments of the performance phase, for musical performances that were judged highly creative objectively by external expert music assessors, we observed an increased activation in the anterior cingulate and medial prefrontal cortex (Brodmann area, BA32), suggesting a maintenance of executive control, and integrating motoric and emotional communication during creativity. Across genre backgrounds, within the performance phase for the interpretation task compared to the improvisation task, there was an increased activity in the insula (BA 13), suggesting a convergent creative task from the linked goal-orientated conscious error-monitoring and audio-visual integration functions. Genre profession also gave rise to differences across phases; jazz pianists presented a decreased parietal (BA7) activity during improvisation tasks suggesting a role for defocussed attention and for classical pianists, both tasks were associated with occipitotemporal (BA 37) activity which is involved in semantic/ metaphorical processing suggesting a close adherence to the visual score. These 3 areas relate the cognitive demands of the creative musical task to the demands of the corresponding genre of music.

HighlightsO_LIEEG activity associated to musical creativity types: Improvisation and Interpretation
C_LIO_LIIncreased activity in Insula (BA 13) for Interpretation suggest convergent creativity
C_LIO_LIDecreased Precuneus (BA7) activity for Improvisation suggest defocussed attention
C_LIO_LIIncreased activity in medial prefrontal cortex (BA32) in highly creative performance
C_LI


title: Dopamine modulates visual threat processing in the superior colliculus via D2 receptors

abstract:
Dopamine (DA) system is intriguing in the aspect that distinct, typically opposing physiological functions are mediated by D1 dopamine receptors (Drd1) and D2 dopamine receptors (Drd2). Both Drd1+ and Drd2+ neurons were identified in superior colliculus (SC), a visuomotor integration center known for its role in defensive behaviors to visual threats. We hypothesized that Drd1+ and Drd2+ neurons in the SC may play a role in promoting instinctive defensive responses.

Optogenetic activation of Drd2+ neurons, but not Drd1+ neurons, in the SC triggered strong defensive behaviors. Chemogenetic inhibition of SC Drd2+ neurons decreased looming-induced defensive behavior, suggesting involvement of SC Drd2+ neurons in defensive responses. To further confirm this functional role of Drd2 receptors, pretreatment with the Drd2+ agonist quinpirole in the SC impaired looming-evoked defensive responses, suggesting an essential role of Drd2 receptors in the regulation of innate defensive behavior. Inputs and outputs of SC Drd2+ neurons were investigated using viral tracing: SC Drd2+ neurons mainly receive moderate inputs from the Locus Coeruleus (LC), whilst we did not find any incoming projections from other dopaminergic structures. Our results suggest a sophisticated regulatory role of DA and its receptor system in innate defensive behavior.


title: Computational anatomy and geometric shape analysis enables analysis of complex craniofacial phenotypes in zebrafish mutants

abstract:
Due to the complexity of fish skulls, previous attempts to classify craniofacial phenotypes have relied on qualitative features or 2D landmarks. In this work we aim to identify and quantify differences in 3D craniofacial phenotypes in adult zebrafish mutants. We first estimate a synthetic  normative zebrafish template using microCT scans from a sample pool of wildtype animals using the Advanced Normalization Tools (ANTs). We apply a computational anatomy (CA) approach to quantify the phenotype of zebrafish with disruptions in bmp1a, a gene implicated in later skeletal development and whose human ortholog when disrupted is associated with Osteogenesis Imperfecta. Compared to controls, the bmp1a fish have larger otoliths and exhibit shape differences concentrated around the operculum, anterior frontal, and posterior parietal bones. Moreover, bmp1a fish differ in the degree of asymmetry. Our CA approach offers a potential pipeline for high throughput screening of complex fish craniofacial phenotypes, especially those of zebrafish which are an important model system for testing genome to phenome relationships in the study of development, evolution, and human diseases.

Summary statementA computational anatomy approach offers a potential pipeline for high throughput screening of complex zebrafish craniofacial phenotypes, an important model system for the study of development, evolution, and human diseases.


title: The Evolution of Chunks in Sequence Learning

abstract: Chunking mechanisms are central to several cognitive processes and notably to the acquisition of visuo-motor sequences. Individuals segment sequences into chunks of items to perform visuo-motor tasks more fluidly, rapidly, and accurately. However, the exact dynamics of chunking processes in the case of extended practice remain unclear. Using an operant conditioning device, eighteen Guinea baboons (Papio papio) produced a fixed sequence of nine movements during 1,000 trials by pointing to a moving target on a touch screen. Response times analyses revealed a specific chunking pattern of the sequence for each baboon. More importantly, we found that these patterns evolved during the course of the experiment, with chunks becoming progressively fewer and longer. We identified two chunk reorganization mechanisms: the recombination of preexisting chunks and the concatenation of two distinct chunks into a single one. These results provide new evidence on chunking mechanisms in sequence learning and challenge current models of associative and statistical learning.

category: animal behavior and cognition


title: StrainFLAIR: Strain-level profiling of metagenomic samples using variation graphs

abstract:
Current studies are shifting from the use of single linear references to representation of multiple genomes organised in pangenome graphs or variation graphs. Meanwhile, in metagenomic samples, resolving strain-level abundances is a major step in microbiome studies, as associations between strain variants and phenotype are of great interest for diagnostic and therapeutic purposes.

We developed StrainFLAIR with the aim of showing the feasibility of using variation graphs for indexing highly similar genomic sequences up to the strain level, and for characterizing a set of unknown sequenced genomes by querying this graph.

On simulated data composed of mixtures of strains from the same bacterial species Escherichia coli, results show that StrainFLAIR was able to distinguish and estimate the abundances of close strains, as well as to highlight the presence of a new strain close to a referenced one and to estimate its abundance. On a real dataset composed of a mix of several bacterial species and several strains for the same species, results show that in a more complex configuration StrainFLAIR correctly estimates the abundance of each strain. Hence, results demonstrated how graph representation of multiple close genomes can be used as a reference to characterize a sample at the strain level.

Availabilityhttp://github.com/kevsilva/StrainFLAIR

category: bioinformatics


title: Benchmarking Association Analyses of Continuous Exposures with RNA-seq in Observational Studies

abstract: Large datasets of hundreds to thousands of individuals measuring RNA-seq in observational studies are becoming available. Many popular software packages for analysis of RNA-seq data were constructed to study differences in expression signatures in an experimental design with well-defined conditions (exposures). In contrast, observational studies may have varying levels of confounding of the transcript-exposure associations; further, exposure measures may vary from discrete (exposed, yes/no) to continuous (levels of exposure), with non-normal distributions of exposure. We compare popular software for gene expression - DESeq2, edgeR, and limma - as well as linear regression-based analyses for studying the association of continuous exposures with RNA-seq. We developed a computation pipeline that includes transformation, filtering, and generation of empirical null distribution of association p-values, and we apply the pipeline to compute empirical p-values with multiple testing correction. We employ a resampling approach that allows for assessment of false positive detection across methods, power comparison, and the computation of quantile empirical p-values. The results suggest that linear regression methods are substantially faster with better control of false detections than other methods, even with the resampling method to compute empirical p-values. We provide the proposed pipeline with fast algorithms in R.


title: Interplay between brassinosteroids and TORC signaling in Arabidopsis revealed by integrated multi-dimensional analysis

abstract: Brassinosteroids (BR) and Target of Rapamycin Complex (TORC) are two major processes coordinating plant growth and stress responses. BRs function through a signaling pathway to extensively regulate gene expression and TORC is known to regulate translation and autophagy. Recent studies revealed that these two pathways crosstalk, but a system-wide view of their interplay is still missing. Thus, we performed transcriptome, proteome, and phosphoproteome profiling of Arabidopsis mutants with altered levels of either BIN2 or RAPTOR1B, two key players in BR and TORC signaling, respectively. We found that perturbation of BIN2 or RAPTOR1B levels affects a common set of gene-products involved in growth and stress responses. Additionally, we performed Multiplexed Assay for Kinase Specificity (MAKS), which provided a system-wide view of direct BIN2 substrates. Furthermore, phosphoproteomic data was used to reconstruct a kinase-signaling network and to identify novel proteins dependent on BR and/or TORC signaling pathways. Loss of function mutants of many of these proteins led to an altered BR response and/or modulated autophagy activity. Altogether, these results provide genome-wide evidence for crosstalk between BR and TORC signaling and established a kinase signaling network that defines the molecular mechanisms of BR and TORC interactions in the regulation of plant growth/stress balance.

category: plant biology


title: Single-cell Visualization and Quantification of Trace Metals in Chlamydomonas Lysosome-Related Organelles

abstract:
The acidocalcisome is an acidic organelle in the cytosol of eukaryotes, defined by its low pH and high calcium and polyphosphate content. It is visualized as an electron-dense object by transmission electron microscopy (TEM) or described with mass-spectrometry (MS)-based imaging techniques or multimodal X-ray fluorescence microscopy (XFM) based on its unique elemental composition. Compared to MS-based imaging techniques, XFM offers the advantage of absolute quantification of trace metal content, since sectioning of the cell is not required and metabolic states can be preserved rapidly by either vitrification or chemical fixation. We employed XFM in Chlamydomonas reinhardtii, to determine single-cell and organelle trace metal quotas within algal cells in situations of trace metal over-accumulation (Fe, Cu). We found up to 70% of the cellular Cu and 80% of Fe sequestered in acidocalcisomes in these conditions, and identified two distinct populations of acidocalcisomes, defined by their unique trace elemental makeup. We utilized the vtc1 mutant, defective in polyphosphate synthesis and failing to accumulate Ca to show that Fe sequestration is not dependent on either. Finally, quantitation of the Fe and Cu contents of individual cells and compartments via XFM, over a range of cellular metal quotas created by nutritional and genetic perturbations, indicated excellent correlation with bulk data from corresponding cell cultures, establishing a framework to distinguish the nutritional status of single cells.

Significance statementTransition metals are of crucial importance for primary productivity; their scarcity limits crop yield in agriculture and carbon sequestration at global scale. Copper (Cu), iron (Fe) and manganese (Mn) are among the most important trace elements that enable the redox chemistry in oxygenic photosynthesis. The single-celled, eukaryotic green alga Chlamydomonas reinhardtii is a choice experimental system for studying trace metal homeostasis in the context of phototrophy, offering all the advantages of a classical microbial system with a well-characterized photosystem and trace metal metabolism machinery of relevance to plants. This project identifies and differentiates different trace metal storage sites in Chlamydomonas and uncovers the dynamics of trace metal storage and mobilization in situations of fluctuating resources.


title: Fighting over defence chemicals disrupts mating behaviour

abstract: Studies on intraspecific contest behaviour predominantly focus on contests between individuals of the same sex, however contest behaviour is also expected to occur between individuals of the opposite sex including possible mates. Here we investigate potential trade-offs between mating and fighting behaviour in the turnip sawfly (Athalia rosae). Adults of this species collect chemical defence compounds (clerodanoids) directly from plants but also indirectly by nibbling on conspecifics that have already obtained clerodanoids themselves, a highly aggressive behavioural interaction. An A. rosae individual without clerodanoids may therefore be the potential mate or attacker of an individual of the opposite sex that has gained clerodanoids. We paired males and females with or without clerodanoid access and manipulated body mass differences between the sexes via the early life starvation of females. We show that asymmetrical clerodanoid acquisition between male-female pairs causes an increase in agonistic nibbling behaviour, irrespective of sex. Moreover, fighting over clerodanoids disrupted mating behaviour, and the frequency of aggressive nibbling behaviour in these pairs was determined by the comparative body mass of the attacking individual. Our study highlights the vital importance of investigating agonistic intersex interactions not only over mating but also over resources.


title: Sterile Inflammation Alters Neutrophil Kinetics in Mice

abstract: Neutrophils play a crucial role in establishing inflammation in response to an infection or injury, but their production rates, as well as blood and tissue residence times, remain poorly characterized under these conditions. Herein, using a biomaterial implant model to establish inflammation followed by in vivo tracking of newly formed neutrophils, we determine neutrophil kinetics under inflammatory conditions. To obtain quantifiable information from our experimental observations, we develop an ordinary differential equation-based mathematical model to extract kinetic parameters. Our data show that in the presence of inflammation resulting in emergency granulopoiesis-like conditions, neutrophil maturation time in the bone marrow and half-life in the blood reduces by about 40%, compared to non-inflammatory conditions. Additionally, neutrophil residence time at the inflammatory site increases by two-fold. Together, these data improve our understanding of neutrophil kinetics under inflammatory conditions, which could pave the way for therapies that focus on modulating in vivo neutrophil dynamics.

category: immunology


title: Development and validation of a mouse model of contemporary cannabis smoke exposure

abstract:
Cannabis is widely used for both recreational and medicinal purposes. Inhalation of combusted cannabis smoke is the most common mode of drug consumption, exposing the lungs to the pharmacologically active ingredients, including tetrahydrocannabinol (THC) and cannabidiol (CBD). While the relationship between cannabis smoke exposure and compromised respiratory health has yet to be sufficiently defined, previous investigations suggest that cannabis smoke may dysregulate pulmonary immunity. Presently, there exists few pre-clinical animal models that have been extensively validated for contemporary cannabis smoke exposure. To address this need, we developed a mouse model with readouts of total particular matter, serum cannabinoid and carboxyhemoglobin levels, lung cellular responses, and immune mediator production.

Using a commercially available smoke exposure system and a cannabis source material of documented THC/CBD composition, we exposed mice to a total particulate matter of 698.89 (SD = 66.09) {micro}g/L and demonstrate increases in serum cannabinoids and carboxyhemoglobin. We demonstrate that cannabis smoke modulates immune cell populations and mediators in both male and female BALB/c mice. This modulation is highlighted by increases in airway and lung tissue macrophage populations, including tissue-resident alveolar macrophages, monocyte-derived alveolar macrophages, and interstitial macrophage subpopulations. No changes in airway or lung tissue infiltration of neutrophils were observed. Immune mediator analysis indicated significant upregulation of MDC, TARC, and VEGF within the lung tissue of cannabis smoke exposed mice. This accessible and reproducible smoke exposure model provides a foundation to explore the impact of chronic cannabis exposures and/or co-exposures with pathogens of clinical relevance, such as influenza.


title: Elucidating Mechanisms of Antitumor Immunity Mediated by Live Oncolytic Vaccinia and Heat-Inactivated Vaccinia

abstract:
BackgroundViral-based immunotherapy has the potential to overcome resistance to immune checkpoint blockade (ICB) and to fill the unmet needs of many cancer patients. Oncolytic viruses (OVs) are defined as engineered or naturally occurring viruses that selectively replicate in and kill cancer cells. OVs also induce antitumor immunity. The purpose of this study is to compare the antitumor effects of live OV-GM expressing murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) versus inactivated OV-GM and elucidate the underlying immunological mechanisms.

MethodsIn this study, we engineered a replication-competent, oncolytic vaccinia virus (OV-GM) by inserting a murine GM-CSF gene into the thymidine kinase (TK) locus of a mutant vaccinia E3L{Delta}83N, which lacks the Z-DNA-binding domain of vaccinia virulence factor E3. We compared the antitumor effects of intratumoral (IT) delivery of live OV-GM vs. heat-inactivated OV-GM (heat-iOV-GM) in a murine B16-F10 melanoma bilateral implantation model.

ResultsHeat-iOV-GM infection of dendritic cells (DCs) and tumor cells in vitro induces type I IFN and pro inflammatory cytokines and chemokines, whereas live OV-GM does not. IT live OV-GM is less effective in generating systemic antitumor immunity compared with heat-iOV-GM. Similar to heat-iOV-GM, the antitumor effects of live OV-GM also require Batf3-dependent CD103+ dendritic cells. IT heat-iOV-GM induces higher numbers of infiltrating activated CD8+ and CD4+ T cells as well as higher levels of type I IFN, proinflammatory cytokines, and chemokines in the distant non-injected tumors, which is dependent on CD8+ T cells. When combined with systemic delivery of ICB, IT heat-iOV-GM is more effective in eradicating tumors compared with live OV-GM.

ConclusionsTumor lysis induced by the replication of oncolytic DNA viruses has a limited effect on the generation of systemic antitumor immunity. The activation of Batf3-dependent CD103+ DCs is critical for antitumor effects induced by both live OV-GM and heat-iOV-GM. Heat-iOV-GM is more potent than live OV-GM in the induction of innate and adaptive immunity in both the injected and distant non-injected tumors. We propose that evaluations of both innate and adaptive immunity induced by IT oncolytic viral immunotherapy at injected and non-injected tumors should be included as potential biomarkers.


title: Sensitivity of infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants to neutralizing antibodies

abstract: SARS-CoV-2 B.1.1.7 and B.1.351 variants emerged respectively in United Kingdom and South Africa and spread in many countries. Here, we isolated infectious B.1.1.7 and B.1.351 strains and examined their sensitivity to anti-SARS-CoV-2 antibodies present in sera and nasal swabs, in comparison with a D614G reference virus. We established a novel rapid neutralization assay, based on reporter cells that become GFP+ after overnight infection. B.1.1.7 was neutralized by 79/83 sera from convalescent patients collected up to 9 months post symptoms, almost similar to D614G. There was a mean 6-fold reduction in titers and even loss of activity against B.1.351 in 40% of convalescent sera after 9 months. Early sera from 19 vaccinated individuals were almost as potent against B.1.1.7 but less efficacious against B.1.351, when compared to D614G. Nasal swabs from vaccine recipients were not neutralizing, except in individuals who were diagnosed COVID-19+ before vaccination. Thus, faster-spreading variants acquired a partial resistance to humoral immunity generated by natural infection or vaccination, mostly visible in individuals with low antibody levels.


title: Single-cell sequencing of plasma cells from COVID-19 patients reveals highly expanded clonal lineages produce specific and neutralizing antibodies to SARS-CoV-2

abstract:
Isolation and characterization of antibodies in COVID-19 patients has largely focused on memory B cells, however it is the antibody-secreting plasma cells that are directly responsible for the production of serum antibodies, which play a critical role in controlling and resolving SARS-CoV-2 infection. To date there is little known about the specificity of plasma cells in COVID-19 patients. This is largely because plasma cells lack surface antibody expression, which complicates their screening. Here, we describe a technology pipeline that integrates single-cell antibody repertoire sequencing and high-throughput mammalian display screening to interrogate the specificity of plasma cells from 16 convalescent COVID-19 patients. Single-cell sequencing allows us to profile antibody repertoire features in these patients and identify highly expanded clonal lineages. Mammalian display screening is employed to reveal that 37 antibodies (out of 132 candidates) derived from expanded plasma cell clonal lineages are specific for SARS-CoV-2 antigens, including antibodies that target the receptor binding domain (RBD) with high affinity and exhibit potent neutralization of SARS-CoV-2.

One Sentence SummarySingle-cell antibody repertoire sequencing and high-throughput screening identifies highly expanded plasma cells from convalescent COVID-19 patients that produce SARS-CoV-2-specific antibodies capable of potent neutralization.


title: TPR1, a novel rifampin derivative demonstrates efficacy alone and in combination with doxycycline against the NIAID Category A Priority Pathogen F. tularensis

abstract:
ObjectivesFrancisella tularensis is a highly virulent and contagious gram-negative intracellular bacterium that causes the disease tularemia in mammals and is classified as a Category A priority pathogen. The high infectivity, difficulty of obtaining a durable cure of disseminated disease and the emergence of drug resistance warrants investigation of new drugs that can be used alone and in combination with existing standard of care clinical drugs.

MethodsWe utilized a systematic analysis of antibacterial potency, extent of dissemination by analysis of bacterial burden in a secondary vital organ, and survival rates to assess the efficacy of a novel rifampicin derivative, TPR1. The efficacy of TPR1 was evaluated alone and in combination with the standard of care drug, doxycycline, against Type A F. tularensis Schu S4 using a lethal pulmonary model of infection in mice.

ResultsTPR1 has an MIC value range of 0.125-4 mg/L against reference laboratory strain SchuS4 and a panel of clinical strains. TPR1 alone reduced the bacterial burden in the lungs and spleen at 40 mg/kg and 80 mg/kg, and no antagonism was observed when co-administered with doxycycline. Dosing at 40 mg/kg doxycycline reduced the bacterial burden by 1 Log10 CFU in the lungs and 4 Log10 CFU spleen in comparison to untreated controls. Co-administration of TPR1 and doxycycline demonstrated efficacy upon treatment withdrawal after 4 days of treatment, and 100% survival.

ConclusionsSignificantly, TPR1 demonstrated efficacy when delivered alone and in combination with doxycycline, which provides compelling evidence of a superior treatment strategy that would normally rely on a single chemotherapeutic for efficacy. In addition, this work substantiates the use of rifampicin derivatives as a platform for the development of novel treatments to other bacterial agents in addition to tularemia.

category: microbiology


title: Direct-from-specimen microbial growth inhibition spectrums under antibiotic exposure and comparison to conventional antimicrobial susceptibility testing

abstract: Increasing global travel and changes in the environment may increase the frequency of contact with a natural host carrying an infection, and therefore increase our chances of encountering microorganisms previously unknown to humans. During an emergency (man-made, natural disaster, or pandemic), the etiology of infection might be unknown at the time of patient treatment. The existing local or global Antimicrobial Stewardship Programs might not be fully prepared for emerging/re-emerging infectious disease outbreaks, especially if they are caused by an unknown organism, engineered bioterrorist attack, or rapidly evolving superbug. We demonstrate an antimicrobial efficacy profiling method that can be performed in hours directly from clinical urine specimens. The antimicrobial potency is determined by the microbial growth inhibition and compared to conventional antimicrobial susceptibility testing (AST) results. The oligonucleotide probe pairs on the sensor were designed to target gram-negative bacteria, specifically Enterobacterales and Pseudomonas aeruginosa. A total of 10 remnant clinical specimens from the CLIA labs of New York-Presbyterian Queens were tested, resulting in 100% categorical agreement with reference AST methods (Vitek and broth microdilution method). The combined categorical susceptibility reporting of 12 contrived urine specimens was 100% for ciprofloxacin, gentamicin, and meropenem over a range of microbial loads from 105 to 108 CFU/mL.


title: Chlorine inactivation of Escherichia coli O157:H7 in fresh produce wash process: effectiveness and modeling

abstract: This study presents a modified disinfection kinetics model to evaluate the potential effect of organic content on the chlorine inactivation coefficient of Escherichia coli O157:H7 in fresh produce wash processes. Results show a significant decrease in the bactericidal efficacy of free chlorine (FC) in the presence of organic load compared to its absence. While the chlorine inactivation coefficient of Escherichia coli O157:H7 is 70.39 {+/-} 3.19 L.mg-1.min-1 in the absence of organic content, it drops by 73% in chemical oxygen demand (COD) level of 600 - 800 mg.L-1. Results also indicate that the initial chlorine concentration and bacterial load have no effect on the chlorine inactivation coefficient. A second-order chemical reaction model for FC decay, which utilizes a percentage of COD as an indicator of organic content in fresh produce wash was employed, yielding an apparent reaction rate of (9.45 {+/-} 0.22) x 10-4 M-1.min-1. This model was validated by predicting FC concentration (R2 = 0.96) in multi-run continuous wash cycles with periodic replenishment of chlorine.


title: Mapping global shifts in Saccharomyces cerevisiae gene expression across asynchronous time trajectories with diffusion maps

abstract:
Grapes grown in a particular geographic region often produce wines with consistent characteristics, suggesting there are site-specific factors driving recurrent fermentation outcomes. However, our understanding of the relationship between site-specific factors, microbial metabolism, and wine fermentation outcomes are not well understood. Here, we used differences in Saccharomyces cerevisiae gene expression as a biosensor for differences among Pinot noir fermentations from 15 vineyard sites. We profiled time series gene expression patterns of primary fermentations, but fermentations proceeded at different rates, making analyzes of these data with conventional differential expression tools difficult. This led us to develop a novel approach that combines diffusion mapping with continuous differential expression analysis. Using this method, we identified vineyard specific deviations in gene expression, including changes in gene expression correlated with the activity of the non-Saccharomyces yeast Hanseniaspora uvarum, as well as with initial nitrogen concentrations in grape musts. These results highlight novel relationships between site-specific variables and Saccharomyces cerevisiae gene expression that are linked to repeated wine fermentation outcomes. In addition, we demonstrate that our analysis approach can extract biologically relevant gene expression patterns in other contexts (e.g., hypoxic response of Saccharomyces cerevisiae), indicating that this approach offers a general method for investigating asynchronous time series gene expression data.

ImportanceWhile it is generally accepted that foods, in particular wine, possess sensory characteristics associated with or derived from their place of origin, we lack knowledge of the biotic and abiotic factors central to this phenomenon. We have used Saccharomyces cerevisiae gene expression as a biosensor to capture differences in fermentations of Pinot noir grapes from 15 vineyards across two vintages. We find that gene expression by non-Saccharomyces yeasts and initial nitrogen content in the grape must correlates with differences in gene expression among fermentations from these vintages. These findings highlight important relationships between site-specific variables and gene expression that can be used to understand, or possibly modify, wine fermentation outcomes. Our work also provides a novel analysis method for investigating asynchronous gene expression data sets that is able to reveal both global shifts and subtle differences in gene expression due to varied cell - environment interactions.


title: Recurrent Vulvovaginal Candidiasis; a dynamic interkingdom biofilm disease of Candida and Lactobacillus

abstract: Vulvovaginal Candidiasis (VVC) is the most prevalent Candida infection in humans affecting 75% of women at least once throughout their lifetime. In its debilitating recurrent form, RVVC is estimated to affect 140 million women annually. Despite this strikingly high prevalence, treatment options for RVVC remain limited with many women experiencing failed clinical treatment with frontline azoles. Further, the cause of onset and recurrence of disease is largely unknown with few studies identifying potential mechanisms of failed treatment. This study aimed to assess a panel of clinical samples from healthy women and those with RVVC to investigate the influence of Candida, vaginal microbiome and antagonism between Candida and Lactobacillus on disease pathology. 16S rRNA sequencing characterised disease by a reduction in specific health-associated Lactobacillus such as L. crispatus, coupled with an increase in L. iners. In vitro analysis showed Candida albicans clinical isolates are capable of heterogeneous biofilm formation and show the presence of hyphae and C. albicans aggregates in vaginal lavage. Additionally, the ability of Lactobacillus to inhibit C. albicans biofilm formation and biofilm-related gene expression was demonstrated. Using RNA sequencing technology, we were able to exploit a possible mechanism by which L. crispatus may aim to re-establish a healthy vaginal environment through amino-acid acquisition from C. albicans. This study suggests RVVC is not entirely due to an arbitrary switch in C. albicans from commensal to pathogen and understanding interactions between the yeast and vaginal Lactobacillus species may be more crucial to elucidating the cause of RVVC and developing appropriate therapies.


title: Genome sequencing of the bacteriophage CL31 and interaction with the host strain 	Corynebacterium glutamicum ATCC 13032

abstract: In this study, we provide a comprehensive analysis of the genomic features of the phage CL31 and the infection dynamics with the biotechnologically relevant host strain Corynebacterium glutamicum ATCC 13032. Genome sequencing and annotation of CL31 revealed a 45-kbp genome composed of 72 open reading frames, mimicking the GC content of its host strain (54.4 %). An ANI-based distance matrix showed the highest similarity of CL31 to the temperate corynephage {Phi}16. While the C. glutamicum ATCC 13032 wild type strain showed only mild propagation of CL31, a strain lacking the cglIR-cglIIR-cglIM restriction-modification system was efficiently infected by this phage. Interestingly, the prophage-free strain C. glutamicum MB001 featured an even accelerated amplification of CL31 compared to the {Delta}resmod strain suggesting a role of cryptic prophage elements in phage defense. Proteome analysis of purified phage particles and transcriptome analysis provide important insights into structural components of the phage and the response of C. glutamicum to CL31 infection. Isolation and sequencing of CL31-resistant strains revealed SNPs in genes involved in mycolic acid biosynthesis suggesting a role of this cell envelope component in phage adsorption. Altogether, these results provide an important basis for further investigation of phage-host interactions in this important biotechnological model organism.


title: Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli

abstract: Bacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of {beta}-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3[->]3 cross-links compared to 4[->]3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify amongst eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4[->]3 (MepM) and 3[->]3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4[->]3 and 3[->]3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4[->]3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of {beta}-lactams.


title: Do size and shape matter? Exploring the interactions and the metabolome of the soil isolate Hylemonella gracilis

abstract:
Microbial community analysis of aquatic environments showed that an important component of microbial diversity consists of bacteria with cell sizes smaller than ~0.1 m. However, so far no study investigated if such bacteria with small cell sizes exist in terrestrial environments as well.

Here, we isolated soil bacteria that passed through a 0.1 m filter, by applying a novel isolation and culturing approach. The complete genome of one of the isolates was sequenced and the bacterium was identified as Hylemonella gracilis. We performed a set of interaction assays with phylogenetically distant soil bacteria with larger cell and genome size. The interaction assays revealed that H. gracilis grows better when interacting with other soil bacteria like Paenibacillus sp. AD87 and Serratia plymuthica. Furthermore, we observed that H. gracilis is able to change the behavior of interacting bacteria without direct cell-cell contact. Transcriptomics and metabolomics analysis was performed with the aim to explain the mechanisms of these interactions.

Our study indicates that soil bacteria that can pass through a 0.1 M filter may have been overlooked in soil microbial communities and that such bacteria are able to induce transcriptional and metabolomics responses in other soil-bacteria. Furthermore, we revealed that the interaction allowed utilization of substrates that are not utilized by monocultures.


title: Salmonella Typhimurium outer membrane protein A (OmpA) renders protection against nitrosative stress by promoting SCV stability in murine macrophage

abstract: Porins are highly conserved bacterial outer membrane proteins involved in the selective transport of charged molecules across the membrane. Despite their significant contributions to the pathogenesis of Gram-negative bacteria, their precise role in salmonellosis remains elusive. In this study, we investigated the role of porins (OmpA, OmpC, OmpD, and OmpF) in Salmonella Typhimurium (STM) pathogenesis. OmpA played a multifaceted role in STM pathogenesis, and a strain deleted for ompA (STM{Delta} ompA) showed enhanced proneness to phagocytosis and compromised proliferation in macrophages. However, in the epithelial cells, despite being invasion deficient, it was hyper-proliferative. The poor colocalization of STM{Delta} ompA with LAMP-1 confirmed impaired stability of SCV membrane around the intracellular bacteria, resulting in its (STM{Delta} ompA) release into the cytosol of macrophages where it is assaulted with reactive nitrogen intermediates (RNI). The cytosolic localization of STM{Delta} ompA was responsible for the downregulation of SPI-2 encoded virulence factor SpiC, which is required to suppress the activity of iNOS. The reduced recruitment of nitrotyrosine on STM in the macrophage cytosol upon ectopically expressing Listeriolysin O (LLO) explicitly supported the pro-bacterial role of OmpA against the host nitrosative stress. Further, we show that the generation of time-dependent redox burst could be responsible for the enhanced sensitivity of STM{Delta} ompA towards nitrosative stress. The absence of OmpA in STM{Delta} ompA resulted in the loss of integrity and enhanced porosity of the bacterial outer membrane, which was attributed to the upregulated expression of ompC, ompD, and ompF. We showed the involvement of OmpF in the entry of excess nitrite in STM{Delta} ompA, thus increasing the susceptibility of the bacteria towards in vitro and in vivo nitrosative stress. In conclusion, we illustrated a mechanism of strategic utilization of OmpA compared to other porins by wildtype Salmonella for combating the nitrosative stress in macrophages.


title: Salmonella Typhimurium outer membrane protein A (OmpA) renders protection against nitrosative stress by promoting SCV stability in murine macrophages


title: Virus transmission by ultrasonic scaler and its prevention by antiviral agent

abstract: During an ultrasonic scaler (USS) operation, droplets and aerosol are generated that may contribute to the transmission of viruses contained in saliva and gingival crevicular fluid. The purpose of this research was to develop an experimental model for testing the spread of viruses during USS instrumentation and to examining the prevention of spreading by replacing the coolant with an antiviral agent. In a virus transmission tunnel, USS operation with saline coolant and delivery of a viral suspension to the vicinity of USS tip generated droplets and aerosol containing Equine Arteritis Virus (EAV). Evaluation of droplet transmission was evaluated with adherent 48h cell culture monolayer RK13 cell lines in standard 48-well-plates positioned at a distance from 30 to 55 cm. The aerosol was collected by a cyclone aero-sampler flow of 100l/min. Antiviral activity of 0.25% sodium hypochlorite or electrolyzed water (EOW) was tested by suspension test. The two tested antiviral agents transmission prevention ability was evaluated by repeating the same experiment as with saline coolant. All experiments were repeated twice. With saline coolant, the cytopathic effect on cells was found in cells up to the distance of 45 cm, with the number of infected wells decreasing with distance. Viral particles were detected in only one AS in a very low concentration ([≤]4.2 TCID50/ml). In suspension test of 0.25% NaOCl and EOW, the TCID50/ml was below detection limit after 5s. With both antiviral agents, no cytopathic effect was found. However, the cytotoxic effect of 0.25% NaOCl was evident up to the distance of 35 cm. By USS activity, EAV could be transmitted by droplets up to a distance of 45 cm. Both antiviral agents could prevent virus droplet transmission. The transmission of EAV by aerosol yielded inconclusive results.


title: Kinetics and topology of DNA associated with circulating extracellular vesicles released during exercise

abstract: Although it is widely accepted that cancer derived extracellular vesicles (EVs) carry DNA cargo, the association of cell-free circulating DNA (cfDNA) and EVs in plasma of healthy humans remains elusive. Using a physiological exercise model, where EVs and cfDNA are synchronously released, we aimed to characterize the kinetics and localization of DNA associated with EVs. EVs were separated from human plasma using size exclusion chromatography or immuno-affinity capture for CD9+, CD63+, and CD81+ EVs. DNA was quantified with an ultra-sensitive qPCR assay targeting repetitive LINE elements, with or without DNase digestion. This model shows that a minute part of circulating cell-free DNA is associated with EVs. During rest and following exercise, only 0.12 % of the total cfDNA occurs in association with CD9+/CD63+/CD81+EVs. DNase digestion experiments indicate that the largest part of EV associated DNA is sensitive to DNase digestion and only ~20 % are protected within the lumen of the separated EVs. A single bout of running or cycling exercise increases the levels of EVs, cfDNA, and EV associated DNA. While EV surface DNA is increasing, DNAse-resistant DNA remains at resting levels, indicating that EVs released during exercise (ExerVs) do not contain DNA. Consequently, DNA is largely associated with the outer surface of circulating EVs. ExerVs recruit cfDNA to their corona, but do not carry DNA in their lumen.

category: molecular biology


title: SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization

abstract: Transmission of SARS-CoV-2 from humans to farmed mink was observed in Europe and the US. In the infected animals viral variants arose that harbored mutations in the spike (S) protein, the target of neutralizing antibodies, and these variants were transmitted back to humans. This raised concerns that mink might become a constant source of human infection with SARS-CoV-2 variants associated with an increased threat to human health and resulted in mass culling of mink. Here, we report that mutations frequently found in the S proteins of SARS-CoV-2 from mink were mostly compatible with efficient entry into human cells and its inhibition by soluble ACE2. In contrast, mutation Y453F reduced neutralization by an antibody with emergency use authorization for COVID-19 therapy and by sera/plasma from COVID-19 patients. These results suggest that antibody responses induced upon infection or certain antibodies used for treatment might offer insufficient protection against SARS-CoV-2 variants from mink.


title: Joint changes in RNA, RNA polymerase II, and promoter activity through the cell cycle identify non-coding RNAs involved in proliferation

abstract: Proper regulation of the cell cycle is necessary for normal growth and development of all organisms. Conversely, altered cell cycle regulation often underlies proliferative diseases such as cancer. Long non-coding RNAs (lncRNAs) are recognized as important regulators of gene expression and are often found dysregulated in diseases, including cancers. However, identifying lncRNAs with cell cycle functions is challenging due to their often low and cell-type specific expression. We present a highly effective method that analyses changes in promoter activity, transcription, and RNA levels for identifying genes enriched for cell cycle functions. Specifically, by combining RNA sequencing with ChIP sequencing through the cell cycle of synchronized human keratinocytes, we identified 1009 genes with cell cycle-dependent expression and correlated changes in RNA polymerase II occupancy or promoter activity as measured by histone 3 lysine 4 trimethylation (H3K4me3). These genes were highly enriched for genes with known cell cycle functions and included 59 lncRNAs. We selected four of these lncRNAs - AC005682.5, RP11-132A1.4, ZFAS1, and EPB41L4A-AS1 - for further experimental validation and found that knockdown of each of the four lncRNAs affected cell cycle phase distributions and reduced proliferation in multiple cell lines. These results show that many genes with cell cycle functions have concomitant cell-cycle dependent changes in promoter activity, transcription, and RNA levels and support that our multi-omics method is well suited for identifying lncRNAs involved in the cell cycle.


title: Quantifying habitat and landscape effects on composition and structure of plant-pollinator networks in the US Northern Great Plains

abstract: Community structure contributes to ecosystem persistence and stability. To understand the mechanisms underlying pollination and community stability of natural areas in a human influenced landscape, a better understanding of the interaction patterns between plants and pollinators in disturbed landscapes is needed. The Northern Great Plains still retain extensive tracts of remnant temperate grassland habitat within a matrix of varying land-uses. We used a network-based approach to quantify how temperate grassland attributes and landscape heterogeneity influence plant-pollinator community structure in natural habitats. We also quantified pollinator diversity and floral diversity to assess the functional role of temperate grassland attributes and the surrounding landscape on the composition of the plant-pollinator communities in natural habitats. We found that the amount of local nectar sugar and increased proportions of certain land-uses contribute to pollinator diversity that in turn influences the structure of interactions between plants and pollinators. Understanding the factors contributing to plant-pollinator network structure can guide management decisions to support resilient plant-pollinator communities and conserve the stability of pollination services.


title: Sexually antagonistic evolution of mitochondrial and nuclear linkage

abstract: Across eukaryotes, genes encoding bioenergetic machinery are located in both mitochondrial and nuclear DNA, and incompatibilities between the two genomes can be devastating. Mitochondria are often inherited maternally, and theory predicts sex-specific fitness effects of mitochondrial mutational diversity. Yet how evolution acts on linkage patterns between mitochondrial and nuclear genomes is poorly understood. Using novel mito-nuclear population genetic models, we show that the interplay between nuclear and mitochondrial genes maintains mitochondrial haplotype diversity within populations, and it selects both for sex-independent segregation of mitochondrion-interacting genes and for paternal leakage. These effects of genetic linkage evolution can eliminate male-harming fitness effects of mtDNA mutational diversity. With maternal mitochondrial inheritance, females maintain a tight mitochondrial-nuclear match, but males accumulate mismatch mutations because of the weak statistical associations between the two genomic components. Sex-independent segregation of mitochondria-interacting loci improves the mito-nuclear match. In a sexually antagonistic evolutionary process, male nuclear alleles evolve to increase the rate of recombination, while females evolve to suppress it. Paternal leakage of mitochondria can evolve as an alternative mechanism to improve the mito-nuclear linkage. Our modelling framework provides an evolutionary explanation for the observed paucity of mitochondrion-interacting genes on mammalian sex chromosomes and for paternal leakage in protists, plants, fungi, and some animals.


title: Strong trait correlation and phylogenetic signal in North American ground beetle (Carabidae) morphology

abstract: Functional traits mediate species responses to and roles within their environment, and are constrained by evolutionary history. While we have a strong understanding of trait evolution for macro-taxa such as birds and mammals, our understanding of invertebrates is comparatively limited. Here we address this gap in North American beetles with a sample of ground beetles (Carabidae), leveraging a large-scale collection and digitization effort by the National Ecological Observatory Network (NEON). For 154 ground beetle species, we measured seven morphological traits, which we placed into a recently-developed effect-response framework that characterizes traits by how they predict species effects on their ecosystems or responses to environmental stressors. We then used cytochrome oxidase one sequences from the same specimens to generate a phylogeny and tested evolutionary tempo and mode of the traits. We found strong phylogenetic signal in, and correlations among, morphological ground beetle traits. These results indicate that, for these species, beetle body shape trait evolution is constrained, and phylogenetic inertia is a stronger driver of beetle traits than (recent) environmental responses. Strong correlations among effect and response traits suggest that future environmental drivers are likely to affect both ecological composition and functioning in these beetles.


title: Profiling of histone H1 variants and changes on genome architecture and gene expression upon histone H1 depletion in breast cancer cells

abstract: Up to seven members of the histone H1 family may contribute to chromatin compaction and its regulation in human somatic cells. In breast cancer cells, knock-down of multiple H1 variants deregulates many genes, promotes the appearance of genome-wide accessibility sites and triggers an interferon response via activation of heterochromatic repeats. However, how these changes in the expression profile relate to the re-distribution of H1 variants as well as to genome conformational changes have not been yet studied. Here, we combined ChIP-seq of five endogenous H1 variants with Chromosome Conformation Capture analysis in wild-type and H1 knock-down T47D cells. The results indicate that H1 variants coexist in the genome in two large groups depending on the local DNA GC content and that their distribution is robust with respect to multiple H1 depletion. Despite the small changes in H1 variants distribution, knock-down of H1 translated into more isolated but de-compacted chromatin structures at the scale of Topologically Associating Domains or TADs. Such changes in TAD structure correlated with a coordinated gene expression response of their resident genes. This is the first report describing simultaneous profiling of five endogenous H1 variants within a cell line and giving functional evidence of genome topology alterations upon H1 depletion in human cancer cells.


title: Coordinated changes in gene expression, H1 variant distribution and genome 3D conformation in response to H1 depletion


title: Non-invasive sex genotyping of paiche Arapaima gigas by qPCR: An applied bioinformatic approach to identify sex differences

abstract:
Arapaima gigas, one of the largest freshwater fish in the world, is suffering from high fishing pressure and habitat loss, which threaten the conservation status of its natural populations. Of great cultural importance to Amazonian people, the paiche or pirarucu A. gigas is in high demand for its meat, ornamental uses and other byproducts such as scales. Aquaculture is a feasible solution to this dilemma. However, the fact that A. gigas presents no sexual dimorphism until it is 5 years old and its long period to sexual maturity are major obstacles for brood-stock management and fingerling production. Thus, the aim of this study was to develop a molecular tool for non-invasive genotypic sexing of paiche throughout its life cycle. We collected samples from gonads, fins and gill mucus of juvenile specimens from local facilities for histological and molecular analysis. Based on the recently available genome sequence of the paiche and making use of current NGS method, we implemented a novel approach, called Genome Differences by Unmapped Reads, to identify DNA sex markers. We found a Male-Specific Region (MSR), identified as MSR_3728, to be present only in males. Next, we designed two specific sets of primers on this region to identify genotypic sex by qPCR assays. Both primer sets, MSR_107 and MSR_129, detected males with 100% accuracy. Then we developed a duplex qPCR reaction for each primer set along a reference gene, analyzed the melting curves and detected males by observing two distinct peaks, one for MSR_107 or MSR_129 and one for the reference, while females only presented the reference peak. The same results were obtained for gonads, fins and interestingly, a non-invasive source from gill mucus samples. Finally, the gonads were evaluated histologically in a double-blind test, showing 100% accuracy with qPCR assay for identifying males and females. Data clearly demonstrated a novel pipeline approach for identifying DNA sex markers, followed by a quick, non-invasive, cost-effective duplex qPCR method for sexing A. gigas. These results may be valuable to efficient paiche aquaculture and conservation studies, helping to reduce the fishing pressure on its natural populations.

Highlights of the manuscript- Implementation of a novel approach, called Genome Differences by Unmapped Reads, to identify DNA sex markers.
- Finding of a Male-Specific Region (MSR), present only in males.
- Development of a duplex qPCR to identify genotypic sex through non-invasive sampling.


title: Kincore: a web resource for structural classification of protein kinases and their inhibitors

abstract: The active form of kinases is shared across different family members, as are several commonly observed inactive forms. We previously performed a clustering of the conformation of the activation loop of all protein kinase structures in the Protein Data Bank (PDB) into 8 classes based on the dihedral angles that place the Phe side chain of the DFG motif at the N-terminus of the activation loop. Our clusters are strongly associated with the placement of the activation loop, the C-helix, and other structural elements of kinases. We present Kincore, a web resource providing access to our conformational assignments for kinase structures in the PDB. While other available databases provide conformational states or drug type but not both, KinCore includes the conformational state and the inhibitor type (Type 1, 1.5, 2, 3, allosteric) for each kinase chain. The user can query and browse the database using these attributes or determine the conformational labels of a kinase structure using the web server or a standalone program. The database and labeled structure files can be downloaded from the server. Kincore will help in understanding conformational dynamics of these proteins and guide development of inhibitors targeting specific states. Kincore is available at http://dunbrack.fccc.edu/kincore.


title: Methane-derived carbon flow through host-virus trophic networks in soil

abstract: The concentration of atmospheric methane continues to increase with microbial communities controlling soil-atmosphere fluxes. While there is substantial knowledge of the diversity and function of organisms regulating methane production and consumption, the frequency and impact of interactions with viruses on their activity in soil is unknown. Metagenomic sequencing of soil microbial communities has enabled identification of linkages between viruses and hosts. However, determining host-virus linkages through sequencing does not determine whether a virus or a host are active. In this study, we identified active individual interactions in situ by following the transfer of assimilated carbon from active hosts to viruses. Using DNA stable-isotope probing combined with metagenomic analyses, we characterized methane-fueled microbial networks in acidic and neutral pH soils, specifically primary and secondary utilisers of carbon, together with the recent transfer of methane-derived carbon to viruses. Sixty-three percent of viral contigs from replicated soil incubations contained genes associated with known methanotrophic bacteria. Genomic sequences from 13C-enriched viruses were present in clustered regularly interspaced short palindromic repeats (CRISPR) arrays of multiple, closely-related Methylocystis populations, revealing differences in their history of viral interaction. Viruses infecting non-methanotrophic methylotrophs and heterotrophic predatory bacteria were also identified through the analysis of shared homologous genes, demonstrating that carbon is transferred to a diverse range of viruses associated with methane-fueled microbial food networks.


title: slim shady is a novel allele of PHYTOCHROMEB present in the T-DNA line SALK_015201

abstract: Auxin is a hormone that is required for hypocotyl elongation during seedling development. In response to auxin rapid changes in transcript and protein abundance occur in hypocotyls and some auxin responsive gene expression is linked to hypocotyl growth. To functionally validate proteomic studies, a reverse genetics screen was performed on mutants in auxin-regulated proteins to identify novel regulators of plant growth. This uncovered a long hypocotyl mutant, which we called slim shady, in an annotated insertion line in IMMUNOREGULATORY RNA-BINDING PROTEIN (IRR). Overexpression of the IRR gene failed to rescue the slim shady phenotype and characterization of a second T-DNA allele of IRR found that it had a wild-type hypocotyl length. The slim shady mutant has an elevated expression of numerous genes associated with the brassinosteroid-auxin-phytochrome (BAP) regulatory module compared to wild-type, including transcription factors that regulate brassinosteroid, auxin and phytochrome pathways. Additionally, slim shady seedlings fail to exhibit a strong transcriptional response to auxin. Using whole genome sequence and transcriptomics data for SALK_015201C we determined that a novel single nucleotide polymorphism in PHYTOCHROME B was responsible for the slim shady phenotype. This is predicted to convert induce a frameshift and premature stop codon at leucine 1125, within the histidine kinase-related domain of the carboxy terminus of PHYB, which is required for phytochrome signaling and function. Genetic complementation analyses with phyb-9 confirmed that slim shady is a mutant allele of PHYB. This study advances our understanding of the molecular mechanisms in seedling development, by furthering our understanding of how light signaling is linked to auxin dependent cell elongation. Furthermore, this study highlights the importance of confirming the genetic identity of research material before attributing phenotypes to known mutations sourced from T-DNA stocks.


title: Whole plant-environment microscopy reveals how Bacillus subtilis utilises the soil pore space to colonise plant roots

abstract: Our understanding of plant-microbe interactions in soil is limited by the difficulty of observing processes at the microscopic scale throughout plants large volume of influence. Here, we present the development of 3D live microscopy for resolving plant-microbe interactions across the environment of an entire seedling growing in a transparent soil in tailor-made mesocosms, maintaining physical conditions for the culture of both plants and microorganisms. A tailor made dual-illumination light-sheet system acquired scattering signals from the plant whilst fluorescence signals were captured from transparent soil particles and labelled microorganisms, allowing the generation of quantitative data on samples approximately 3600 mm3 in size with as good as 5 m resolution at a rate of up to one scan every 30 minutes. The system tracked the movement of Bacillus subtilis populations in the rhizosphere of lettuce plants in real time, revealing previously unseen patterns of activity. Motile bacteria favoured small pore spaces over the surface of soil particles, colonising the root in a pulsatile manner. Migrations appeared to be directed towards the root cap, the point "first contact", before subsequent colonisation of mature epidermis cells. Our findings show that microscopes dedicated to live environmental studies present an invaluable tool to understand plant-microbe interactions.


title: Plant-environment microscopy tracks interactions of Bacillus subtilis with plant roots across the entire rhizosphere


title: Unravelling cotton RNAseq repositories to the fiber development specific modules and their alliance with the fiber-related traits

abstract: Cotton fiber development is still an intriguing question to understand the fiber commitment and development. Here, we remapped >350 publicly available cotton RNA sequencing data on recently published cotton genome with [~]400 fold coverage. The differentially expressed genes were clustered in six modules whose functions are specific to commitment, initiation, elongation and Secondary Cell Wall (SCW) fiber development stages. Gene Ontology analysis of commitment and initiation specific modules suggests enrichment of genes involved in organ development. The modules specific for elongation and SCW showed significant enrichment of hydroxyproline-rich proteins and hydrolases. Transcription factors (TFs) binding frequency of defined modules suggested that homeodomain, MYB and NAC expresses at commitment stages but their expression was governed by other TFs. We also mined the stage-specific transcriptional biomarker and Exclusively Expressed Transcripts (EETs) for fiber. These EETs were positively selected during fiber evolution and cotton domestication. The extensive expression profiling of six EETs in 100 cotton genotypes at different fiber developmental stages using nCounter assay and their correlation with eight fiber-related suggests that several EETs are correlated with different fiber quality-related traits. Thus, our study reveals several important genes and pathways that may be important for cotton fiber development and future improvement of cotton.


title: Tooth chipping patterns in Paranthropus do not support regular hard food mastication

abstract: The paranthropines, including Paranthropus boisei and Paranthropus robustus, have often been considered hard-food specialists. The large post-canine teeth, thick enamel, and robust craniofacial features are often suggested to have evolved to cope with habitual mastication of hard foods. Yet, direct evidence for Paranthropus feeding behaviour often challenges these morphological interpretations. The main exception being antemortem tooth chipping which is still regularly used as evidence of habitual mastication of hard foods in this genus. In this study, data were compiled from the literature for six hominin species (including P. boisei and P. robustus) and 17 extant primate species, to analyse Paranthropus chipping patterns in a broad comparative framework. Severity of fractures, position on the dentition, and overall prevalence were compared among species. The results indicate that both Paranthropus species had a lower prevalence of tooth fractures compared to other fossil hominin species (P. boisei: 4%; P. robustus: 11%; Homo naledi: 37%; Australopithecus africanus: 17%; Homo neanderthalensis: 45%; Epipalaeolithic Homo sapiens: 29%); instead, their frequencies are similar to apes that masticate hard items in a non-regular frequency, including chimpanzees, gibbons, and gorillas (4%, 7% and 9% respectively). The prevalence is several times lower than in extant primates known to habitually consume hard items, such as sakis, mandrills, and sooty mangabeys (ranging from 28% to 48%). Comparative chipping analysis suggests that both Paranthropus species were unlikely habitual hard object eaters, at least compared to living durophage analogues.

category: paleontology


title: Systematic beach monitoring as a health assessment tool: cetacean morbillivirus under non-epizootic circumstances in stranded dolphins

abstract:
Cetacean morbillivirus (CeMV) was identified as the etiologic agent of several epizootic episodes worldwide. Most of these studies are based on unusual mortality events or identification of new viral strains. We investigated the occurrence of CeMV under non-epizootic circumstances at a world heritage in Southern Brazil by a combination of pathologic, immunohistochemical and molecular assays. From 325 stranded cetaceans, 40 were included. Guiana dolphin (Sotalia guianensis) was the species most frequent. Interstitial pneumonia and non-suppurative encephalitis were the main pathologic findings associated to CeMV infection. Intracytoplasmic immunolabeling anti-CeMV was observed mainly in lungs and lymph nodes. All samples were negative in RT-PCR assay. Diagnosis of CeMV is challenging in areas where epizootic episodes have not been recorded and due to postmortem changes. We observed a CeMV prevalence of 27.5%. The results described here increase the knowledge about CeMV under non-epizootic conditions in Brazil.

Article Summary LineWe observed a prevalence of 27.5% of CeMV in a World Heritage site of Paranas coast. The results indicate an increase in the prevalence of CeMV at this region and, possibly, a degradation of marine ecosystem. Marine mammals are sentinels of marine environment and the ocean health is inextricably linked to human health on a global scale.

category: pathology


title: Conserved residues in the extracellular loop 2 regulate Stachel-mediated activation of ADGRG2

abstract: Cleavage and dissociation of a large N-terminal fragment and the consequent unmasking of a short sequence (Stachel) remaining on the N-terminus have been proposed as mechanisms of activation of some members of the adhesion G protein-coupled receptor (aGPCR) family. However, the identity of residues that play a role in the activation of aGPCRs by the cognate Stachel remains largely unknown. Protein sequence alignments revealed a conserved stretch of residues in the extracellular loop 2 (ECL2) of all 33 members of the aGPCR family. ADGRG2, an orphan aGPCR, plays a major role in male fertility, Ewing sarcoma cell proliferation, and parathyroid cell function. We used ADGRG2 as a model aGPCR and generated mutants of the conserved residues in the ECL2 via site-directed mutagenesis. We show that tryptophan and isoleucine in the ECL2 are required for receptor stability and surface expression in the HEK293 cells. By adjusting the receptor surface expression levels, we show that the ECL2 mutation ablates the Stachel-mediated activation of multiple signaling pathways of ADGRG2. This study provides a novel understanding of the role of the ECL2 in Stachel-mediated signaling and degradation of ADGRG2, which may lay the foundation for the rational design of therapeutics to target aGPCRs.

category: pharmacology and toxicology


title: Synergistic application of continuous granulation and selective laser sintering 3D printing for the development of pharmaceutical dosage forms with enhanced dissolution rates and physical properties

abstract:
This study demonstrated the first case of combining novel continuous granulation with powder-based pharmaceutical 3-dimensional (3D) printing processes to enhance the dissolution rate and physical properties of a poorly water-soluble drug. Powder bed fusion (PBF) and binder jetting 3D printing processes have gained much attention in pharmaceutical dosage form manufacturing in recent times. Although powder bed-based 3D printing platforms have been known to face printing and uniformity problems due to the inherent poor flow properties of the pharmaceutical physical mixtures (feedstock). Moreover, techniques such as binder jetting currently do not provide any solubility benefits to active pharmaceutical ingredients (APIs) with poor aqueous solubility (>40% of marketed drugs). For this study, a hot-melt extrusion-based versatile granulation process equipped with UV-Vis process analytical technology (PAT) tools for the in-line monitoring of critical quality attributes (i.e., solid-state) of indomethacin was developed. The collected granules with enhanced flow properties were mixed with vinylpyrrolidone-vinyl acetate copolymer and a conductive excipient for efficient sintering. These mixtures were further characterized for their bulk properties observing an excellent flow and later subjected to a PBF-3D printing process. The physical mixtures, processed granules, and printed tablets were characterized using conventional as well as advanced solid-state characterization. These characterizations revealed the amorphous nature of the drug in the processed granules and printed tablets. Further, the in vitro release testing of the tablets with produced granules as a reference standard depicted a notable solubility advantage (100% drug released in 5 minutes at >pH 6.8) over the pure drug and the physical mixture. Our developed system known as DosePlus combines innovative continuous granulation and PBF-3D printing process which can potentially improve the physical properties of the bulk drug and formulations in comparison to when used in isolation. This process can further find application in continuous manufacturing of granules and additive manufacturing of pharmaceuticals to produce dosage forms with excellent uniformity and solubility advantage.

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title: Modest changes in Spi1 dosage reveal the potential for altered microglial function as seen in Alzheimers disease

abstract: Genetic association studies have identified multiple variants at the SPI1 locus that modify risk and age of onset for Alzheimers Disease (AD). Reports linking risk variants to gene expression suggest that variants denoting higher SPI1 expression are likely to have an earlier AD onset, and several other AD risk genes contain PU.1 binding sites in the promoter region. Overall, this suggests altered levels of SPi1 may alter microglial phenotype potentially impacting AD. This study determined how the mouse microglial transcriptome was altered following modest changes to Spi1 expression in primary microglia. RNA-sequencing was performed on microglia with reduced or increased Spi1/PU.1 expression to provide an unbiased approach to determine transcriptomic changes affected by Spi1. In summary, a reduction in microglial Spi1 resulted in the dysregulation of transcripts encoding proteins involved in DNA replication pathways while an increased Spi1 results in an upregulation of genes associated with immune response pathways. Additionally, a subset of 194 Spi1 dose-sensitive genes was identified and pathway analysis suggests that several innate immune and interferon response pathways are impacted by the concentration of Spi1. Together these results suggest Spi1 levels can alter the microglial transcriptome and suggests interferon pathways may be altered in individuals with AD related Spi1 risk SNPs.