title: Dynamic Synchronization between Hippocampal Spatial Representations and the Stepping Rhythm

abstract: Summary paragraphThe hippocampus is a vertebrate brain structure that expresses spatial representations1 and is critical for navigation2,3. Navigation in turn intricately depends on locomotion; however, current accounts suggest a dissociation between hippocampal spatial representations and the details of locomotor processes. Specifically, the hippocampus is thought to primarily represent higher-order cognitive and locomotor variables like position, speed, and direction of movement4-7, while the limb movements that propel the animal are thought to be computed and represented primarily in subcortical circuits, including the spinal cord, brainstem, and cerebellum8-11. Whether hippocampal representations are actually decoupled from the detailed structure of locomotor processes remains unknown. To address this question, we simultaneously monitored hippocampal spatial representations and ongoing limb movements underlying locomotion at fast timescales. We found that the forelimb stepping cycle in freely behaving rats is rhythmic and peaks at ~8 Hz during movement, matching the ~8 Hz organization of information processing in the hippocampus during locomotion12. We also discovered precisely timed coordination between the time at which the forelimbs touch the ground ( plant times of the stepping cycle) and the hippocampal representation of space. Notably, plant times coincide with hippocampal representations closest to the actual position of the animal, while in-between these plant times, the hippocampal representation progresses towards possible future locations. This synchronization was specifically detectable when animals approached upcoming spatial decisions. Taken together, our results reveal profound and dynamic coordination on a timescale of tens of milliseconds between central cognitive representations and peripheral motor processes. This coordination engages and disengages rapidly in association with cognitive demands and is well suited to support rapid information exchange between cognitive and sensory-motor circuits.


title: Investigation on How Dynamic Effective Connectivity Patterns Encode the Fluctuating Pain Intensity in Chronic Migraine

abstract:
Chronic migraine is characterised by persistent headaches for more than 15 days per month; the intensity of the pain is fluctuating over time. Here, we explored the dynamic interplay of connectivity patterns between regions known to be related to pain processing and their relation to the ongoing dynamic pain experience. We recorded EEG from 80 sessions (20 chronic migraine patients in 4 separate sessions of 25 minutes). The patients were asked to continuously rate the intensity of their endogenous headache. On different time-windows, a dynamic causal model (DCM) of cross spectral responses was inverted to estimate connectivity strengths. For each patient and session, the evolving dynamics of effective connectivity were related to pain intensities and to pain intensity changes by using a Bayesian linear model. Hierarchical Bayesian modelling was further used to examine which connectivity-pain relations are consistent across sessions and across patients.

The results reflect the multi-facetted clinical picture of the disease. Across all sessions, each patient with chronic migraine exhibited a distinct pattern of pain intensity-related cortical connectivity. The diversity of the individual findings are accompanied by inconsistent relations between the connectivity parameters and pain intensity or pain intensity changes at group level. This suggests a rejection of the idea of a common neuronal core problem for chronic migraine.

category: neuroscience


title: Genetic deletion of Kiaa1109 in mice leads to developmental defects at the neuromuscular junction

abstract:
KIAA1109 (4932438A13Rik) is a novel gene linked to Alkuraya-Kucinska Syndrome, an autosomal recessive disorder with severe brain malformations and arthrogryposis in humans. The role of KIAA1109 in mammalian development and function is unknown. Here, we characterize mutant mice deficient in Kiaa1109 (Kiaa1109-/-). We report that Kiaa1109-/- mice died during perinatal stages. These Kiaa1109-/- embryos exhibited impaired intramuscular nerve growth and reduced sizes of the neuromuscular junction (NMJ) compared with their littermate controls. Electrophysiological analysis further revealed defects in neuromuscular synaptic transmission in Kiaa1109-/- embryos. Notably, the frequency of spontaneous neurotransmitter release was markedly increased, whereas evoked neurotransmitter release and quantal content were reduced. Furthermore, neuromuscular synapses in Kiaa1109-/- embryos failed to respond to a repetitive, low frequency stimulation (10Hz). These results demonstrate that KIAA1109 is required for survival in mice and for proper development and function of the NMJ.

Significance StatementThis is the first report characterizing the phenotype of mutant mice deficient in KIAA1109 (4932438A13Rik), a novel gene in mammals. We show that KIAA1109 is required for survival in mice and that KIAA1109 plays important roles in normal development and function of the NMJ in mice.


title: Phospho-proteomic analysis of CAR-T cell signaling following activation by antigen-presenting cancer cells

abstract: Chimeric antigen receptors (CARs) are synthetic biomolecules comprised of an extracellular antigen recognition domain and intracellular signaling domains. When expressed in immune cells, CARs direct their host cells to kill diseased cells expressing the antigen recognized by the CAR. Although signaling pathways downstream of CAR activation control the cytotoxic function of CAR-expressing cells, phospho-proteomic studies of CAR signaling have been limited. Most approaches have used antibodies or soluble ligands, rather than cell-displayed antigens, to activate CAR signaling. Here, we demonstrate an efficient and cost-effective label-free phospho-proteomic approach to analyze CAR signaling in immune cells stimulated with antigen-presenting cancer cells. Following co-culture of CAR-T cells with cancer cells, we first preserve phospho-signaling by cross-linking proteins with formalin. Then, we use magnet-activated cell sorting (MACS) to isolate CAR-T cells from the co-culture. Validation experiments demonstrated that formalin fixation did not alter the phospho-proteome and that MACS achieved >90% CAR-T cell purity. Next, we compared the phospho-proteome in CAR-T cells stimulated with either CD19-expressing or non-CD19-expressing SKOV3 ovarian cancer cells. This analysis revealed that CAR signaling activated known pathways including the mitogen- activated protein kinases (MAPKs) ERK1/2. Bioinformatic approaches further showed that CAR activation induced other signaling pathways including the MAPK p38, protein kinase A, and checkpoint kinase 1 (CHK1). Taken together, this work presents an easy and inexpensive method to better understand CAR immunotherapy by label-free phospho-proteomic analysis of CAR signaling in immune cells stimulated by antigen- presenting cancer cells.

category: immunology


title: Differential recognition of computationally optimized H3 hemagglutinin influenza vaccine candidates by human antibodies

abstract:
Among circulating influenza viruses in humans, H3N2 viruses typically evolve faster than other subtypes and have caused severe illness and deaths in millions of people since emerging in 1968. Computationally optimized broadly reactive antigen (COBRA) technology is one strategy to broaden vaccine-elicited antibody responses among influenza subtypes. In this study, we determined the structural integrity of an H3N2 COBRA HA, TJ5, and, as nearly all humans have pre-existing immunity to H3N2 influenza viruses, we probed the antigenic profile of several H3N2 COBRA HAs by assessing recognition of these immunogens by human B cells and monoclonal antibodies (mAbs). Of three recently described COBRA H3 HA antigens (TJ5, NG2, and J4), we determined that TJ5 and J4 HA proteins recognize pre-existing B cells (from the 2017-2018 vaccine season) more effectively than NG2 HA and a wild type Hong Kong/4801/2014 protein. H3 HA-specific human mAbs recognize wild type and COBRA HA proteins, and have functional activity against a broad panel of H3N2 viruses. mAb TJ5-5 recognizes TJ5 and J4 HA proteins, but has poor recognition of NG2 HA, similar to the global B cell analysis. To probe these recognition differences and to verify the structural integrity of the TJ5 HA protein, we determined a 3.4 [A] structure via cryo-electron microscopy of TJ5-5 complexed with the TJ5 HA, which revealed residues important to the differential binding. Overall, these studies determined that COBRA H3 HA proteins have correct antigenic and structural features, and are recognized by B cells and mAbs isolated from seasonally vaccinated humans.

ImportanceVaccine development for circulating influenza viruses, particularly for the H3N2 subtype, remains challenging due to consistent antigenic drift. Computationally optimized broadly reactive antigen (COBRA) technology has proven effective for broadening influenza hemagglutinin (HA) elicited antibody responses compared to wild type immunogens. Here we determined the structural features and antigenic profiles of H3 COBRA HA proteins. Two H3 COBRA HA proteins, TJ5 and J4, are better recognized by pre-existing B cells and monoclonal antibodies from the 2017-2018 vaccine season compared to COBRA NG2 and a wild type A/Hong Kong/2014 HA protein. We determined a cryo-EM structure of one mAb that poorly recognizes NG2, mAb TJ5-5, in complex with the TJ5 COBRA HA protein and identified residues critical to mAb recognition. As NG2 is more effective than TJ5 for a recent Hong Kong/2019 virus, these data provide insights into the diminished effectiveness of influenza vaccines across vaccine seasons.


title: Development of duplex real time PCR for quick detection of Cryptosporidia in goats

abstract:
Cryptosporidium spp. is the most important foodborne and waterborne pathogens and the leading cause of mortality from foodborne and waterborne gastrointestinal disease. In neonates of domestic animals it is associated with consistent diarrhoea and dehydration. Cryptosporidium infection begins with the ingestion of sporulated oocytes disseminated by carrier animals that consistently contaminate the environment. Many diagnostic tests are available including microscopy, antigen trap ELISA, but none of the diagnostic tests available currently cannot differentiate between active and passive infection in the host. In the current study, to address this challenge an mRNA based duplex TaqMan(R) probe PCR (dRT-qPCR) was developed to target the Cryptosporidium oocyst wall protein (COWP) gene and 18ssu rRNA gene in a single tube that can detect metabolically active Cryptosporidial oocysts. The mRNA transcripts are the direct indicator of any actively replicating cell and it will help decipher the active stages of its lifecycle in host. This diagnostic assay was standardized by computing transcript copy number-based limit of detection. For COWP and 188ssu rRNA genes the limit of detection was 7.08x1004 and 5.95x1005 respectively. During active infections the oocyst wall protein will be active and so its COWP gene transcripts will act as marker for active infection. While transcripts for 18SSU rRNA are constitutively expressing in Cryptosporidial life cycle. This current diagnostic assay will be a quantitative marker that will help assess active stages of Cryptosporidium infection in neonates. The disease dynamics will help better understand to formulate the control strategies and contain infection among the healthy animals.

ImportanceCryptosporidiosis is an important neonatal disease affecting goats causing diarrhoea, dehydration and stunted growth. For diagnosing this condition, many diagnostic tests are available including microscopy, immunological tests, but none of the diagnostic tests available currently can differentiate between active and passive infection in the host. The mRNA transcripts are the direct indicator of any actively replicating cell and especially in intracellular parasites it will help decipher the infective stages of a lifecycle in the host, and hence the test was developed in a reverse transcriptional format in a duplex mode. The currently developed diagnostic assay for cryptosporidiosis was evaluated for sensitivity using Limit of detection (LOD). This diagnostic test will act as a quantitative marker to aid in detecting active stages of Cryptosporidium infection in neonatal goats and will eventually lead to better control strategies for managing cryptosporidial infections in the future.

category: molecular biology


title: Copepod functional traits and groups show contrasting biogeographies in the global ocean

abstract:
AimTo define global zooplankton functional groups (FGs) and to estimate their environmental niche and habitat distribution. We model the spatial patterns of copepod FGs habitat and identify regions sharing similar functional trait expression at the community level.

TaxonMarine planktonic Neocopepoda.

LocationGlobal ocean.

MethodsFactor analysis on mixed data and hierarchical clustering were used to identify copepod FGs based on five species-level functional traits. An ensemble of species distribution models was used to estimate the environmental niches of the modelled species, project the mean annual habitat suitability of the FGs, and to estimate the community weighted mean values of the traits studied. Ocean regions were defined based on their community-level mean trait expression using a principal component analysis and hierarchical clustering.

ResultsEleven global copepod FGs were identified. They displayed contrasting latitudinal patterns in mean annual habitat suitability that could be explained by differences in environmental niche preferences: two FGs were associated with polar conditions, one followed the global temperature gradient, five were associated with tropical oligotrophic gyres, and the remaining three with boundary currents and counter currents. Four main regions of varying community weighted mean trait values emerged: the Southern Ocean, the northern and southern high latitudes, the tropical gyres, and the boundary currents and upwelling systems.

ConclusionsWe build on an exhaustive species trait dataset to put forward novel FGs that will improve the representation of zooplankton in global marine ecosystem models. Our results contribute to our understanding of the spatial patterns and drivers of marine plankton trait biogeography and will serve as a basis for studying the links between zooplankton biodiversity and ecosystem functioning and how they might evolve in the context of climate change.

category: ecology


title: Direct and delayed synergistic effects of marine heatwaves, metals and food limitation on tropical reef-associated fish larvae

abstract:
Tropical fish are fast-growing and high energetic-demand organisms, which can be highly vulnerable to long-lasting effects of heat stress and pollution, particularly under food shortages. We tested this by assessing highly complex direct and delayed interactive effects of an extreme temperature (32{degrees}C) from a simulated marine heatwave (MHW), copper (Cu, 0, 100, 150 and 175 {micro}g L-1) and food availability (limited and saturated food) on larvae of a tropical, reef-associated seaperch (Psammoperca waigiensis). Cu, MHW, and food limitation independently reduced survival and growth, partly explained by reduced feeding. The negative effect of Cu on fish survival was more substantial under MHW, particularly under limited food. Delayed interactive effects of Cu, MHW, and food limitation were still lethal to fish larvae during the post-exposure period. These results indicate that reef-associated fish larvae are highly vulnerable to these dominant stressors, impairing their ecological function as predators in the coral reefs.

Graphical abstract

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title: Intentional stocking undermines ecological stability

abstract:
The past decades have witnessed efforts to unveil ecological risks associated with massive releases of captive-bred individuals ("stock enhancement"). Still, we may underestimate the negative impact because current schemes rarely consider the consequences of stock enhancement for the whole ecological community. Here, we use theory and long-term data of Japanese stream fish communities to show that stock enhancement undermines community stability. Our theory predicted that stock enhancement destabilizes community dynamics by facilitating competitive exclusion. Consistent with this prediction, fish communities showed greater temporal fluctuations and fewer species richness in rivers with the intensive stocking of hatchery masu salmon - a major freshwater resource in the region. Our findings paint a bleak picture for stock enhancement, reinforcing the recurrent calls for alternative management strategies.

One Sentence SummaryLarge-scale releases of captive-bred organisms undermine the long-term persistence of ecological communities by disrupting the stable coexistence of competing species.


title: xcore: an R package for inference of gene expression regulators

abstract:
Elucidating the Transcription Factors (TFs) that drive the gene expression changes in a given experiment is one of the most common questions asked by researchers. The existing methods rely on the predicted Transcription Factor Binding Site (TFBS) to model the changes in the motif activity. Such methods only work for TFs that have a motif and assume the TF binding profile is the same in all cell types. Given the wealth of the ChIP-seq data available for a wide range of the TFs in various cell types, we propose that the modeling can be done using ChiP-seq "signatures" directly, effectively skipping the motif finding and TFBS prediction steps. We present xcore, an R package that allows Transcription Factor activity modeling based on their ChiP-seq signatures and users gene expression data. We also provide xcoredata a companion data package that provides a collection of preprocessed ChiP-seq signatures. We demonstrate that xcore leads to biologically relevant predictions using TGF-beta induced epithelial-mesenchymal transition and rinderpest infection time-course CAGE data as examples.

Availability and Implementationxcore and xcoredata R packages are freely available at www.github.com/bkaczkowski/xcore and www.github.com/mcjmigdal/xcoredata.

xcore user guide is available www.bkaczkowski.github.io/xcore/articles/xcore_vignette.html.

Contactb.kaczkowski@gmail.com

category: bioinformatics


title: Time-lagged Flux in the Transition Path Ensemble: Flux Maximization and Relation to Transition Path Theory

abstract: Transition path ensemble is of special interest in reaction coordinate identification as it consists of reactive trajectories that start from the reactant state and end in the product one. As a theoretical framework for describing the transition path ensemble, the transition path theory has been introduced more than ten years ago and so far its applications have been only illustrated in several low-dimensional systems. Given the transition path ensemble, expressions for calculating flux, current (a vector field), and principal curve are derived here in the space of collective variables from the transition path theory and they are applicable to time-series obtained from molecular dynamics simulations of high-dimensional systems, i.e., the position coordinates as a function of time in the transition path ensemble. The connection of the transition path theory is made to a density-weighted average flux, a quantity proposed in a previous work to appraise the relevance of a coordinate to the reaction coordinate [W. Li, J. Chem. Phys. 156, 054117 (2022)]. Most importantly, as an extension of the existing quantities, time-lagged quantities such as flux and current are also proposed. The main insights and objects provided by these time-lagged quantities are illustrated in the application to the alanine peptide in vacuum.

category: biophysics


title: Western diet unmasks transient low-level vinyl chloride exposure-induced tumorigenesis; potential role of the (epi-)transcriptome

abstract:
Background & AimsVinyl chloride (VC) monomer is a volatile organic compound commonly used in industry to produce polyvinyl chloride (PVC). At high exposure levels, VC causes liver cancer and toxicant-associated steatohepatitis. However, lower exposure levels (i.e., < regulatory exposure limits) that do not directly damage the liver, enhance injury caused by Western diet (WD). Although these lower exposure levels are considered  safe, it is unknown if the long-term impact of transient low-concentration VC enhances the risk of liver cancer development. Low-concentration VC is especially a concern given that fatty liver disease is in and of itself a risk factor for the development of liver cancer.

MethodsC57Bl/6J mice were fed WD or control diet (CD) for 1 year. During the first 12 weeks of feeding only, mice were also exposed to VC via inhalation at sub-regulatory limit concentrations (<1 ppm) or air for 6 hours/day, 5 days/week.

ResultsFeeding WD for 1 year caused significant hepatic injury, including steatohepatitis and moderate fibrosis, which was exacerbated by VC. Additionally, VC increased the number of tumors which ranged from moderately to poorly differentiated hepatocellular carcinoma (HCC). Transcriptomic analysis demonstrated VC-induced changes in metabolic but also ribosomal processes. Epitranscriptomic analysis showed a VC-induced shift of the modification pattern that has been associated with metabolic disease, mitochondrial dysfunction, and cancer.

ConclusionsThese data indicate that VC sensitizes the liver to other stressors (e.g., WD), resulting in enhanced tumorigenesis. These data raise concerns about a potential interaction between VC exposure and WD. Furthermore, it also emphasizes that current safety restrictions may be insufficient to account for other factors that can influence hepatotoxicity.

category: cancer biology


title: New insights into 4,000 years of resource economy across Greenland using ancient DNA

abstract: The success and failure of past cultures across the Arctic was tightly coupled to the ability of past people to exploit the full range of resources available to them, and to adapt to fluctuations in resource availability. There is substantial evidence for the hunting of birds, caribou and a wide range of marine mammals in pre-historic Greenland from bone remains preserved in ancient middens. However, the extent to which these communities relied on marine resources such as fish and large cetaceans is understudied because of the taphonomic processes and bias that affect how these taxa present themselves in the archaeological record. To address this, we analyse DNA from bulk bone samples from 12 archaeological sites across Greenland dating from Paleo-Inuit to Neo-Inuit periods. Using a combination of metabarcoding and shotgun metagenomics we identify an assemblage of 43 species consisting of birds, fish, and both marine and terrestrial mammals. We find genetic evidence of five different whale species, of which the bowhead whale (Balaena mysticetus) was the most commonly detected. Furthermore, we detect nine fish species, of which four have not previously been identified in any of the studied sites. Lastly, we identify a novel haplotype in caribou (Rangifer tarandus) at the 3,000-year-old site Itinnera, suggesting the presence of a distinct lineage of (now extinct) dwarfed caribou that colonised Greenland after the last ice age 9,000 years ago. Collectively, these findings provide a rare insight into whaling and fishing practices in Greenland and demonstrate that prehistoric Greenlandic communities had the social and technological capacity to target the largest whales available in the waters around them.

category: paleontology


title: A robust modular automated neuroimaging pipeline for model inputs to TheVirtualBrain

abstract: TheVirtualBrain, an open-source platform for large-scale network modelling, can be personalized to an individual using a wide range of neuroimaging modalities. With the growing number and scale of neuroimaging data sharing initiatives of both healthy and clinical populations comes an opportunity to create large and heterogeneous sets of dynamic network models to better understand individual differences in network dynamics and their impact on brain health. Here we present TheVirtualBrain-UK Biobank pipeline, a robust, automated and open-source brain image processing solution to address the expanding scope of TheVirtualBrain project. Our pipeline generates connectome-based modelling inputs compatible for use with TheVirtualBrain. We leverage the existing multimodal MRI processing pipeline from the UK Biobank made for use with a variety of brain imaging modalities. We add various features and changes to the original UK Biobank implementation specifically for informing large-scale network models, including user-defined parcellations for the construction of matching whole-brain functional and structural connectomes. Changes also include detailed reports for quality control of all modalities, a streamlined installation process, modular software packaging, updated software versions, and support for various publicly available datasets. The pipeline has been tested on both healthy and clinical populations and is robust to the morphological changes observed in aging and dementia. In this paper, we describe these and other pipeline additions and modifications in detail, as well as how this pipeline fits into the TheVirtualBrain ecosystem.


title: Dissecting the effects of adaptive encoding and predictive inference on a single perceptual estimation

abstract: The human visual system exploits continuity in the visual environment, which induces two contrasting perceptual phenomena: repulsive aftereffects and attractive serial dependences. Recent studies have theorized that these two effects concurrently arise in perceptual processing, but empirical evidence that directly supports this hypothesis lacks. Here we show that the two effects concurrently modulate estimation behavior in a typical sequence of perceptual tasks. We first demonstrate that observers estimation error as a function of both the previous stimulus and response cannot be fully described by either attractive or repulsive bias but is instead well captured by a summation of repulsion from the previous stimulus and attraction toward the previous response. We then reveal that the repulsive bias is centered on the observers sensory encoding of the previous stimulus, which is again repelled away from the preceding trials, whereas the attractive bias is centered precisely on the previous response, which is the observers best prediction about the incoming stimuli. Our findings provide strong evidence that sensory coding is shaped by dynamic tuning of the system to the past stimuli, inducing repulsive aftereffects, and followed by inference incorporating the prediction from the past estimation, leading to attractive serial dependence.


title: Rich Experience Boosts Functional Connectome and High-Dimensional Coding in Hippocampal Network

abstract: Challenging the brain with experiential richness creates tissue-level changes and synaptic plasticity, but the interjacent network level has not been accessible. We here show that environmental enrichment has unexpectedly far-reaching effects on network connectivity and multi-dimensional coding in the hippocampus. We present direct evidence that experience impacts local and global network connectivity, synchrony, and rhythmic dynamics. For this, we investigated the hippocampi from standard-housed mice (SD) and mice living in an enriched environment (ENR) using large-scale ex vivo recordings with a high-density microelectrode sensing array that - with the unprecedented spatiotemporal resolution-allowed simultaneous electrophysiological assessment across the entire circuit. In the absence of extrinsic electrical network stimulation, we found enhanced functional connectivity and high-dimensional coding in hippocampal-cortical networks of ENR mice. The mapped connectome illustrated a scale-free smallworld topology and an ENR-induced resilience to random failures. ENR enhanced large-scale spatiotemporal firing patterns, which facilitated efficient pattern separation and boosted the information encoded in the firing phases of slow oscillatory rhythms. Given that essentially all electrophysiological studies on network behaviors have been done on animals housed in stimulus-poor conditions, our SD mice showed the expected normal functionality. The literature consequently underestimates the extent of spontaneous network activity and connectivity under truly physiological conditions. Our results pave the way to unveil fundamental mechanisms of experience-dependent enhancement in the hippocampal network underlying high brain functions and provide markers for large-scale network remodeling and metaplasticity.


title: Age-Related Central Gain with Degraded Neural Synchrony in the Auditory Brainstem of Mice and Humans

abstract: Aging is associated with auditory nerve (AN) functional deficits and decreased inhibition in the central auditory system, amplifying central responses in a process known as central gain. Although central gain enhances response amplitudes, central gain may not restore disrupted response timing. In this translational study, we measured responses from the AN and auditory midbrain in younger and older mice and humans. We hypothesized that older mice and humans exhibit central gain without an improvement in inter-trial synchrony in the midbrain. Our data demonstrated greater age-related deficits in AN response amplitudes than auditory midbrain response amplitudes, as shown by significant interactions between neural generator and age group, indicating central gain in auditory midbrain. However, synchrony decreases with age in both the AN and midbrain responses. These results reveal age-related central gain without concomitant improvements in synchrony, consistent with those predictions based on decreases in inhibition. Persistent decreases in synchrony may contribute to auditory processing deficits in older mice and humans.


title: Astrocytic Ca2+ signals partake in inhibitory neurovascular coupling in a brain state-dependent manner.

abstract: Neurovascular coupling (NVC) modulates cerebral blood flow to match increased metabolic demand during neuronal excitation. Activation of inhibitory interneurons also increase blood flow, but the basis for this inhibitory NVC is unclear. We performed two-photon microscopy in awake mice to examine the correlation between astrocytic Ca2+ and NVC, evoked by activity in either all (VGATIN) or parvalbumin-positive GABAergic interneurons (PVIN). Optogenetic stimulation of VGATIN and PVIN in the somatosensory cortex triggered astrocytic Ca2+ increases that were abolished by anaesthesia. PVIN evoked astrocytic Ca2+ responses with a short latency that preceded NVC, whereas VGATIN evoked Ca2+ increases that were delayed relative to the NVC response. The early onset in PVIN evoked Ca2+ increases dependent on noradrenaline release from locus coeruleus, which also affected inhibitory NVC. Therefore, NVC mechanisms should be studied in awake mice and, though the relationship between interneuron activity and astrocytic Ca2+ is complex, we found a correlation between astrocyte activity and NVC in PVIN.


title: A role for thalamic projection GABAergic neurons in circadian responses to light

abstract:
The thalamus is an important hub for sensory information and participates in sensory perception, regulation of attention, arousal and sleep. These functions are executed primarily by glutamatergic thalamocortical neurons that extend axons to the cortex and initiate cortico-thalamocortical connectional loops. However, the thalamus also contains projection GABAergic neurons that do not engage in direct communication with the cortex. Here, we have harnessed recent insight into the development of the intergeniculate (IGL), the ventrolateral geniculate (LGv) and the perihabenula (pHB) to specifically target and manipulate thalamic projection GABAergic neurons in female and male mice. Our results show that thalamic GABAergic neurons of the IGL and LGv receive retinal input from diverse classes of ipRGCs, but not from the M1 ipRGC type, while those in the pHB lack direct retinal input. We describe the synergistic role of the photoreceptor melanopsin and the thalamic neurons of the IGL/LGv in circadian entrainment to dim light. We identify a requirement for the thalamic IGL/LGv in the rapid changes in vigilance states associated with circadian light transitions. Furthermore, we map a previously undescribed thalamic network of developmentally related GABAergic neurons in the IGL/LGv complex and the pHB potentially involved in light-dependent mood regulation.

Significance statementThe intergeniculate leaflet and ventral geniculate nucleus are part of the extended circadian system and mediate some non-image-forming visual functions. Here we show that each of these structures has a thalamic (dorsal) as well as prethalamic (ventral) developmental origin. We map the retinal input to thalamus-derived cells in the IGL/LGv complex and discover that while ipRGC input is dominant, this is not likely to originate from M1-ipRGCs. We describe the extent of similarity in synaptic input to developmentally related cells in the IGL/LGv and in the perihabenula nucleus (pHB). We implicate thalamic cells in the IGL/LGv in vigilance state transitions at circadian light changes and in overt behavioural entrainment to dim light, the latter exacerbated by concomitant loss of melanopsin expression.


title: Load Non-Linearly Modulates Movement Reflex Gain in an Insect Leg via a Distributed Network of Identified Nonspiking Interneurons

abstract: Producing context-specific motor acts requires sensorimotor neural networks to integrate multiple sensory modalities. Some of this integration occurs via presynaptic interactions between proprioceptive afferent neurons themselves,1,2 other by afferents of different modalities targeting appropriate motor neurons (MNs).3-5 How the interneuronal network typically interposed between sensory afferents and MNs contributes to this integration, particularly at single-neuron resolution, is much less understood. In stick insects, this network contains nonspiking interneurons (NSIs) converging onto the posture-controlling slow extensor tibiae motor neuron (SETi). We analyzed how load altered movement signal processing by tracing the interaction of movement (femoral chordotonal organ, fCO) and load (tibial campaniform sensilla, tiCS) signals from the afferents through the NSI network to the motor output. On the afferent level, load reduced movement signal gain by presynaptic inhibition; tiCS stimulation elicited primary afferent depolarization and reduced fCO afferent action potential amplitude. In the NSI network, graded responses to movement and load inputs summed nonlinearly and increased the gain of NSIs opposing movement-induced reflexes. The gain of SETi and muscle movement reflex responses consequently decreased. Gain modulation was movement parameter-specific and required presynaptic inhibition; pharmacologically blocking presynaptic inhibition abolished load-dependent tuning of SETi responses. These data describe sensorimotor gain control at the sensory, premotor, and motor levels. Presynaptic inhibition-mediated nonlinear integration allowed the NSI network to respond to movement sensory input in a context (load)-dependent manner. These findings show how gain changes can allow premotor networks to integrate multiple sensory modalities and thus generate context-appropriate motor activity.


title: A Geometric View of Signal Sensitivity Metrics in multi-echo fMRI

abstract: In multi-echo fMRI (ME-fMRI), two metrics have been widely used to measure the performance of various acquisition and analysis approaches. These are temporal SNR (tSNR) and differential contrast-to-noise ratio (dCNR). A key step in ME-fMRI is the weighted combination of the data from multiple echoes, and prior work has examined the dependence of tSNR and dCNR on the choice of weights. However, most studies have focused on only one of these two metrics, and the relationship between the two metrics has not been examined. In this work, we present a geometric view that offers greater insight into the relation between the two metrics and their weight dependence. We identify three major regimes: (1) a tSNR robust regime in which tSNR is robust to the weight selection with most weight variants achieving close to optimal performance, whereas dCNR shows a pronounced dependence on the weights with most variants achieving suboptimal performance; (2) a dCNR robust regime in which dCNR is robust to the weight selection with most weight variants achieving close to optimal performance, while tSNR exhibits a strong dependence on the weights with most variants achieving significantly lower than optimal performance; and (3) a within-type robust regime in which both tSNR and dCNR achieve nearly optimal performance when the form of the weights are variants of their respective optimal weights and exhibit a moderate decrease in performance for other weight variants. Insight into the behavior observed in the different regimes is gained by considering spherical representations of the weight dependence of the components used to form each metric.


title: Organotopic organisation of the cervical vagus nerve

abstract:
The functional anatomy of fascicles evident in human and large mammal cervical vagus nerve is unknown. Organ-specific organization in the pig cervical nerve has been observed for cardiac, recurrent laryngeal and pulmonary function using anatomical tracing with X-ray microCT, and functional connectivity with selective stimulation or fast neural Electrical Impedance Tomography (EIT) with a custom nerve cuff. Electrical stimulation of the vagus nerve (VNS) is currently used to treat drug-resistant epilepsy and depression but current practice of stimulation of the entire nerve causes undesired side effects. These findings pave the way for improved outcomes in VNS as unwanted side effects could be reduced by targeted selective stimulation of identified organspecific fascicles.

One-Sentence SummaryParasympathetic innervation to the heart, lungs and larynx is organized in an organ-specific way in the cervical vagus nerve.


title: Organotopic organization of the cervical vagus nerve


title: The Development, but not Expression, of Alcohol Front-loading in C57BL/6J Mice Maintained on LabDiet 5001 is Abolished by Maintenance on Teklad 2920x Rodent Diet

abstract: Excessive alcohol (ethanol) consumption, such as binge-drinking, is extremely commonplace and represents a major health concern. Through modeling excessive drinking in rodents, we are beginning to uncover the neurobiological and neurobehavioral causes and consequences of this pattern of ethanol intake. One important factor for modeling binge drinking in mice is that subjects reliably drink to blood ethanol concentrations (BECs) of 80 mg/dl or higher. Drinking-in-the-dark (DID) is a commonly used mouse model of binge drinking, and we have shown these methods reliably result in robust ethanol front-loading and binge-level BECs in C57BL/6J (B6) mice as well as other ethanol-preferring mouse strains/lines. However, establishing the DID model in a new vivarium space forced us to consider the use of rodent diet formulations we had not previously used. The current set of experiments were designed to investigate the role of two standard rodent diet formulations on binge drinking and the development of ethanol front-loading using DID. We found that BECs in animals maintained on LabDiet 5001 (LD01) were double those found in mice maintained on Teklad 2920x (TL20). Interestingly, this effect was paralleled by differences in the degree of front-loading, such that LD01-fed mice consumed approximately twice as much ethanol in the first 15 minutes of the 2-hour DID sessions compared to TL20-fed mice. Surprisingly however, mice that developed front-loading during maintenance on the LD01 diet continued to display front-loading behavior after being switched to the TL20 diet. These data emphasize the importance of choosing and reporting diet formulations when conducting voluntary drinking studies and support the need for further investigation into the mechanisms behind diet-induced differences in binge drinking, particularly front-loading.


title: An open, analysis-ready, and quality controlled resource for pediatric brain white-matter research

abstract: We created resources to facilitate research on the role of human brain microstructure in the development of mental health disorders, based on openly-available diffusion MRI (dMRI) data from the Healthy Brain Network (HBN) study. First, we curated the HBN dMRI data (N=2747) into the Brain Imaging Data Structure and preprocessed it according to best-practices, including denoising and correcting for motion effects, susceptibility-related distortions, and eddy currents. Preprocessed, analysis-ready data was made openly available. Data quality plays a key role in the analysis of dMRI, and we provide automated quality control (QC) scores for every scan, as part of the data release. To scale QC to this large dataset, we trained a neural network through the combination of a small data subset scored by experts and a larger set scored by community scientists. The network performs QC highly concordant with that of experts on a held out set (ROC-AUC = 0.947). A further analysis of the neural network demonstrates that it relies on image features with relevance to QC. Altogether, this work both delivers a resource for transdiagnostic research in brain connectivity and pediatric mental health and serves as a novel tool for automated QC of large datasets.


title: T cells use distinct topological and membrane receptor scanning strategies that individually coalesce during receptor recognition

abstract:
During immune surveillance, CD8 T cells scan the surface of antigen presenting cells using dynamic microvillar palpation and movements as well as by having their receptors pre-concentrated into patches. Here, we use real-time lattice light sheet microscopy to demonstrate the independence of microvillar and membrane receptor patch scanning. While T cell receptor (TCR) patches can distribute to microvilli, they do so stochastically and not preferentially as for other receptors such as CD62L. The distinctness of TCR patch movement from microvillar movement extends to many other receptors that form patches that also scan independently of the TCR. An exception to this is the CD8 co-receptor which largely co-migrates in patches that overlap with or are closely adjacent to those containing TCRs. Microvilli that assemble into a synapse contain various arrays of the engaged patches, notably of TCRs and the inhibitory receptor PD-1, creating a pastiche of occupancies that vary from microvillar contact to contact. In summary, this work demonstrates that localization of receptor patches within the membrane and on microvillar projections is stochastic prior to antigen detection and that such stochastic variation may play into the generation of many individually-composed receptor patch compositions at a single synapse.

Significance statementMotile T cell microvilli palpate surfaces to facilitate surface scanning in a pattern that is independent of the movement of pre-formed patches of transmembrane antigen-receptors across those microvilli; once T cell receptors engage, the microvilli act to scaffold multiple receptors within a microvillar close-contact.


title: ABIOTIC STRESS GENE 1 mediates aroma volatiles accumulation by activating MdLOX1a in apple

abstract:
Fruit aroma is an important organoleptic quality, which influences consumer preference and market competitiveness. Aroma compound synthesis pathways in plants have been widely identified of which the lipoxygenase pathway is crucial for fatty acid catabolism to form esters in apple. However, the regulatory mechanism of this pathway remains elusive. In this study, linear regression analysis and transgene verification revealed that the lipoxygenase MdLOX1a participates in ester biosynthesis. Yeast one-hybrid library screening indicated that a novel abiotic stress gene, MdASG1 (ABIOTIC STRESS GENE 1), was a positive regulator of the MdLOX1a promoter and ester production based on yeast one-hybrid and dual-luciferase assays, and correlation analysis among eight apple cultivars. Overexpression of MdASG1 in apple and tomato stimulated the lipoxygenase pathway and increased the fatty acid-derived volatile content, whereas the latter was decreased by MdASG1 silencing. Furthermore, MdASG1 overexpression enhanced the salt-stress tolerance of tomato and apple  Orin calli accompanied by a higher content of fatty acid-derived volatiles compared with that of non-stressed transgenic tomato fruit. Collectively, these findings indicate that MdASG1 activates MdLOX1a expression and participates in the lipoxygenase pathway, subsequently increasing the accumulation of aroma compounds especially under moderate salt stress treatment. The results also provide insight into the regulation of aroma production, and the potential strategy of prudent development and utilization of saline-alkali land to produce high-quality fruit, thereby reducing pressure on arable land and ensuring national food security.

One-sentence SummaryMdASG1 directly activates MdLOX1a expression to promote aroma volatiles accumulation especially under moderate salt stress.


title: Evolutionary emergence of alternative stable states in shallow lakes

abstract: Ecosystems under stress may respond abruptly and irreversibly through tipping points. Although much is explored on the mechanisms that affect tipping points and alternative stable states, little is known on how ecosystems with alternative stable states could have emerged in the first place. Here, we explore whether evolution by natural selection can lead to the emergence of ecosystems with tipping points using a toy model inspired by shallow lakes. Shallow lakes are the best-known example where a shift from a submersed macrophyte dominated state to a floating macrophyte dominated state in response to excess nutrient loading corresponds to a tipping point between alternative stable states. We model the evolution of the macrophytes living depth in the lake, we identify the conditions under which the ancestor macrophyte population diversifies, and investigate whether alternate stable states dominated by different macrophyte phenotypes occur. Given the trade-off between access to light and nutrient along the water column, we show that asymmetry in competition for light is required for diversification, while alternative stable states require an additional competitive asymmetry, a priority effect to nutrient access. We find that eco-evolutionary dynamics may produce alternative stable states, but only under a restrictive range of conditions. Our analysis suggests that competitive asymmetries along opposing resource gradients may allow alternative stable states to emerge by natural selection.


title: Database-independent analysis of probable post-translational modifications of soil proteins across ecosystems

abstract: The identification rate of measured peptide spectra to proteins barely scratches 1% in best-case scenarios. Hundreds of thousands of valuable spectra are lost as no viable match in the database is found. Here, we apply the delta m/z plot that was previously implemented in MSnbase as tool for quality control to 63 soil samples from three ecosystems with different vegetation (39 forests, 11 grasslands, and 13 shrublands) with the aim to extract probable post-translational modifications (PTM) without the need of a reference database. The validity of the approach was verified with amino acids proposed for their respective 1 Da mass interval and compared to their relative abundance in proteins. We found that the average probable PTM and most known PTMs proposed for the mass intervals are similar across ecosystems. Otherwise, 11 mass intervals changed significantly in relative abundance in the three ecosystems but only for one an annotation could be proposed. Our approach not only highlights the opportunity of the database-independent analysis in soil metaproteomics but paves the way for targeted analysis of the yet unknown PTMs.


title: The impacts of transfer learning, phylogenetic distance, and sample size on big-data bioacoustics

abstract: Vocalizations in animals, particularly birds, are critically important behaviors that influence their reproductive fitness, but automatically extracting vocalization data from existing large databases has only recently gained traction and has yet to be evaluated with respect to accuracy of different approaches. Here, we use a recently-published machine learning framework to extract syllables from six bird species ranging in their phylogenetic relatedness from 1-85 million years, comparing how phylogenetic relatedness impacts accuracy as well as the utility of applying trained models to novel species. Model performance is best on conspecifics, with accuracy progressively decreasing as phylogenetic distance increases between taxa; however, using models trained on multiple distantly related species can recover the lost accuracy. When planning big-data bioacoustics studies, care must be taken in sample design to maximize sample size and minimize human labor without sacrificing accuracy.

category: evolutionary biology


title: High coverage of single cell genomes by T7-assisted enzymatic methyl-sequencing

abstract: Conventional approaches to studying 5mC marks in single cells or samples with picogram input DNA amounts usually suffer from low genome coverage due to DNA degradation. Many methods have been developed to optimize the library construction efficiency for bisulfite-treated DNA. However, most of these approaches ignored the amplification bias of bisulfite-treated DNA, which leads to shallow genome coverage. In this study, we developed the T7-assisted enzymatic methyl-sequencing method (TEAM-seq), which adopts enzymatic conversion to minimize DNA degradation and T7 polymerase-assisted unbiased amplification. We demonstrate that TEAM-seq delivered, to the best of our knowledge, the highest reported coverage(70% for 100pg, 35% for 20pg) of single cell genomes in whole-genome 5mC sequencing.

category: genomics


title: Methods in Cognitive Pupillometry: Design, Preprocessing, and Statistical Analysis

abstract: Cognitive pupillometry is the measurement of pupil size to investigate cognitive processes such as attention, mental effort, working memory, and many others. Currently, there is no commonly agreed-upon methodology for conducting cognitive-pupillometry experiments, and approaches vary widely between research groups and even between different experiments from the same group. This lack of consensus makes it difficult to know which factors to consider when conducting a cognitive-pupillometry experiment. Here we provide a comprehensive, hands-on guide to methods in cognitive pupillometry, with a focus on trial-based experiments in which the measure of interest is the task-evoked pupil response to a stimulus. We cover all methodological aspects of cognitive pupillometry: experimental design; preprocessing of pupil-size data; and statistical techniques to deal with multiple comparisons when testing pupil-size data. In addition, we provide code and toolboxes (in Python) for preprocessing and statistical analysis, and we illustrate all aspects of the proposed workflow through an example experiment and example scripts.

category: animal behavior and cognition


title: Wheat Panache - a pangenome graph database representing presence/absence variation across 16 bread wheat genomes

abstract:
Bread wheat is one of humanitys most important staple crops, characterized by a large and complex genome with a high level of gene presence/absence variation between cultivars, hampering genomic approaches for crop improvement. With the growing global population and the increasing impact of climate change on crop yield, there is an urgent need to apply genomic approaches to accelerate wheat breeding. With recent advances in DNA sequencing technology, a growing number of high-quality reference genomes are becoming available, reflecting the genetic content of a diverse range of cultivars. However, information on the presence or absence of genomic regions has been hard to visualize and interrogate due to the size of these genomes and the lack of suitable bioinformatics tools. To address this limitation, we have produced a wheat pangenome graph maintained within an online database to facilitate interrogation and comparison of wheat cultivar genomes. The database allows users to visualize regions of the pangenome to assess presence/absence variation between bread wheat genomes.

Database URL: http://www.appliedbioinformatics.com.au/wheat_panache


title: Model-free analysis in the spectral domain of postmortem mouse brain EPSI reveals inconsistencies with model-based analyses of the free induction decay

abstract:
PurposeDysmyelinating disorders lead to abnormalities in myelin structure that produce detectable effects in an echo-planar spectroscopic imaging (EPSI) signal. To estimate the voxel-wise proportion of myelin, data are typically fit to compartmental models in the time domain. This work characterizes limitations in these models by comparing high-resolution water spectra measured in postmortem fixed mouse brains to spectra predicted from time-domain models fit to the same data, specifically by comparing spectra from control and shiverer mice, a model for dysmyelination.

MethodsPerfusion-fixed, resected control (n = 5) and shiverer (n = 4) mouse brains were imaged using 3D EPSI with 100 {micro}m isotropic resolution. The free induction decay (FID) was sampled every 2.74 ms over 192 echoes and Fourier transformed to produce water spectra with 1.9 Hz resolution. FIDs were also fit to two biophysical models and the resulting fits were converted to spectra with a Fourier transform. Spectral asymmetry was computed and compared before and after fitting the data to models.

ResultsSpectra derived from both models did not show the magnitude of asymmetric broadening observed in the raw data. Correlations between data- and model-derived asymmetries and estimated frequency shifts are weak, leading to a reduction in spectral sensitivity to changes in white-matter structure after fitting the data to models.

ConclusionThe results demonstrate spectral inconsistencies between biophysical model predictions and measured data, promoting the further incorporation of spectral analysis methods to develop and benchmark new model-based approaches.


title: Resource requirements for ecotoxicity testing: A comparison of traditional and new approach methods

abstract: Toxicity testing is under transformation as it aims to harness the potential of New Approach Methods (NAMs) as alternative test methods that may be less resource intensive (i.e., fewer animals, cheaper costs, quicker assays) than traditional approaches while also providing more data and information. While many stakeholders are of the opinion that this unfolding transformation holds significant promise as a more efficient and ethical way forward, few studies have compared the resources required for NAMs versus those needed for traditional animal-based toxicity tests, particularly in the field of ecotoxicology. The objective was to compare resources needed for traditional animal-based ecotoxicity tests versus alternative tests using emergent NAMs. From a bibliometric review, we estimate that traditional tests for a single chemical cost $118,000 USD, require 135 animals, and take 8 weeks. In comparison, alternative tests cost $2,600, require 20 animals (or none), and take up to 4 weeks to test 16 (to potentially hundreds of) chemicals. Based on our analysis we conclude that NAMs in ecotoxicology can be more advantageous than traditional methods in terms of resources required (i.e., monetary costs, number of animals needed, and testing times). We note, however, that the evidence underpinning these conclusions is relatively sparse. Moving forward, groups developing and applying NAMs should provide more detailed accounts of the resources required. In addition, there is also a need for carefully designed case studies that demonstrate the domain of applicability of NAMs (and make comparisons to traditional tests) to ultimately build confidence among the user community.

category: pharmacology and toxicology


title: Association Mapping Across a Multitude of Traits Collected in Diverse Environments Identifies Pleiotropic Loci in Maize.

abstract: Classical genetic studies have identified many cases of pleiotropy where mutations in individual genes alter many different phenotypes. Quantitative genetic studies of natural genetic variants frequently examine one or a few traits, limiting their potential to identify pleiotropic effects of natural genetic variants. Widely adopted community association panels have been employed by plant genetics communities to study the genetic basis of naturally occurring phenotypic variation in a wide range of traits. High-density genetic marker data - 18M markers - from two partially overlapping maize association panels comprising 1,014 unique genotypes grown in field trials across at least seven US states and scored for 162 distinct trait datasets enabled the identification of of 2,154 suggestive marker-trait associations and 697 confident associations in the maize genome using a resampling-based genome-wide association strategy. The precision of individual marker-trait associations was estimated to be three genes based a reference set of genes with known phenotypes. Examples were observed of both genetic loci associated with variation in diverse traits (e.g. above-ground and below-ground traits), as well as individual loci associated with the same or similar traits across diverse environments. Many significant signals are located near genes whose functions were previously entirely unknown or estimated purely via functional data on homologs. This study demonstrates the potential of mining community association panel data using new higher density genetic marker sets combined with resampling-based genome-wide association tests to develop testable hypotheses about gene functions, identify potential pleiotropic effects of natural genetic variants, and study genotype by environment interaction.

category: plant biology


title: The estrous cycle modulates early-life adversity effects on mouse avoidance behavior through progesterone signaling

abstract: Early-life adversity (ELA) predisposes individuals to develop neuropsychiatric conditions, which are more prevalent in women than men. Efforts to model this sex difference in rodents have produced mixed results, with some studies paradoxically showing stronger phenotypes in males than females. Since changes in reproductive hormone levels can increase the likelihood of anxiety disorders in women, we examined the effects of ELA on adult female mice across the estrous cycle. We found that during diestrus, when the ratio of progesterone to estrogen is relatively high, ELA mice exhibit increased avoidance behavior, altered activity levels in specific contexts, and increased theta oscillation power in the ventral hippocampus. Ovariectomy, which eliminates circulating estrogen but not progesterone, unexpectedly preserved some of the effects present in diestrus ELA mice. Progesterone receptor antagonism in diestrus normalized avoidance behavior in ELA mice, while treatment with a negative allosteric modulator of the progesterone metabolite allopregnanolone promoted avoidance behavior in control mice. These results suggest that altered progesterone and allopregnanolone signaling during diestrus increases avoidance behavior in ELA mice


title: Single-deletion-mutant, third-generation rabies viral vectors allow nontoxic retrograde targeting of projection neurons with greatly increased efficiency

abstract: Rabies viral vectors have become important components of the systems neuroscience toolkit, allowing both direct retrograde targeting of projection neurons and monosynaptic tracing of inputs to defined postsynaptic populations, but the rapid cytotoxicity of first-generation ({Delta}G) vectors limits their use to short-term experiments. We recently introduced second-generation, double-deletion-mutant ({Delta}GL) rabies viral vectors, showing that they efficiently retrogradely infect projection neurons and express recombinases effectively but with little to no detectable toxicity; more recently, we have shown that {Delta}GL viruses can be used for monosynaptic tracing with far lower cytotoxicity than the first-generation system. Here we introduce third-generation ({Delta}L) rabies viral vectors, which, like first-generation vectors, have only a single gene deleted from their genomes (in this case the viral polymerase gene L) but which appear to be as nontoxic as second-generation ones: using longitudinal structural and functional two-photon imaging in mouse visual cortex in vivo, we found that they did not kill labeled neurons or noticeably perturb their response properties over the entire months-long courses of imaging. Although third-generation vectors are therefore phenotypically very similar to second-generation ones, we show that they have the major advantage of growing to much higher titers, and this key difference results in 25% - 525% increased numbers of retrogradely labeled neurons in vivo. These {Delta}L rabies viral vectors therefore constitute a new state of the art for minimally perturbative, pathway-specific expression of recombinases and transactivators in mammalian neurons selected on the basis of their axonal projections. Because replication of deletion-mutant rabies viruses within complementing cells is precisely the process that underlies monosynaptic tracing, the higher replication efficiency of this new class of rabies viral vectors furthermore suggests the potential to provide the foundation of an improved nontoxic monosynaptic tracing system.


title: The circ_0002538/miR-138-5p/PLLP axis regulates Schwann cell migration and myelination in diabetic peripheral neuropathy

abstract: Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes, but the underlying molecular pathogenesis remains unclear. Accumulating evidence indicates that circular RNAs (circRNAs) play vital roles in DPN, while their expressions and functions in Schwann cell (SC) are rarely reported. Here, we performed protein profiling and circRNA sequencing on the peripheral nerves of patients with or without DPN. A total of 265 differentially expressed proteins were identified in DPN by protein profiling, which mainly enrich in myelination according to Gene Ontology analysis. Further, 15637 circRNAs were identified by circRNA sequencing, of which 11 were verified to be dysregulated. Among them, circ_0002538 was found to be downregulated in DPN and chosen for further investigation. Functional experiments revealed that circ_0002538 overexpression promoted SC migration and relieved demyelination in DPN. Mechanistic studies revealed that circ_0002538 could promote PLLP expression by sponging miR-138-5p, while lack of circ_0002538 led to PLLP deficiency, which further suppressed SC migration and caused demyelination. The present research provided novel insights into the pathogenesis of DPN, in which the circ_0002538/miR-138-5p/PLLP axis was suppressed in SCs and hence caused demyelination. These findings expanded the role of circRNAs in DPN and provided potential therapeutic targets for DPN.


title: Automated Analysis Pipeline for Extracting Saccade, Pupil, and Blink Parameters Using Video-Based Eye Tracking

abstract: The tremendous increase of video-based eye tracking has made it possible to collect data from thousands of participants. Traditional manual detection and classification procedures for saccades and trial categorization is not viable for the large data sets being collected. Additionally, high-speed video-based eye trackers now allow for the novel analysis of pupil responses and blink behavior. Here we present a detailed description of our pipeline for collecting data, storing data, organizing participant codes, and cleaning data, which are fairly lab-specific but nonetheless important precursory steps in establishing standardized pipelines. More importantly, we also include descriptions of the automated detection and classification of saccades, blinks,  blincades (blinks occurring during saccades), and boomerang saccades (two saccades in opposite directions that occur almost simultaneously so that speed-based algorithms fail to split them), which are almost entirely task-agnostic and can be used on a wide variety of data. We additionally describe novel findings about post-saccadic oscillations, and provide a method to get more accurate estimates for end-points of saccades. Lastly, we describe the automated behavior classification for the Interleaved Pro- and Anti-Saccade Task (IPAST), a well-known task that probes voluntary and inhibitory control. This pipeline was evaluated using data collected from 592 human participants between 5 and 93 years of age, making it robust enough to handle large clinical patient data sets as well. In sum, this pipeline has been optimized to consistently handle large data sets obtained from diverse study cohorts (i.e., development, aging, clinical), and collected across multiple laboratory sites.


title: Identification of HIV-Reservoir Cells with Reduced Susceptibility to Antibody-Dependent Immune Response

abstract:
HIV establishes a persistent infection in heterogeneous cell reservoirs, which can be maintained by different mechanisms including cellular proliferation, and represent the main obstacle to curing the infection. The expression of the Fc{gamma} receptor CD32 has been identified as a marker of the active cell reservoirs in people on antiretroviral therapy, but if its expression has any role in conferring advantage for viral persistence is unknown. Here, we report that HIV-infected cells expressing CD32 have reduced susceptibility to natural killer (NK) antibody-dependent cell cytotoxicity (ADCC) by a mechanism compatible with the suboptimal binding of HIV-specific antibodies. Infected CD32 cells have increased proliferative capacity in the presence of immune complexes, and are more resistant to strategies directed to potentiate NK function. Remarkably, reactivation of the latent reservoir from antiretroviral-treated people living with HIV increases the pool of infected CD32 cells, which are largely resistant to the ADCC immune mechanism. Thus, we report the existence of reservoir cells that evade part of the NK immune response through the expression of CD32.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: Variation in supplemental carbon dioxide requirements defines lineage-specific antibiotic resistance acquisition in Neisseria gonorrhoeae

abstract: The evolution of the obligate human pathogen Neisseria gonorrhoeae has been shaped by selective pressures from diverse host niche environments1,2 as well as antibiotics3,4. The varying prevalence of antibiotic resistance across N. gonorrhoeae lineages5 suggests that underlying metabolic differences may influence the likelihood of acquisition of specific resistance mutations6,7. We hypothesized that the requirement for supplemental CO2, present in approximately half of isolates8, reflects one such example of metabolic variation. Here, using a genome-wide association study and experimental investigations, we show that CO2-dependence is attributable to a single substitution in a {beta}-carbonic anhydrase, canB. CanB19E is necessary and sufficient for growth in the absence of CO2, and the hypomorphic CanB19G variant confers CO2-dependence. Furthermore, ciprofloxacin resistance is correlated with CanB19G in clinical isolates, and the presence of CanB19G increases the likelihood of acquisition of ciprofloxacin resistance. Together, our results suggest that metabolic variation has impacted the acquisition of fluoroquinolone resistance.

category: microbiology


title: Highly divergent white-tailed deer SARS-CoV-2 with potential deer-to-human transmission

abstract: Wildlife reservoirs of SARS-CoV-2 can lead to viral adaptation and spillback from wildlife to humans (Oude Munnink et al., 2021). In North America, there is evidence of spillover of SARS-CoV-2 from humans to white-tailed deer (Odocoileus virginianus), but no evidence of transmission from deer to humans (Hale et al., 2021; Kotwa et al., 2022; Kuchipudi et al., 2021). Through a multidisciplinary research collaboration for SARS-CoV-2 surveillance in Canadian wildlife, we identified a new and highly divergent lineage of SARS-CoV-2. This lineage has 76 consensus mutations including 37 previously associated with non-human animal hosts, 23 of which were not previously reported in deer. There were also mutational signatures of host adaptation under neutral selection. Phylogenetic analysis revealed an epidemiologically linked human case from the same geographic region and sampling period. Together, our findings represent the first evidence of a highly divergent lineage of SARS-CoV-2 in white-tailed deer and of deer-to-human transmission.


title: Stable iPSC-derived NKX2-1+ Lung Bud Tip Progenitor Organoids Give Rise to Airway and Alveolar Cell Types

abstract:
Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. The current work aimed to develop an iPSC organoid model enriched with stable NKX2-1+ BTP-like cells. Building on prior work, we optimized a directed differentiation paradigm to generate spheroids with robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs can be purified and expanded as induced bud tip organoids (iBTO), which maintain an enriched population of bud tip progenitors. When iBTOs are directed to differentiate into airway or alveolar cell types using well-established methods, they give rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model to study human lung development and differentiation.

SUMMARY STATEMENTiPSC-derived lung bud tip progenitors emerge in organoid culture, can be isolated and expanded, are transcriptionally similar to primary bud tip progenitors, and can differentiate into airway or alveolar organoids.

category: developmental biology


title: Scrub encroachment promotes biodiversity in wetland restoration under eutrophic conditions

abstract:
Wetlands are important habitats, often threatened by drainage, eutrophication and suppression of ungulate grazing. In many countries, considerable resources are spent combatting scrub encroachment. Here, we hypothesize that encroachment may benefit biodiversity - especially under eutrophic conditions where asymmetric competition among plants compromises conservation targets.

We studied the effects of scrub cover, nutrient levels and soil moisture on richness of vascular plants, bryophytes, soil fungi and microbes in open and overgrown wetlands. We also tested the effect of encroachment, eutrophication and soil moisture on indicators of conservation value (red-listed species, indicator species and uniqueness).

Plant and bryophyte species richness peaked at low soil fertility, whereas soil fertility promoted soil microbes. Soil fungi responded negatively to increasing soil moisture. Lidar-derived variables reflecting degree of scrub cover had predominantly positive effects on species richness measures.

Conservation value indicators had a negative relationship to soil fertility and a positive to encroachment. For plant indicator species, the negative effect of high nutrient levels was offset by encroachment, supporting our hypothesis of competitive release under shade. The positive effect of soil moisture on indicator species was strong in open habitats only.

Nutrient poor mires and meadows host many rare species and require conservation management by grazing and natural hydrology. On former arable lands, where restoration of infertile conditions is unfeasible, we recommend rewilding with opportunities for encroachment towards semi-open willow scrub and swamp forest, with the prospect of high species richness in bryophytes, fungi and soil microbes and competitive release in the herb layer.


title: Bimodal breeding phenology in the parsley frog Pelodytes punctatus as a bet-hedging strategy in an unpredictable environment despite strong priority effects

abstract: When environmental conditions are unpredictable, expressing alternative phenotypes spreads the risk of failure, a mixed strategy called bet-hedging. In the southern part of its range, the Parsley frog Pelodytes punctatus breeds from autumn to spring. Our aim was to study the breeding phenology and reproductive success associated with the use of those two seasonal niches to understand how this breeding strategy can be maintained. Field surveys revealed that breeding phenology was typically bimodal with a higher breeding effort in autumn. More importantly, in spring, the survival rate of offspring was severely reduced by the presence of autumn tadpoles, indicating a clear priority effect. However, the autumn cohort often failed to survive over winter, in which case spring cohorts were often successful. Based on those results, we constructed a model in which females can allocate a variable portion of eggs to each season and added a priority effect. We conclude that the existence of the two breeding seasons may indeed constitute a bet-hedging strategy.


title: Caecilian genomes reveal molecular basis of adaptation and convergent evolution of limblessness in snakes and caecilians.

abstract: We present genome sequences for Geotrypetes seraphini (3.8Gb) and Microcaecilia unicolor (4.7Gb) caecilians, a limbless, mostly soil-dwelling amphibian clade with reduced eyes, and unique putatively chemosensory tentacles. We identify signatures of positive selection unique to caecilians in 1,150 orthogroups, with enrichment of functions for olfaction and detection of chemical signals. All our caecilian genomes are missing the ZRS enhancer of Sonic Hedgehog, shown by in vivo deletions to be required for limb development in mice and also absent in snakes, thus revealing a shared molecular target implicated in the independent evolution of limblessness in snakes and caecilians.


title: Human HspB1, HspB3, HspB5 and HspB8: Shaping these Disease Factors during Vertebrate Evolution

abstract: Small heat shock proteins (sHSPs) emerged early in evolution and occur in all domains of life and nearly in all species, including humans. Mutations in four sHSPs (HspB1, HspB3, HspB5, HspB8) are associated with neuromuscular disorders. The aim of this study is to investigate the evolutionary forces shaping these sHSPs during vertebrate evolution. We performed comparative evolutionary analyses on a set of orthologous sHSP sequences, based on the ratio of non-synonymous: synonymous substitution rates for each codon. We found that these sHSPs had been historically exposed to different degrees of purifying selection, decreasing in this order: HspB8 > HspB1, HspB5 > HspB3. Within each sHSP, regions with different degrees of purifying selection can be discerned, resulting in characteristic selective pressure profiles. The conserved -crystallin domains were exposed to the most stringent purifying selection compared to the flanking regions, supporting a  dimorphic pattern of evolution. Thus, during vertebrate evolution the different sequence partitions were exposed to different and measurable degrees of selective pressures. Among the disease-associated mutations, most are missense mutations primarily in HspB1 and to a minor extent in the other sHSPs. Our data provide an explanation for this disparate incidence. Contrary to the expectation, most missense mutations cause dominant disease phenotypes. Theoretical considerations support a connection between the historic exposure of these sHSP genes to a high degree of purifying selection and the unusual prevalence of genetic dominance of the associated disease phenotypes. Our study puts the genetics of inheritable sHSP-borne diseases into the context of vertebrate evolution.


title: Novel mtDNA Imparts the Connective Tissue Disorder of a Tourette Pedigree

abstract: Mitochondrial dysfunction is associated with a range of clinical manifestations including neuropsychiatric and metabolic disorder. Here, we reanalyzed a family with an L-Histidine Decarboxylase (HDC) variant previously linked to Tourette syndrome but with associated connective tissue and metabolic features of unknown etiology. We identified a mitochondrial haplogroup J-defining mutation on the haplogroup H background that functionally interacts with the L-Histidine Decarboxylase variant via calcium homeostasis. Our findings establish how a common mtDNA variant on a different mtDNA background can result in mitochondrial dysfunction, demonstrate a role for histaminergic signaling in modifying mitochondrial phenotypes, and link mitochondria dysfunction to connective tissue phenotypes.

category: genetics


title: Quality assessment and refinement of chromatin accessibility data using a sequence-based predictive model

abstract: Chromatin accessibility assays are central to the genome-wide identification of gene regulatory elements associated with transcriptional regulation. However, the data have highly variable quality arising from several biological and technical factors. To surmount this problem, we use the predictability of open-chromatin peaks from DNA sequence-based machine-learning models to evaluate and refine chromatin accessibility data. Our framework, gapped k-mer SVM quality check (gkmQC), provides the quality metrics for a sample based on the prediction accuracy of the trained models. We tested 886 samples with DNase-seq from the ENCODE/Roadmap projects to demonstrate that gkmQC can effectively identify high-quality samples underperforming owing to marginal read depths. Peaks identified in high-quality samples by gkmQC are more accurately aligned at functional regulatory elements, show greater enrichment of regulatory elements harboring functional variants from genome-wide association studies (GWAS), and explain greater heritability of phenotypes from their relevant tissues. Moreover, gkmQC can optimize the peak-calling threshold to identify additional peaks, especially for single-cell chromatin accessibility data as well as bulk data. Here we provide a standalone open-source toolkit (https://github.com/Dongwon-Lee/gkmQC) for such analyses and share improved regulatory maps using gkmQC. These resources will contribute to the functional interpretation of disease-associated regulatory genetic variation.


title: Deficiency of the RNA-binding protein Cth2 extends yeast replicative lifespan by alleviating its repressive effects on mitochondrial function

abstract: Iron dyshomeostasis contributes to aging, but little information is available about the molecular mechanisms. Here, we provide evidence that, in Saccharomyces cerevisiae, aging is associated with altered expression of genes involved in iron homeostasis. We further demonstrate that defects in the conserved mRNA-binding protein Cth2, which controls stability and translation of mRNAs encoding iron-containing proteins, increase lifespan by alleviating its repressive effects on mitochondrial function. Mutation of the conserved cysteine residue in Cth2 that inhibits its RNA-binding activity is sufficient to confer longevity, whereas Cth2 gain-of-function shortens replicative lifespan. Consistent with its function in RNA degradation, we demonstrate that Cth2 deficiency relieves Cth2-mediated post-transcriptional repression of nuclear-encoded components of the electron transport chain. Our findings uncover a major role of the RNA-binding protein Cth2 in the regulation of lifespan and suggest that modulation of iron starvation signaling can serve as a target for potential aging interventions.


title: Towards a New Model for the TREX1 Exonuclease

abstract: The 3 [->] 5 exonuclease, TREX1, functions in vivo to degrade DNA and prevent chronic immune activation by the cGAS-STING pathway. Current literature describes TREX1 as a nonprocessive enzyme with little preference for ssDNA versus dsDNA, and several reports propose sources of ssDNA as TREX1s principle in vivo substrate. In addition, there exist minimal published data rationalizing TREX1s obligate homodimer structure. We used a combination of biochemical assays, molecular dynamics simulations, and robust kinetic modeling to determine TREX1s relative affinities for ss- versus dsDNA substrates, to quantify its processivity, and to interrogate the possibility of inter-protomer communication in the TREX1 homodimer. Our findings demonstrate that TREX1 is a semi-processive exonuclease with significantly greater affinity for dsDNA than ssDNA, predicted at several orders of magnitude. Furthermore, we find extensively correlated dynamics between TREX1s protomers that involve previously described as well as unstudied substrate-adjacent regions of the TREX1 enzyme. We infer from these and other findings that TREX1 has evolved as a semi-processive exonuclease to degrade a dsDNA species in vivo, and we propose that its dimeric structure facilitates a mechanism for efficient binding and catabolism of nicked dsDNA. Overall, these studies identify potentially critical regions of the TREX1 enzyme hitherto unstudied, and they provide compelling evidence that the prevailing model of TREX1 exonuclease activity should be critically re-evaluated.

category: biochemistry


title: A new paradigm of multiheme cytochrome evolution by grafting and pruning protein modules

abstract: Multiheme cytochromes play key roles in diverse biogeochemical cycles, but understanding the origin and evolution of these proteins is a challenge due to their ancient origin and complex structure. Up until now, the evolution of multiheme cytochromes composed by multiple redox modules in a single polypeptide chain was proposed to occur by gene fusion events. In this context, the pentaheme nitrite reductase NrfA and the tetraheme cytochrome c554 were previously proposed to be at the origin of the extant octa- and nonaheme cytochromes c involved in metabolic pathways that contribute to the nitrogen, sulfur and iron biogeochemical cycles by a gene fusion event. Here, we combine structural and character-based phylogenetic analysis with an unbiased root placement method to refine the evolutionary relationships between these multiheme cytochromes. The evidence show that NrfA and cytochrome c554 belong to different clades, which suggests that these two multiheme cytochromes are products of truncation of ancestral octaheme cytochromes related to extant ONR and MccA, respectively. From our phylogenetic analysis, the last common ancestor is predicted to be an octaheme cytochrome with nitrite reduction ability. Evolution from this octaheme framework led to the great diversity of extant multiheme cytochromes analised here by pruning and grafting of protein modules and hemes. By shedding light into the evolution of multiheme cytochromes that intervene in different biogeochemical cycles, this work contributes to our understanding about the interplay between biology and geochemistry across large time scales in the history of Earth.


title: Structure of IgG-Fc hexamer reveals a mutual lock-and-key mode of Fc-Fc interaction

abstract: IgG exists mainly as monomer, but recent studies suggest that IgG forms hexamer to mediate antibody functions. Although many structures of IgG monomer and its fragments are determined, there is no IgG hexamer structure at atomic level due to the weak Fc-Fc interactions. Here we engineered a hexameric IgG with IgM tailpiece fusion and determined the structure by cryo-EM. IgG-Fc hexamer forms hexagon symmetry with the six Fcs lie in a plane with Fc-Fc interaction in a mutual lock-and-key mode. This structure provides structural insights into Fc-Fc interaction of IgG and reveals molecular basis for its function.


title: DLGAP1-AS2-Mediated Phosphatidic Acid Synthesis Confers Chemoresistance via Activation of YAP Signaling

abstract:
Squamous cell carcinomas (SCCs) constitute a group of human malignancies that originate from the squamous epithelium. Most SCC patients experience treatment failure and relapse and have a poor prognosis due to de novo and acquired resistance to first-line chemotherapeutic agents. To identify chemoresistance mechanisms and explore novel chemosensitizer targets, we performed whole-transcriptome sequencing of paired resistant/parental SCC cells. We identified DLGAP1 antisense RNA 2 (D-AS2) as a crucial noncoding RNA that contributes to chemoresistance in SCC. Mechanistically, D-AS2 associates with histones to regulate the distal elements of FAM3 metabolism regulating signaling molecule D (FAM3D) and reduces extracellular FAM3D protein secretion. FAM3D interacts with Gi-coupled G protein-coupled receptor (GPCR) formyl peptide receptor (FPR) 1 and FPR2 to suppress phospholipase D (PLD) activity; thus, reduced FAM3D activates PLD signaling. Moreover, activated PLD promotes phosphatidic acid (PA) production and subsequent yes-associated protein (YAP) nuclear translocation. Accordingly, in vivo administration of a D-AS2-targeting antisense oligonucleotide sensitizes SCC to cisplatin treatment. In summary, our study reveals that D-AS2/FAM3D-mediated PLD/PA lipid signaling is essential in SCC chemoresistance and that D-AS2 can be targeted to sensitize SCC to cytotoxic chemotherapeutic agents.

SignificanceThis study identifies D-AS2 as a targetable lipid-related lncRNA that activates YAP signaling via PLD/PA axis to trigger chemoresistance in SCC.


title: Construction of a constitutively active type III secretion system for heterologous protein secretion

abstract:
Proteins comprise a multibillion-dollar industry in enzymes and therapeutics, but bacterial protein production can be costly and inefficient. Proteins of interest (POIs) must be extracted from lysed cells, purified, and resolubilized. The Salmonella pathogenicity island 1 type III secretion system (T3SS) is a protein secretion complex in Salmonella that has been engineered to secrete heterologous proteins and addresses the problems associated with bacterial protein production. However, the current best practices method of T3SS pathway activation for secretion is not ideal for industrial scaleup. Previously, the T3SS was activated by plasmid-based overexpression of the T3SS transcriptional regulator, hilA, which requires the addition of a small molecule inducer to the culture media and adds significant cost to the production media. Plasmid-based expression is also subject to instability in large-scale fermentation. Here, we show that we can constitutively activate the T3SS by modulating the upstream transcriptional regulator, hilD, either through knocking out hilE, a repressor of HilD, or by adding transcriptional fusions to hilD. Finally, we combine the two most promising genomic modifications to build a constitutively active T3SS capable of secreting a range of heterologous proteins at titers comparable to those reported with synthetic induction of hilA. These improvements further our goal of making an industrially competitive protein production strain that reduces the challenges associated with plasmid induction and maintenance.

ImportanceProteins are used in our everyday lives as therapeutics (insulin), industrial enzymes (laundry detergent), and bio-based materials (spider silk). Current industrial protein production in bacteria is costly because it requires purification of the target protein from the other proteins inside the cell. We solve this problem by engineering the Salmonella pathogenicity island 1 type III secretion system (T3SS) to export the target protein into the cell growth media. This makes the protein purification process more efficient and cheaper. However, this system currently requires an expensive inducer reagent to activate it which significantly increases the cost of the production media. We show here the creation of a constitutively active T3SS, meaning the T3SS pathway is always on. In doing so, we successfully created a Salmonella strain that eliminates the need for the inducer reagent and exports proteins at levels comparable to the inducer-activated system, ultimately reducing the cost of T3SS protein production.

category: synthetic biology


title: Generation of PRKN and PINK1-KO and double KO cell lines from healthy induced pluripotent stem cells using CRISPR/Cas9 editing

abstract: Autosomal recessive mutations in either PRKN or PINK1 are associated with early-onset Parkinsons disease. The corresponding proteins, PRKN, an E3 ubiquitin ligase, and the mitochondrial serine/threonine-protein kinase PINK1 play a role in mitochondrial quality control. Using CRISPR/CAS9 technology we generated three human iPSC lines from the well characterized AIW002-02 control line. These isogenic iPSCs contain homozygous knockouts of PRKN (PRKN-KO, CBIGi001-A-1), PINK1 (PINK1-KO, CBIGi001-A-2) or both PINK1 and PRKN (PINK1-KO/PRKN-KO, CBIGi001-A-3). The knockout lines display normal karyotypes, express pluripotency markers and upon differentiation into relevant brain cells or midbrain organoids may be valuable tools to model Parkinsons disease.

category: neuroscience


title: T Cell Deficiency Precipitates Antibody Evasion and Emergence of Neurovirulent Polyomavirus

abstract: JC polyomavirus (JCPyV) causes Progressive Multifocal Leukoencephalopathy (PML), a life-threatening brain disease in T cell immunosuppressed patients. PML patients often carry mutations in the JCPyV VP1 capsid protein. These mutations confer resistance to neutralizing VP1 antibodies (Ab). We found that T cell insufficiency during persistent infection, in the setting of monospecific VP1 Ab, was required for outgrowth of VP1 Ab-escape viral variants. CD4 T cells were primarily responsible for preventing resurgent virus infection in the kidney and checking emergence of these mutant viruses. T cells also provided a second line of defense against Ab-escape VP1 mutant viruses. A virus with two capsid mutations, one conferring Ab-escape yet impaired infectivity and a second compensatory mutation, yielded a highly neurovirulent variant. These findings link T cell deficiency and evolution of Ab-escape polyomavirus VP1 variants with neuropathogenicity.

category: immunology


title: Microbial Community Dynamics Provide Evidence for Hypoxia During a Coral Reef Mortality Event

abstract:
In July 2016, a severe coral reef invertebrate mortality event occurred approximately 200km southeast of Galveston, Texas at the East Flower Garden Bank wherein upwards of 80% of corals in a 0.06 km2 area died. Based on surveys of dead corals and other invertebrates shortly after this mortality event, responders hypothesized that localized hypoxia was the most likely direct cause. However, no dissolved oxygen data were available to test this hypothesis because oxygen is not continuously monitored within the Flower Garden Banks sanctuary. Here we quantify microbial plankton community diversity based on four cruises over two years at the Flower Garden Banks, including a cruise just 5-8 days after the mortality event was first observed. In contrast with observations collected during baseline conditions, microbial plankton communities in the thermocline were differentially enriched with taxa known to be active and abundant in oxygen minimum zones or that have known adaptations to oxygen limitation shortly after the mortality event (e.g. SAR324, Thioglobaceae, Nitrosopelagicus, and Thermoplasmata MGII). Unexpectedly, these enrichments were not localized to the East Bank, but were instead prevalent across the entire study area, suggesting there was a widespread depletion of dissolved oxygen concentrations in the thermocline around the time of the mortality event. Hydrographic analysis revealed the southern East Bank coral reef (where the localized mortality event occurred) was uniquely within the thermocline at this time. Our results demonstrate how temporal monitoring of microbial communities can be a useful tool to address questions related to past environmental events.

IMPORTANCEIn the northwestern Gulf of Mexico in July 2016, upwards of 80% of corals in a small area of the East Flower Garden Bank coral reef suddenly died without warning. Oxygen depletion is believed to have been the cause. However, there was considerable uncertainty as no oxygen data is available from the time of the event. Microbes are sensitive to changes in oxygen and can be used as bioindicators of oxygen loss. In this study, we analyze microbial communities in water samples collected over several years at the Flower Garden Banks, including shortly after the mortality event. Our findings indicate that compared to normal conditions, oxygen depletion was widespread in the deep-water layer during the mortality event. Hydrographic analysis of water masses further revealed some of this low oxygen water likely upwelled onto the coral reef.


title: Congenital Rift Valley fever in Sprague Dawley rats is associated with diffuse infection and pathology of the placenta

abstract: Rift Valley fever (RVF) is a disease of animals and humans associated with abortions in ruminants and, more recently, late-gestation miscarriages in African women. Here, we use a rat model of congenital RVF to identify cellular tropism, pathology, and local immune responses occurring in the placenta during vertical transmission. Infection of pregnant rats during late gestation (embryonic day 14) resulted in vertical transmission to the placenta and widespread viral infection throughout the decidua, basal zone, and labyrinth zone. Some pups delivered from infected dams appeared normal while others had gross signs of teratogenicity including fetal death. Histopathological lesions were detected in placenta from pups regardless of teratogenicity, while teratogenic pups had more widespread hemorrhage throughout multiple placenta layers. Teratogenic events were also associated with significant increases in pro-inflammatory cytokines, type I interferons, and chemokines within the placental tissue. Thus, we found that RVFV displays a high degree of tropism for all placental tissue layers, and that the degree of hemorrhage and inflammatory mediator production is highest in placenta from pups with adverse outcomes. This is the first detailed pathological study of the mechanisms of RVFV infection of placental tissue in a tractable rodent model of congenital RVF disease.


title: Rif-Correct, a software tool for bacterial mRNA half-life estimation

abstract:
BackgroundGenome-wide measurement of bacterial mRNA lifetimes using the antibiotic rifampicin has provided new insights into the control of bacterial mRNA decay. However, for long polycistronic mRNAs, the estimation of mRNA half-life can be confounded with transcriptional runoff caused by rifampicins inhibition of initiating RNA polymerases, and not elongating RNA polymerases.

ResultsWe present the Rif-correct software package, a free open-source tool that uses transcriptome models of transcript architecture to allow for more accurate mRNA half-life estimates that account for the transcriptional runoff. Rif-correct is implemented as a customizable python script that allows for users to control all the analysis parameters to achieve improved mRNA half-life estimates.

ConclusionsRif-correct is the first free open-source computational analysis pipeline for Rif-seq dataset mRNA half-life estimation. It is simple to run, fast, and easy to run with a detailed instruction manual and example datasets.


title: The lipid raft-associated protein stomatin is required for accumulation of dectin-1 in the phagosomal membrane and for full activity of macrophages against Aspergillus fumigatus

abstract: Alveolar macrophages are among the first cells to come into contact with inhaled fungal conidia of the human pathogenic fungus Aspergillus fumigatus. In lung alveoli, they contribute to phagocytosis and elimination of conidia. As a counter defense, conidia contain a grey-green pigment allowing them to survive in phagosomes of macrophages for some time. Previously, we showed that this conidial pigment interferes with the formation of flotillin-dependent lipid-rafts in the phagosomal membrane thereby preventing the formation of functional phagolysosomes. In this study, the role of the lipid raft-associated protein stomatin in macrophages during antifungal defense was investigated. To determine the function of this integral membrane protein, a stomatin-deficient macrophage cell line was generated by CRISPR/Cas9 gene editing. Immunofluorescence and flow cytometry revealed that stomatin contributes to the phagocytosis of conidia and is important for recruitment of both the {beta}-glucan receptor dectin-1 and the vATPase to the membrane of phagosomes. In the stomatin knockout cell line, fusion of phagosomes and lysosomes was reduced when infected with pigmentless pksP conidia. Thus, our data suggest that stomatin is involved in maturation of phagosomes via fostering fusion of phagosomes with lysosomes.