title: TRACER: A toolkit to register and visualize anatomical coordinates in the rat brain

abstract: The need for anatomical registration and visualization tools is greater than ever thanks to novel technologies that allow users to record from thousands of neurons across multiple brain regions simultaneously. The vast majority of digital reconstruction toolkits for rodent models were developed using mouse brain atlases, leaving few options for those using rats. Retrofitting rat atlases into extant software is possible, but this demands effort and programming skills which most end-users lack. We therefore developed an open-source, python-based Toolkit for Reconstructing Anatomical CoordinatEs in Rats, TRACER, which allows users to reconstruct the trajectories of recording electrodes (e.g Neuropixels) or to visualize virus expression or other features in a comprehensively annotated 3D volume, the Waxholm Space (WHS) rat brain atlas. The WHS atlas is made from high-resolution magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) scans, and contains >200 regional delineations. Beyond post-hoc reconstructions of electrodes or virus expression, TRACER can be used to generate coordinates for targeting brain regions prior to surgery. TRACER is available openly on GitHub and is compatible with Windows, macOS and Linux operating systems.


title: BioKIT: a versatile toolkit for processing and analyzing diverse types of sequence data

abstract: Bioinformatic analysis--such as genome assembly quality assessment, alignment summary statistics, relative synonymous codon usage, paired-end aware quality trimming and filtering of sequencing reads, file format conversion, and processing and analysis--is integrated into diverse disciplines in the biological sciences. Several command-line pieces of software have been developed to conduct some of these individual analyses; however, the lack of a unified toolkit that conducts all these analyses can be a barrier in workflows. To address this obstacle, we introduce BioKIT, a versatile toolkit for the UNIX shell environment with 40 functions, several of which were community-sourced, that conduct routine and novel processing and analysis of genome assemblies, multiple sequence alignments, coding sequences, sequencing data, and more. To demonstrate the utility of BioKIT, we assessed the quality and characteristics of 901 eukaryotic genome assemblies, calculated alignment summary statistics for 10 phylogenomic data matrices, determined relative synonymous codon usage across 171 fungal genomes including those that use alternative genetic codes, and demonstrate that a novel metric, gene-wise relative synonymous codon usage, can accurately estimate gene-wise codon optimization. BioKIT will be helpful in facilitating and streamlining sequence analysis workflows. BioKIT is freely available under the MIT license from GitHub (https://github.com/JLSteenwyk/BioKIT), PyPi (https://pypi.org/project/jlsteenwykbiokit/), and the Anaconda Cloud (https://anaconda.org/jlsteenwyk/jlsteenwyk-biokit). Documentation, user tutorials, and instructions for requesting new features are available online (https://jlsteenwyk.com/BioKIT).

category: bioinformatics


title: Gamma Entrainment Improves Synchronization Deficits Caused by Dementia

abstract: Non-invasive gamma entrainment has shown promising results in alleviating cognitive symptoms of Alzheimers disease in mice and humans. In this study, we examine improvements in the synchronization characteristics of the brains oscillations induced by 40Hz auditory stimulation based on electroencephalography data recorded from a group of dementia patients. We observed that when the quality of entrainment surpasses a certain level, several indicators of brain synchronization significantly improve. Specifically, the entrained oscillatory activity maintains temporal phase stability in the frontal, parietal, and occipital regions, and persistent spatial phase coupling between them. In addition, notable theta-gamma phase-amplitude coupling is observed in these areas. Interestingly, a high theta power at rest predicts the quality of entrainment. We identify differentiating attributes of temporal/spatial synchronization and cross-frequency coupling in the data of two groups with entrained and non-entrained responses which point to enhanced network synchronization caused by entrainment and can explain its potential therapeutic effects.

category: neuroscience


title: Gamma Entrainment Improves Synchronization Deficits in Dementia Patients


title: Local brain-state dependency of effective connectivity: evidence from TMS-EEG

abstract:
BackgroundSpontaneous cortical oscillations have been shown to modulate cortical responses to transcranial magnetic stimulation (TMS). If not controlled for, they might increase variability in responses and mask meaningful changes in the signals of interest when studying the brain with TMS combined with electroencephalography (TMS-EEG). To address this challenge in future closed-loop stimulation paradigms, we need to understand how spontaneous oscillations affect TMS-evoked responses.

ObjectiveTo describe the effect of the pre-stimulus phase of cortical mu (8-13 Hz) and beta (13-30 Hz) oscillations on TMS-induced effective connectivity patterns.

MethodsWe applied TMS to the left primary motor cortex and right pre-supplementary motor area of three subjects while recording EEG. We classified trials off-line into positive- and negative-phase classes according to the mu and beta rhythms. We calculated differences in the global mean-field amplitude (GMFA) and compared the cortical spreading of the TMS-evoked activity between the two classes.

ResultsPhase had significant effects on the GMFA in 11 out of 12 datasets (3 subjects x 2 stimulation sites x 2 frequency bands). Seven of the datasets showed significant differences in the time range 15-50 ms, nine in 50-150 ms, and eight after 150 ms post-stimulus. Source estimates showed complex spatial differences between the classes in the cortical spreading of the TMS-evoked activity.

ConclusionsTMS-evoked effective connectivity appears to depend on the phase of local cortical oscillations at the stimulated site. This may be crucial for efficient design of future brain-state-dependent and closed-loop stimulation paradigms.


title: Presaccadic attention enhances contrast sensitivity, but not at the upper vertical meridian

abstract: Human visual performance is not only better at the fovea and decreases with eccentricity, but also has striking radial asymmetries around the visual field: At a fixed eccentricity, it is better along (1) the horizontal than vertical meridian and (2) the lower than upper vertical meridian. These asymmetries are not alleviated by covert exogenous or endogenous attention, but have been studied exclusively during eye fixation. However, a major driver of everyday attentional orienting is saccade preparation, during which visual attention automatically shifts to the future eye fixation. This presaccadic shift of attention is considered strong and compulsory, and relies on fundamentally different neural computations and substrates than covert attention. Given these differences, we investigated whether presaccadic attention can compensate for the ubiquitous performance asymmetries observed during eye fixation. Our data replicate polar performance asymmetries during fixation and document the same asymmetries during saccade preparation. Crucially, however, presaccadic attention enhanced contrast sensitivity at the horizontal and lower vertical meridian, but not at the upper vertical meridian. Thus, instead of attenuating polar performance asymmetries, presaccadic attention exacerbates them.


title: A mechanochemical model recapitulates distinct vertebrate gastrulation modes

abstract: Gastrulation is a critical event in vertebrate morphogenesis driven by cellular processes, and characterized by coordinated multi-cellular movements that form the robust morphological structures. How these structures emerge in a developing organism and vary across vertebrates remains unclear. Inspired by experiments on the chick, we derive a theoretical framework that couples actomyosin activity to tissue flow, and provides a basis for the dynamics of gastrulation morphologies. Our model predicts the onset and development of observed experimental patterns of wild-type and perturbations of chick gastrulation as a spontaneous instability of a uniform state. Varying the initial conditions and a parameter in our model, allows us to recapitulate the phase space of gastrulation morphologies seen across vertebrates, consistent with experimental observations in the accompanying paper. All together, this suggests that early embryonic self-organization follows from a minimal predictive theory of active mechano-sensitive flows.

category: developmental biology


title: A new Mendelian Randomization method to estimate causal effects of multivariable brain imaging exposures

abstract: The advent of simultaneously collected imaging-genetics data in large study cohorts provides an unprecedented opportunity to assess the causal effect of brain imaging traits on externally measured experimental results (e.g., cognitive tests) by treating genetic variants as instrumental variables. However, classic Mendelian Randomization methods are limited when handling high-throughput imaging traits as exposures to identify causal effects. We propose a new Mendelian Randomization framework to jointly select instrumental variables and imaging exposures, and then estimate the causal effect of multivariable imaging data on the outcome. We validate the proposed method with extensive data analyses and compare it with existing methods. We further apply our method to evaluate the causal effect of white matter microstructure integrity on cognitive function. The findings suggest that our method achieved better performance regarding sensitivity, bias, and false discovery rate compared to individually assessing the causal effect of a single exposure and jointly assessing the causal effect of multiple exposures without dimension reduction. Our application results indicated that WM measures across different tracts have a joint causal effect that significantly impacts the cognitive function among the participants from the UK Biobank.


title: Macroscopic dynamics of gene regulatory networks revealed by individual entropy

abstract: Complex systems are usually high-dimensional with intricate interactions among internal components, and may display complicated dynamics under different conditions. While it is difficult to measure detail dynamics of each component, proper macroscopic description of a complex system is crucial for quantitative studies. In biological systems, each cell is a complex system containing a huge number of molecular components that are interconnected with each other through intricate molecular interaction networks. Here, we consider gene regulatory networks in a cell, and introduce individual entropy as a macroscopic variable to quantify the transcriptional dynamics in response to changes in random perturbations and/or network structures. The proposed individual entropy measures the information entropy of a system at each instant with respect to a basal reference state, and may provide temporal dynamics to characterize switches of system states. Individual entropy provides a method to quantify the stationary macroscopic dynamics of a gene set that is dependent on the gene regulation connections, and can be served as an indicator for the evolution of network structure variation. Moreover, the individual entropy with reference to a preceding state enable us to characterize different dynamic patterns generated from varying network structures. Our results show that the proposed individual entropy can be a valuable macroscopic variable of complex systems in characterizing the transition processes from order to disorder dynamics, and to identify the critical events during the transition process.

category: systems biology


title: Detection of the 40-Hz Auditory Steady-state Response with Optically Pumped Magnetometers

abstract: Magnetoencephalography (MEG) is a functional neuroimaging technique that noninvasively detects the brain magnetic field from neuronal activations. Conventional MEG measures brain signals using superconducting quantum interference devices (SQUIDs). SQUID-MEG requires a cryogenic environment involving a bulky non-magnetic dewar and the consumption of liquid helium, which restricts the variability of the sensor array and the gap between the cortical sources and sensors. Recently, miniature optically pumped magnetometers (OPMs) have been developed and commercialized. OPMs do not require cryogenic cooling and can be placed within millimeters from the scalp. In the present study, we arranged six OPM sensors on the temporal area to detect auditory-related brain responses in a two-layer magnetically shielded room. We presented the auditory stimuli of 1-kHz pure-tone bursts with 200-ms duration and obtained the M50 and M100 components of auditory evoked fields. We delivered the periodic stimuli with a 40-Hz repetition rate and observed the gamma-band power changes and inter-trial phase coherence of auditory steady-state responses at 40 Hz. We found that the OPM sensors have a performance comparable to that of conventional SQUID-MEG sensors, and our results suggest the feasibility of using OPM sensors for functional neuroimaging and brain-computer interface applications.


title: Episodic timing: how spontaneous alpha clocks, retrospectively

abstract:
We seldom time life events intently yet recalling the duration of events is lifelike. Is episodic time the outcome of a rational after-thought or of physiological clocks keeping track of time without our conscious awareness of it? To answer this, we recorded human brain activity with magnetoencephalography (MEG) during quiet wakefulness. Unbeknownst to participants, we asked them after the MEG recording to guess its duration. In the absence of overt attention to time, the relative amount of time participants alpha brain rhythms ( ~10 Hz) were in bursting mode predicted participants retrospective duration estimate. This relation was absent when participants prospectively measured elapsed time during the MEG recording. We conclude that  bursts embody discrete states of awareness for episodic timing.

One-Sentence SummaryIn the human brain, the relative number of alpha oscillatory bursts at ~10 Hz can tell time when the observer does not attend to it.


title: Stimulating music supports attention in listeners with attentional difficulties

abstract: Background music is widely used to sustain attention, but little is known about what musical properties aid attention. This may be due to inter-individual variability in neural responses to music. We test the hypothesis that music can sustain attention by affecting oscillations via acoustic amplitude modulation, differentially for those with varying levels of attentional difficulty. We first show that heavily-modulated music improves sustained attention for participants with more ADHD symptoms. FMRI showed this music elicited greater activity in attentional networks in this group only, and EEG showed greater stimulus-brain coupling for this group in response to the heavily-modulated music. Finally, we parametrically manipulated the depth and rate of amplitude modulations inserted in otherwise-identical music, and found that beta-range modulations helped more than other frequency ranges for participants with more ADHD symptoms. Results suggest the possibility of an oscillation-based neural mechanism for targeted music to support improved cognitive performance.


title: A ROLE FOR CORTICAL DOPAMINE IN THE PARADOXICAL CALMING EFFECTS OF PSYCHOSTIMULANTS

abstract: Attention deficit hyperactivity disorder (ADHD) affects young children and manifests symptoms such as hyperactivity, impulsivity and cognitive disabilities. Psychostimulants, which are the primary treatment for ADHD, target monoamine transporters and have a paradoxical calming effect, but their mechanism of action, is unclear. Studies using the dopamine (DA) transporter (DAT) knockout mice, which have elevated striatal DA levels and are considered an animal model of ADHD, have suggested that the paradoxical calming effect of psychostimulants might be through the actions on serotonin neurotransmission. On the other hand, newer non-stimulant class of drugs such as atomoxetine and Intuniv suggest that targeting the norepinephrine (NE) system in the PFC might explain this paradoxical calming effect. We sought to decipher the mechanism of this paradoxical effect of psychostimulants through an integrated approach using ex vivo monoamine efflux experiments, monoamine transporter knockout mice, drug infusions and behavior. Our ex vivo efflux experiments reveal that NE transporter (NET) blocker desipramine elevates both norepinephrine and dopamine but not serotonin levels, in PFC tissue slices from wild-type and DAT-KO but not NET KO mice. However, serotonin (5-HT) transporter (SERT) inhibitor fluoxetine elevates only serotonin in all three genotypes. Systemic administration of both desipramine and fluoxetine but local PFC infusion of only desipramine and not fluoxetine inhibits hyperactivity in the DAT-KO mice. In contrast, pharmacological norepinephrine depletion but dopamine elevation using Nepicastat also inhibits hyperactivity in DATKO mice. Together, these data suggest that elevation of PFC dopamine and not norepinephrine or serotonin as a convergent mechanism for the paradoxical psychostimulant effects observe in ADHD therapy.


title: Distinct signatures of loss of consciousness during Focal Impaired Awareness (FIA) versus Focal to Bilateral Tonic-Clonic (FBTC) seizures

abstract:
Loss of consciousness (LOC) is a hallmark of many epileptic seizures and carries risks of serious injury and sudden death. While cortical sleep-like activities accompany LOC during focal impaired awareness (FIA) seizures, the mechanisms of LOC during focal to bilateral tonic-clonic (FBTC) seizures remain unclear. Quantifying differences in markers of cortical activation and ictal recruitment between FIA and FBTC seizures may also help to understand their different consequences for clinical outcomes and to optimize neuromodulation therapies.

We quantified clinical signs of LOC and intracranial EEG (iEEG) activity during 129 FIA and 50 FBTC from 41 patients. We characterized iEEG changes both in the seizure onset zone (SOZ) and in areas remote from SOZ with a total of 3386 electrodes distributed across brain areas. First, we compared the dynamics of iEEG sleep-like activities: slow-wave activity (SWA; 1-4 Hz) and beta/delta ratio (B/D; a validated marker of cortical activation) during FIA vs. FBTC. Second, we quantified differences between FBTC and FIA for a marker validated to detect ictal cross-frequency coupling: phase-locked high-gamma (PLHG; high gamma phased locked to low frequencies) and a marker of ictal recruitment: the epileptogenicity index (i.e. the number of channels crossing an energy ratio threshold for high vs. low frequency power). Third, we assessed changes in iEEG activity preceding and accompanying behavioral generalization onset and their correlation with electromyogram (EMG) channels. In addition, we analyzed human cortical multi-unit activity recorded with Utah arrays during three FBTC.

Compared to FIA, FBTC seizures were characterized by deeper LOC and by stronger increases in SWA in parieto-occipital cortex. FBTC also displayed more widespread increases in cortical activation (B/D), ictal cross-frequency coupling (PLHG) and ictal recruitment (epileptogenicity index). Even before generalization, FBTC displayed deeper LOC; this early LOC was accompanied by a paradoxical increase in B/D in fronto-parietal cortex. Behavioral generalization coincided with complete loss of responsiveness and a subsequent increase in high-gamma in the whole brain, which was especially synchronous in deep sources and could not be explained by EMG. Similarly, multi-unit activity analysis of FBTC revealed sustained increases in cortical firing rates during and after generalization onset in areas remote from the SOZ.

Unlike during FIA, LOC during FBTC is characterized by a paradoxical increase in cortical activation and neuronal firing. These findings suggest differences in the mechanisms of ictal LOC between FIA and FBTC and may account for the more negative prognostic consequences of FBTC.


title: Distinct signatures of loss of consciousness during Focal Impaired Awareness versus Focal to Bilateral Tonic-Clonic seizures


title: Direct brain recordings reveal continuous encoding of structure in random stimuli

abstract: The brain excels at processing sensory input, even in rich or chaotic environments. Mounting evidence attributes this to the creation of sophisticated internal models of the environment that draw on statistical structures in the unfolding sensory input. Understanding how and where this modeling takes place is a core question in statistical learning and predictive processing. In this context, we address the role of transitional probabilities as an implicit structure supporting the encoding of a random auditory stream. Leveraging information-theoretical principles and the high spatiotemporal resolution of intracranial electroencephalography, we analyzed the trial-by-trial high-frequency activity representation of transitional probabilities. This unique approach enabled us to demonstrate how the brain continuously encodes structure in random stimuli and revealed the involvement of a network outside of the auditory system, including hippocampal, frontal, and temporal regions. Linking the frame-works of statistical learning and predictive processing, our work illuminates an implicit process that can be crucial for the swift detection of patterns and unexpected events in the environment.


title: tACS competes with ongoing oscillations for control of spike-timing in the primate brain.

abstract: Transcranial alternating current stimulation (tACS) is commonly used to enhance brain rhythms, in the hopes of improving behavioral performance. Unfortunately, these interventions often yield highly variable results. Here, we identify a key source of this variability by recording from single neurons in alert non-human primates. We find that, rather than enhancing rhythmic activity, tACS appears to compete with the brains endogenous oscillations for control of spike timing. Specifically, when the strength of stimulation is weak relative to endogenous oscillations, tACS actually decreases the rhythmicity of spiking. However, when stimulation is comparatively stronger, tACS imposes its own rhythm on spiking activity. Thus the effect of tACS depends categorically on the strength of neural entrainment to endogenous oscillations, which varies greatly across behavioral states and brain regions. Without carefully considering these factors, attempts to impose external rhythms on specific brain regions may often yield precisely the opposite of the intended effect.


title: Brain stimulation competes with ongoing oscillations for control of spike timing in the primate brain


title: Role of the Topoisomerase IIα Chromatin Tether domain in Nucleosome Binding & Chromosome Segregation

abstract:
Due to the intrinsic nature of DNA replication, replicated genomes retain catenated genomic loci that must be resolved to ensure faithful segregation of sister chromatids in mitosis. Type II DNA Topoisomerase (TopoII) decatenates the catenated genomic DNA through its unique Strand Passage Reaction (SPR). Loss of SPR activity results in anaphase chromosome bridges and formation of Polo-like Kinase Interacting Checkpoint Helicase (PICH)-coated ultra-fine DNA bridges (UFBs) whose timely resolution is required to prevent micronuclei formation. Vertebrates have two TopoII isoforms- TopoII and TopoII{beta}, that share a conserved catalytic core. However, the essential mitotic function of TopoII cannot be compensated by TopoII{beta}, due to differences in their catalytically inert C-terminal domains (CTDs). Using genome-edited human cells, we show that specific binding of TopoII to methylated histone, tri-methylated H3K27 (H3K27me3), via its Chromatin Tether (ChT) domain within the CTD contributes critically to avoid anaphase UFB formation. Reducing H3K27 methylation prior to mitosis increases UFBs, revealing a requirement for proper establishment of H3K27me3 after DNA replication to facilitate TopoII-ChT dependent UFB prevention. We propose that interaction of the TopoII-ChT with H3K27me3 is a key factor that ensures the complete resolution of catenated loci to permit faithful chromosome segregation in human cells.

Summary StatementGenomic catenations originating from the DNA replication process must be resolved by DNA Topoisomerase II (TopoII) to permit sister chromatid disjunction. The results show that specific recognition of methylated histone containing chromatin by TopoII is critical for complete resolution of the genome.

category: cell biology


title: Self-association configures the NAD+-binding site of plant NLR TIR domains

abstract: TIR domains are signalling domains present in plant nucleotide-binding leucine-rich repeat receptors (NLRs), with key roles in plant innate immunity. They are required for the induction of a hypersensitive response (HR) in effector-triggered immunity, but the mechanism by which this occurs is not yet fully understood. It has been recently shown that the TIR domains from several plant NLRs possess NADase activity. The oligomeric structure of TIR-containing NLRs ROQ1 and RPP1 reveals how the TIR domains arrange into an active conformation, but low resolution around the NAD+ binding sites leaves questions unanswered about the molecular mechanisms linking self-association and NADase activity. In this study, a number of crystal structures of the TIR domain from the grapevine NLR RUN1 reveal how self-association and enzymatic activity may be linked. Structural features previously proposed to play roles involve the "AE interface" (mediated by helices A and E), the "BB-loop" (connecting {beta}-strand B and helix B in the structure), and the "BE interface" (mediated by the BB-loop from one TIR and the "DE surface" of another). We demonstrate that self-association through the AE interface induces conformational changes in the NAD+-binding site, shifting the BB-loop away from the catalytic site and allowing NAD+ to access the active site. We propose that an intact "DE surface" is necessary for production of the signalling product (variant cyclic ADPR), as it constitutes part of the active site. Addition of NAD+ or NADP+ is not sufficient to induce self-association, suggesting that NAD+ binding occurs after TIR self-association. Our study identifies a mechanistic link between TIR self-association and NADase activity.

category: biochemistry


title: A NYN domain protein directly interacts with DCP1 and is required for phyllotactic pattern in Arabidopsis.

abstract:
In eukaryotes, general mRNA decay requires the decapping complex. The activity of this complex depends on its catalytic subunit, DCP2 and its interaction with decapping enhancers, including its main partner DCP1. Here, we report that in Arabidopsis, DCP1 also interacts with a NYN domain endoribonuclease, hence named DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1). Interestingly, we find DNE1 predominantly associated with DCP1 but not with DCP2 and reciprocally, suggesting the existence of two distinct protein complexes. We also show that the catalytic residues of DNE1 are required to repress the expression of mRNAs in planta upon transient expression. The overexpression of DNE1 in transgenic lines leads to growth defects and transcriptomic changes related to the one observed upon inactivation of the decapping complex. Finally, the combination of dne1 and dcp2 mutations, revealed a functional redundancy between DNE1 and DCP2 in controlling phyllotactic pattern formation in Arabidopsis. Our work identifies DNE1, a hitherto unknown DCP1 protein partner highly conserved in the plant kingdom and identifies its importance for developmental robustness.

One-sentence summaryDNE1, a NYN domain protein interacts with the decapping activator DCP1 and, together with DCP2, specify phyllotactic patterns in Arabidopsis.

category: plant biology


title: A multi-organ metabolic model of tomato predicts plant responses to nutritional and genetic perturbations

abstract:
Predicting and understanding plant responses to perturbations requires integrating the interactions between nutritional sources, genes, cell metabolism and physiology in the same model. This can be achieved using metabolic modeling calibrated by experimental data. In this study, we developed a multi-organ metabolic model of a tomato plant during vegetative growth, named VYTOP (Virtual Young TOmato Plant) that combines genome-scale metabolic models of leaf, stem and root and integrates experimental data acquired from metabolomics and high-throughput phenotyping of tomato plants. It is composed of 6689 reactions and 6326 metabolites. We validated VYTOP predictions on five independent use cases. The model correctly predicted that glutamine is the main organic nutrient of xylem sap. The model estimated quantitatively how stem photosynthetic contribution impact exchanges between the different organs. The model was also able to predict how nitrogen limitation affects the plant vegetative growth, and to predict the metabolic behavior of transgenic tomato lines with altered expressions of core metabolic enzymes. The integration of different components such as a metabolic model, physiological constraints and experimental data generates a powerful predictive tool to study plant behavior, which will be useful for several other applications such as plant metabolic engineering or plant nutrition.

One sentence summaryA multi-organ metabolic model of tomato gives biological insights into the functioning of a plant such as xylem composition, the role of the stem and the response to environmental or genetic perturbation.


title: Cell wall synthesis and remodeling dynamics determine bacterial division site architecture and cell shape

abstract:
The bacterial division apparatus builds daughter cell poles by catalyzing the synthesis and remodeling of the septal peptidoglycan (sPG) cell wall. Understanding of this essential process has been limited by the lack of native three-dimensional visualization of developing septa. Here, we used state-of-the-art cryogenic electron tomography (cryo-ET) and fluorescence microscopy to understand the division site architecture and sPG biogenesis dynamics of the Gram-negative bacterium Escherichia coli. Our results with mutant cells altered in the regulation of sPG biogenesis revealed a striking and unexpected similarity between the architecture of E. coli septa with those from Gram-positive bacteria, suggesting a conserved morphogenic mechanism. Furthermore, we found that the cell elongation and division machineries are in competition and that their relative activities determine the shape of cell constrictions and the poles they form. Overall, our results highlight how the activity of the division system can be modulated to generate the diverse array of morphologies observed in the bacterial domain.

HighlightsO_LIThe division site architecture of E. coli can be modulated to resemble that of diverse bacteria.
C_LIO_LICell wall degradation at the division site activates septal cell wall synthesis.
C_LIO_LIAssembly of the cytoskeletal ring at the division site is modulated by cell wall remodeling.
C_LIO_LIBalance between the activities of the elongation and division systems modulates cell shape.
C_LI

category: microbiology


title: Genetic basis of body shape variation along the benthic-pelagic axis in cichlid fishes

abstract: Divergence along the benthic-pelagic axis is one of the most widespread and repeated patterns of morphological variation in fishes, producing body shape diversity associated with ecology and swimming mechanics. This ecological shift is also the first stage of the explosive adaptive radiation of cichlid fishes in the East African Rift Lakes. We use two hybrid crosses of cichlids (Metriaclima sp. x Aulonocara sp. and Labidochromis sp. x Labeotropheus sp., >975 animals total) along the benthic-pelagic ecomorphological axis to determine the genetic basis of body shape diversification. Using a series of both linear and geometric shape measurements, we identify 55 quantitative trait loci (QTL) that underlie various aspects of body shape variation associated with benthic-pelagic divergence. These QTL are spread throughout the genome, each explain 3.0-7.2% of phenotypic variation, and are largely modular. Further, QTL are distinct both between these two crosses of Lake Malawi cichlids and compared to previously identified QTL for body shape in fishes such as sticklebacks. We find that body shape is controlled by many genes of small effects. In all, we find that convergent benthic and pelagic body phenotypes commonly observed across fish clades are most likely due to distinct genetic and molecular mechanisms.

category: evolutionary biology


title: SUMM4 complex couples insulator function and DNA replication timingcontrol

abstract:
The mechanisms that establish DNA replication timing programs in eukaryotes remain incompletely understood. Drosophila SNF2-related factor SUUR imparts under-replication (UR) of late-replicating intercalary heterochromatin (IH) in polytene chromosomes. We developed a proteomics technique termed MERCI to isolate a native complex SUMM4 comprising SUUR and chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates the ATPase activity of SUUR and is required for its normal spatiotemporal distribution in vivo. Both SUMM4 subunits mediate the activities of gypsy insulator disrupting enhancer-promoter interactions and establishing chromatin barriers. Furthermore, SuUR or mod(mdg4) mutations reverse UR of IH. Our findings uncover a critical role for architectural proteins in attenuating replication fork progression and suggest an alternative mechanism for DNA replication timing that does not depend on an asynchronous firing of replication origins.

One-Sentence SummaryA stable protein complex comprising an insulator factor and a SNF2-like ATPase imparts late replication of heterochromatin.


title: SUMM4 biochemically complex couples insulator function and DNA replication timingcontrol


title: Multimodal Imaging Mass Spectrometry of Murine Gastrointestinal Tract with Retained Luminal Content Shows Molecular Localization Patterns

abstract: Digestive diseases impact 62 million people a year in the United States. Despite the central role of the gut to human health, past imaging mass spectrometry (IMS) investigations into the gastrointestinal tract are incomplete. The gastrointestinal tract, including luminal content, harbors a complex mixture of microorganisms, host dietary content, and immune factors. Existing imaging approaches remove luminal content, and images focus on small regions of tissue. Here, we demonstrate the use of a workflow to collect multimodal imaging data for both intestinal tissue and luminal content. This workflow for matrix-assisted laser desorption/ionization imaging mass spectrometry retains luminal content and expands the amount of tissue imaged on one slide. Results comparing tissue and luminal content show unique molecular distributions using multimodal imaging modalities including protein, lipid, and elemental imaging. Leveraging this method to investigate intestinal tissue infected with Clostridioides difficile compared to control tissue shows clear differences in lipid abundance of various lipid classes in luminal content during infection. These data highlight the potential for this approach to detect unique biological and markers of infection in the gut.


title: New insights into the human 26S proteasome function and regulation

abstract: The 26S proteasome is a protease complex essential for proteostasis and strict regulation of diverse critical physiological processes, the mechanisms of which are still not fully described. The human 26S proteasome purification was optimized without exogenous nucleotides, to preserve the endogenous nucleotide occupancy and conformation of its AAA-ATPase subunits. This unveiled important effects on the proteasome function and structure resulting from exposure to Ca2+ or Mg2+, with important physiological implications. This sample, with an added model degron designed to mimic the minimum canonical ubiquitin signal for proteasomal recognition, was analysed by high-resolution cryo-EM. Two proteasome conformations were resolved, with only one capable of degron binding. The structural data show that this occurs without major conformation rearrangements and allows to infer into the allosteric communication between ubiquitin degron binding and the peptidase activities. These results revise existing concepts on the 26S proteasome function and regulation, opening important opportunities for further research.


title: The multivalency of the glucocorticoid receptor ligand-binding domain explains its manifold physiological activities

abstract: The glucocorticoid receptor (GR) is a ubiquitously expressed transcription factor that controls metabolic and homeostatic processes essential for life. Although numerous crystal structures of the GR ligand-binding domain (GR-LBD) have been reported, the functional oligomeric state of the full-length receptor, which is essential for its transcriptional activity, remains disputed. Here we present five new crystal structures of agonist-bound GR-LBD, along with a thorough analysis of previous structural work. Biologically relevant homodimers were identified by studying a battery of GR point mutants including crosslinking assays in solution and quantitative fluorescence microscopy in living cells. Our results highlight the relevance of non-canonical dimerization modes for GR, especially of contacts made by loop L1-3 residues such as Tyr545. Our work unveils likely pathophysiologically relevant quaternary assemblies of the nuclear receptor with important implications for glucocorticoid action and drug design.


title: Two-color super-resolution localization microscopy via joint encoding of emitter location and color

abstract: Multi-color super-resolution localization microscopy (SRLM) provides great opportunities for studying the structural and functional details of biological samples. However, current multi-color SRLM methods either suffer from medium to high crosstalk, or require a dedicated optical system and a complicated image analysis procedure. To address these problems, here we propose a completely different method to realize multi-color SRLM. This method is built upon a customized RGBW camera with a repeated pattern of filtered (Red, Green, Blue and Near-infrared) and unfiltered (White) pixels. With a new insight that RGBW camera is advantageous for color recognition instead of color reproduction, we developed a joint encoding scheme of emitter location and color. By combing this RGBW camera with the joint encoding scheme and a simple optical set-up, we demonstrated two-color SRLM with [~]20 nm resolution and < 2% crosstalk (which is comparable to the best reported values). This study significantly reduces the complexity of two-color SRLM (and potentially multi-color SRLM), and thus offers good opportunities for general biomedical research laboratories to use multi-color SRLM, which is currently mastered only by well-trained researchers.

category: biophysics


title: Systematic discovery of mutation-directed neo-protein-protein interactions in cancer

abstract: Comprehensive sequencing of patient tumors reveals numerous genomic mutations across tumor types that enable tumorigenesis and progression. A subset of oncogenic driver mutations results in neomorphic activity where the mutant protein mediates functions not engaged by the parental molecule. Here, we identify prevalent variant-enabled neomorph-protein-protein interactions (neoPPI) with a quantitative High Throughput differential Screening (qHT-dS) platform. Coupling of highly sensitive BRET biosensors with miniaturized co-expression in an ultra-HTS format allows large-scale monitoring of interactions of wild-type and mutant variant counterparts with a library of cancer-associated proteins in live cells. Screening of 13,392 interactions with 1,474,560 data points revealed a landscape of gain-of-interactions encompassing both oncogenic and tumor suppressor mutations. For example, the recurrent BRAF V600E lesion mediates KEAP1 neoPPI, rewiring a BRAFV600E-KEAP1 signaling axis and creating collateral vulnerability to NQO1 substrates, offering a combination therapeutic strategy. Thus, cancer genomic alterations can create neo-interactions, informing variant-directed therapeutic approaches for precision medicine.

category: cancer biology


title: In vivo CRISPRi screen reveals the differential requirement for mitochondrial respiratory chain function between in vivo and in vitro tumor growth

abstract: The Warburg effect, aerobic glycolysis, is a hallmark feature of cancer cells grown in culture. However, the relative roles of glycolysis and respiratory metabolism in supporting in vivo tumor growth and significant processes such as tumor dissemination and metastases remain poorly understood, particularly on a systems level. Using a CRISPRi mini-library enriched for mitochondrial ribosomal protein and respiratory chain genes in multiple human lung cancer cell lines we analyzed in vivo metabolic requirements in xenograft tumors grown in distinct anatomic contexts. While knockdown of mitochondrial ribosomal protein and respiratory chain genes (mito-respiratory genes) has little impact on growth in vitro, tumor cells depend heavily on these genes when grown in vivo as either flank or primary orthotopic lung tumor xenografts. In contrast, respiratory function is comparatively dispensable for metastatic tumor growth. RNA-Seq and metabolomics analysis of tumor cells expressing individual sgRNAs against mito-respiratory genes indicate overexpression of glycolytic genes and increased sensitivity of glycolytic inhibition compared to control when grown in vitro, but when grown in vivo as primary tumors these cells downregulate glycolytic mechanisms. These studies demonstrate that discrete perturbations of mitochondrial metabolism impact in vivo tumor growth in a context-specific manner and provides systems-level evidence that respiratory function modulates tumor growth in vivo, suggesting that ATP limits growth and metastatic potential.


title: Antibody-mediated depletion of human CLEC-2 in a novel humanised mouse model

abstract: Platelet C-type lectin-like receptor 2 (CLEC-2) has been proposed as a potential anti-thrombotic target as genetic or antibody-mediated receptor deficiency prevents occlusive thrombus formation in mice. This occurs through interaction with an unknown ligand as the endogenous ligand podoplanin is not present in the vasculature. However, the CLEC-2-podoplanin interaction does have an important role in tumour metastasis. There are currently no methods to test potential human therapeutics targeting CLEC-2, such as antibodies, in vivo. We have therefore generated and characterised a humanised CLEC-2 mouse (hCLEC-2KI) and developed a novel monoclonal anti-human CLEC-2 antibody, HEL1, for in vivo testing. hCLEC-2KI mice were phenotypically normal and had comparable platelet glycoprotein receptor expression, activation and aggregation to wildtype platelets. hCLEC-2KI mice had both comparable bleeding and vessel occlusion times to WT mice. Challenging hCLEC-2KI mice with HEL1 or a second monoclonal anti-hCLEC-2 antibody, AYP1, resulted in transient thrombocytopenia as well as CLEC-2 depletion for more than 2 weeks but had no effect on haemostasis. This illustrates the power of the humanised CLEC-2 mouse model in evaluating novel therapeutics in vivo, including antibodies that target CLEC-2, as well as the limited effect on haemostasis when targeting CLEC-2.

category: physiology


title: Sex chromosomes, sex ratios and sex gaps in longevity in plants

abstract: In animals, males and females can display markedly different longevity (also called sex gap in longevity, SGLs). Recent work has revealed that sex chromosomes contribute to establishing these SGLs. X-hemizygosity and toxicity of the Y chromosomes are two mechanisms that have been suggested to reduce male longevity (Z-hemizygosity and W toxicity in females in ZW systems). In plants, SGLs are known to exist but the role of sex chromosomes remains to be established. Here, by using adult sex ratio as a proxy for measuring SGLs, we explored the relationship between sex chromosome and SGLs across 43 plant species. Based on the knowledge recently accumulated in animals, we specifically asked whether: (i) species with XY systems tend to have female-biased sex ratios (reduced male longevity) and species with ZW ones tend to have male-biased sex ratios (reduced female longevity), and (ii) this patterns was stronger in heteromorphic systems compared to homomorphic ones. Our results tend to support these predictions although we lack statistical power because of a small number of ZW systems and the absence of any heteromorphic ZW system in the dataset. We discuss the implications of these findings, which we hope will stimulate further research on sex-differences in lifespan and ageing across plants.


title: CHD8 regulates gut epithelial cell function and affects autism-related behaviours through the gut-brain axis

abstract: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by early onset deficits in social behavior and repetitive behavior. Chromodomain helicase DNA binding protein (CHD8) is one of the genes with the strongest association to autism. Alongside with the core symptoms of ASD, individuals with ASD are reported to have gastrointestinal (GI) problems, and a majority of individuals with CHD8 mutations display GI problems. However, the relationship between autism related genes, such as CHD8, gastrointestinal function, and autism related behaviours are yet very unclear. In the current study, we found that mice haploinsufficient for CHD8 have leaky gut, a dysregulated transcriptome in gut epithelial cells, decreased gut tuft cells and goblet cells, and an increase in microbial load. Specific deletion of CHD8 in gut epithelial cells induced an increase in anxiety-related behaviours in, a phenotype that is often observed in autism and full body knockdown of CHD8, in addition to decreased tuft cells. In addition, antibiotic treatment of CHD8 haploinsufficient mice attenuates sociability deficits. Therefore, the current study determines a pathway for autism-related GI deficits, and how these deficits may play a direct role in the development of autism-related behaviours.


title: Solid optical clearing agents based through-Intact-Skull (TIS) window technique for long-term observation of cortical structure and function in mice

abstract: In vivo cortical optical imaging needs to overcome the scattering of skull. Compared to the traditional transcranial surgery-based open-skull glass window and thinned-skull preparation, chemical tissue optical clearing techniques can provide a skull-remained optical access to the brain while maintaining its original environment. However, previously demonstrated skull optical clearing windows could only maintain transparency for a couple of hours and hardly capable for high-resolution monitoring of awake animals. Here, we developed a convenient and easy-handling chronic skull optical clearing technique, named "Through-Intact-Skull (TIS) window", which was compatible with long-term observation at high resolution, and yielded large imaging depth of 900 m for cortical neurovascular visualization. In addition, our TIS window could monitor neuron activity in awake mice for a long term. Therefore, our bio-compatible and non-invasive TIS window is a new promising approach for intravital brain microscopy with great potential for basic research in neuroscience.


title: Mechanically active integrins direct cytotoxic secretion at the immune synapse

abstract: The secretory output of cell-cell interfaces must be tightly controlled in space and time to ensure functional efficacy. This is particularly true for the cytotoxic immune synapse (IS), the stereotyped junction formed between a cytotoxic lymphocyte and the infected or transformed target cell it aims to destroy1. Cytotoxic lymphocytes kill their targets by channeling a mixture of granzyme proteases and the pore forming protein perforin directly into the IS2, 3. The synaptic secretion of these toxic molecules constrains their deleterious effects to the target cell alone, thereby protecting innocent bystander cells in the surrounding tissue from collateral damage. Despite the importance of this process for immune specificity, the molecular and cellular mechanisms that establish secretory sites within the IS remain poorly understood. Here, we identified an essential role for integrin mechanotransduction in cytotoxic secretion using a combination of single cell biophysical measurements, ligand micropatterning, and functional assays. Upon ligand-binding, the L{beta}2 integrin LFA-1 functioned as a spatial cue, attracting lytic granules containing perforin and granzyme and inducing their fusion at closely adjacent sites within the synaptic membrane. LFA-1 molecules were subjected to pulling forces within these secretory domains, and genetic or pharmacological suppression of these forces abrogated cytotoxicity. We conclude that lymphocytes employ an integrin-dependent mechanical checkpoint to enhance both the potency and the security of their cytotoxic output.

category: immunology


title: Th2 single-cell heterogeneity and clonal interorgan distribution in helminth-infected mice

abstract: Th2 cells provide effector functions in type 2 immune responses to helminths and allergens. Despite knowledge about molecular mechanisms of Th2 cell differentiation, there is little information on Th2 cell heterogeneity and clonal distribution between organs. To address this, we performed combined single-cell transcriptome and TCR clonotype analysis on murine Th2 cells in mesenteric lymph nodes (MLN) and lung after infection with Nippostrongylus brasiliensis (Nb) as a human hookworm infection model. We find organ-specific expression profiles, but also populations with conserved migration or effector/resident memory signatures that unexpectedly cluster with potentially regulatory Il10posFoxp3neg cells. A substantial MLN subpopulation with an interferon response signature suggests a role for interferon-signaling in Th2 differentiation or diversification. Further RNA-inferred developmental directions indicate proliferation as a hub for differentiation decisions. We also link long noted Cxcr3 expression in the Th2 compartment to a population of Il4pos NKT cells. Although the TCR repertoire is highly heterogeneous, we identified expanded clones and CDR3 motifs. Clonal relatedness between distant organs confirmed effective exchange of Th2 effector cells, although locally expanded clones dominated the response. These results provide new insights in Th2 cell subset diversity and clonal relatedness in distant organs.


title: Immunometabolic hijacking of immune cells by a Pseudomonas aeruginosa quorum-sensing signal

abstract: Macrophages utilize metabolic pathways to generate energy and metabolites that may be vulnerable to pathogen hijacking to favor pathogen survival and persistence. It is unclear how bacterial pathogens alter metabolic pathways in immune cells for their benefit and persistence in the infected host. We have shown that the Pseudomonas aeruginosa quorum sensing (QS) signal molecule 2-aminoacetophenone (2-AA) allows pathogen persistence in host tissues by triggering host tolerization via histone deacetylase (HDAC)1-mediated epigenetic reprogramming. Here, we provide strong evidence that 2-AA-meditated persistence is linked to specific metabolic pathway alterations that reduce energy availability and biosynthetic macromolecules involved in host immune responses. 2-AA promotes a Warburg-like metabolic reprogramming effect, thereby increasing levels of lactate, which repressed inflammatory signaling in macrophages. Moreover, it interferes with pyruvate translocation to mitochondria, reducing mitochondrial (mt)-oxidative phosphorylation (OXPHOS) due to down-regulation of estrogen-regulated receptor (ERR) and mitochondrial pyruvate carrier (MPC)-1. This metabolic reprogramming dampened energy production, reduced the acetyl-CoA pool, and generated an anti-inflammatory milieu that favors P. aeruginosa persistence. These findings provide evidence of first-in-class metabolic reprogramming in immune cells mediated by a QS signaling molecule. The specific metabolic programs affected provide insights that may guide the design and development of therapeutics and protective interventions against pathogen-induced immunometabolic alterations and persistence factors.


title: Actin capping protein regulates actomyosin contractility to maintain germline architecture in C. elegans

abstract: Actin dynamics play an important role in the morphogenesis of cells and tissues, yet the control of actin filament growth takes place at the molecular level. A challenge in the field is to link the molecular function of actin regulators with their physiological function. Here, we report the in vivo role of the actin capping protein CAP-1 in the C. elegans germline. We show that CAP-1 is associated with actomyosin structures in the cortex and rachis, where it keeps the level of contractility in check. A 60% reduction in the level of CAP-1 leads to a 2-fold increase in F-actin and non-muscle myosin II and only a 30% increase in Arp2/3. CAP-1 depletion leads to severe structural defects in the syncytial germline and oocytes, which can be rescued by reducing myosin activity. Thus, we uncover a physiological role for actin capping protein in maintaining C. elegans fertility by regulating the level of actomyosin contractility.


title: Identifying a novel role for the master regulator Tal1 in the Endothelial to Hematopoietic Transition

abstract: Recent progress in the generation of bona-fide Hematopoietic Stem and Progenitor Cells (HSPCs) in vitro and ex vivo has been built on the knowledge of developmental hematopoiesis, underscoring the importance of understanding in detail this developmental process. Here, we sought to elucidate the function of the hematopoietic regulators Tal1, Lmo2 and Lyl1 in the Endothelial to Hematopoietic Transition (EHT), the process through which HSPCs are generated from endothelial precursors during embryogenesis. We used a mouse embryonic-stem cell (mESC)-based differentiation system to model hematopoietic development, and combined gain-of-function experiments in sorted vascular smooth muscle cells (VSM) with multi-omics to obtain mechanistic insights into the mode of action of Tal1, Lmo2 and Lyl1. We found that these factors promote the silencing of the VSM transcriptional program and the activation of the hematopoietic one. Through this approach and the use of a Tet-on system to control the expression of Tal1 during hematopoietic specification from mESCs, we discovered that its expression in endothelial cells is crucial for the EHT to occur.


title: Mechanisms of damage prevention, signalling, and repair impact the ability of Drosophila to tolerate enteric bacterial infection

abstract: The insect gut is frequently exposed to pathogenic threats and must not only clear these potential infections, but also tolerate relatively high microbe loads. In contrast to the mechanisms that eliminate pathogens, we currently know less about the mechanisms of disease tolerance. We investigated how well-described mechanisms that either prevent, signal, control, or repair damage during infection contribute to the phenotype of disease tolerance. We established enteric infections with the bacterial pathogen Pseudomonas entomophila in transgenic lines of Drosophila melanogaster fruit flies affecting dcy (a major component of the peritrophic matrix), upd3 (a cytokine-like molecule), irc (a negative regulator of reactive oxygen species) and egfr1 (epithelial growth factor receptor). Flies lacking dcy experienced the highest susceptibility, while loss of function of either irc or upd3 reduced tolerance in both sexes. The disruption of egfr1 resulted in a severe loss in tolerance in male flies but had no substantial effect on the ability of female flies to tolerate P. entomophila infection, despite carrying greater microbe loads than males. Together, our findings provide evidence for the role of damage limitation mechanisms in disease tolerance and highlight how sex differences in these mechanisms could generate sexual dimorphism in disease tolerance.


title: Mechanisms of damage prevention, signalling, and repair impact disease tolerance


title: Dietary vitamin B12 regulates chemosensory receptor gene expression via the MEF2 transcription factor in Caenorhabditis elegans

abstract: Dynamic changes in chemoreceptor gene expression levels in sensory neurons is one strategy that an animal can use to modify their responses to dietary changes. However, the mechanisms underlying diet-dependent modulation of chemosensory gene expression are unclear. Here, we show that the expression of the srh-234 chemoreceptor gene localized in a single ADL sensory neuron type of C. elegans is downregulated when animals are fed a Comamonas bacterial diet, but not on an E. coli diet. Remarkably, this diet-modulated effect on srh-234 gene expression levels is dependent on the micronutrient vitamin B12 endogenously produced by Comamonas bacteria. Excess propionate and genetic perturbations in the canonical and shunt propionate breakdown pathways are able to override the repressing effects of vitamin B12 on srh-234 expression. The vitamin B12-mediated regulation of srh-234 expression levels in ADL requires the MEF-2 transcription factor, providing a potential mechanism by which dietary vitamin B12 may transcriptionally tune individual chemoreceptor genes in a single sensory neuron type, which in turn may change animal responses to biologically relevant chemicals in their diet.

category: genetics


title: Fine-mapping of nuclear compartments using ultra-deep Hi-C shows that active promoter and enhancer elements localize in the active A compartment even when adjacent sequences do not

abstract: Megabase-scale intervals of active, gene-rich and inactive, gene-poor chromatin are known to segregate, forming the A and B compartments. Fine mapping of the contents of these A and B compartments has been hitherto impossible, owing to the extraordinary sequencing depths required to distinguish between the long-range contact patterns of individual loci, and to the computational complexity of the associated calculations. Here, we generate the largest published in situ Hi-C map to date, spanning 33 billion contacts. We also develop a computational method, dubbed PCA of Sparse, SUper Massive Matrices (POSSUMM), that is capable of efficiently calculating eigenvectors for sparse matrices with millions of rows and columns. Applying POSSUMM to our Hi-C dataset makes it possible to assign loci to the A and B compartment at 500 bp resolution. We find that loci frequently alternate between compartments as one moves along the contour of the genome, such that the median compartment interval is only 12.5 kb long. Contrary to the findings in coarse-resolution compartment profiles, we find that individual genes are not uniformly positioned in either the A compartment or the B compartment. Instead, essentially all (95%) active gene promoters localize in the A compartment, but the likelihood of localizing in the A compartment declines along the body of active genes, such that the transcriptional termini of long genes (>60 kb) tend to localize in the B compartment. Similarly, nearly all active enhancers elements (95%) localize in the A compartment, even when the flanking sequences are comprised entirely of inactive chromatin and localize in the B compartment. These results are consistent with a model in which DNA-bound regulatory complexes give rise to phase separation at the scale of individual DNA elements.

category: genomics


title: OMA1 High-throughput Screen Reveals Protease Activation by Kinase Inhibitors

abstract: Mitochondrial proteases are interesting but challenging drug targets for multifactorial diseases, such as neurodegeneration and cancer. The mitochondrial inner membrane protease OMA1 is a bona fide drug target for heart failure supported by data from human linkage analysis and animal disease models, but presumably relevant for more indications. OMA1 acts at the intersection of energy metabolism and stress signaling. The protease cleaves the structural protein OPA1, which organizes the cristae, as well as the signaling peptide DELE1, which can stimulate the integrated stress response. OMA1 shows little activity under physiological conditions but hydrolyzes OPA1 in mitochondria destined for mitophagy and during apoptosis. Little is known about OMA1, its structure has not been solved, let alone its context-dependent regulation. Autocatalytic processing and the lack of OMA1 inhibitors are thereby creating the biggest roadblocks. This study introduces a scalable, cellular OMA1 protease assay suitable for high-throughput drug screening. The assay utilizes an engineered luciferase targeted to the inner membrane as artificial OMA1 substrate, whereby the reporter signal inversely correlates to OMA1 activity. Testing different screening protocols and sampling different compound collections validated the reporter and demonstrated that both OMA1 activators as well as OMA1 inhibitors can be identified with the assay. Ten kinase-targeted cancer drugs triggered OMA1 in the assays, which suggests--considering cardiotoxicity as a rather common side-effect of this class of drugs--cross-reactivity with the OMA1 pathway.


title: DeepUMQA: Ultrafast Shape Recognition-based Protein Model Quality Assessment using Deep Learning

abstract:
MotivationProtein model quality assessment is a key component of protein structure prediction. In recent research, the voxelization feature was used to characterize the local structural information of residues, but it may be insufficient for describing residue-level topological information. Design features that can further reflect residue-level topology when combined with deep learning methods are therefore crucial to improve the performance of model quality assessment.

ResultsWe developed a deep-learning method, DeepUMQA, based on Ultrafast Shape Recognition (USR) for the residue-level single-model quality assessment. In the framework of the deep residual neural network, the residue-level USR feature was introduced to describe the topological relationship between the residue and overall structure by calculating the first moment of a set of residue distance sets and then combined with 1D, 2D, and voxelization features to assess the quality of the model. Experimental results on test datasets of CASP13, CASP14, and CAMEO show that USR could complement the voxelization feature to comprehensively characterize residue structure information and significantly improve the model assessment accuracy. DeepUMQA outperformed the state-of-the-art single-model quality assessment methods, including ProQ2, ProQ3, ProQ3D, Ornate, VoroMQA, and DeepAccNet.

AvailabilityThe source code and executable are freely available at https://github.com/iobio-zjut/DeepUMQA.

Contactzgj@zjut.edu.cn


title: F-actin bending facilitates net actomyosin contraction by inhibiting expansion with plus-end-located myosin motors

abstract: Contraction of actomyosin networks underpins important cellular processes including motility and division. The mechanical origin of actomyosin contraction is not fully-understood. We investigate whether contraction arises on the scale of individual filaments, without needing to invoke network-scale interactions. We derive discrete force-balance and continuum partial differential equations for two symmetric, semi-flexible actin filaments with an attached myosin motor. Assuming the system exists within a homogeneous background material, our method enables computation of the stress tensor, providing a measure of contractility. After deriving the model, we use a combination of asymptotic analysis and numerical solutions to show how F-actin bending facilitates contraction on the scale of two filaments. Rigid filaments exhibit polarity-reversal symmetry as the motor travels from the minus to plus-ends, such that contractile and expansive components cancel. Filament bending induces a geometric asymmetry that brings the filaments closer to parallel as a myosin motor approaches their plus-ends, decreasing the effective spring force opposing motor motion. The reduced spring force enables the motor to move faster close to filament plus-ends, which reduces expansive stress and gives rise to net contraction. Bending-induced geometric asymmetry provides both new understanding of actomyosin contraction mechanics, and a hypothesis that can be tested in experiments.


title: Increased remodeling and impaired adaption to endurance exercise in desminopathy

abstract: Desminopathy the most common intermediate filament disease in humans. Desmin is an essential part of the filamentous network that aligns myofibrils, anchors nuclei and mitochondria, and connects the z-discs and the sarcolemma. We created a rat model with a mutation in R349P DES, analog to the most frequent R350P DES missense mutation in humans. To examine the effects of a chronic, physiological exercise stimulus on desminopathic muscle, we subjected R349P DES rats and their wildtype (WT) and heterozygous littermates to a treadmill running regime. We saw significantly lower running capacity in DES rats that worsened over the course of the study. We found indicators of increased autophagic and proteasome activity with running in DES compared to WT. Stable isotope labeling and LC-MS analysis displayed distinct adaptations of the proteomes of WT and DES animals at baseline as well as with exercise: While key proteins of glycolysis, mitochondria and thick filaments increased their synthetic activity with running in WT, these proteins were higher at baseline in DES and did not change with running. The results suggest an impairment in adaption to chronic exercise in DES muscle and a subsequent exacerbation in the functional and histopathological phenotype.


title: Arabidopsis Apoplastic Fluid Contains sRNA- and Circular RNA-Protein Complexes that Are Located Outside Extracellular Vesicles

abstract:
Previously, we have shown that apoplastic wash fluid purified from Arabidopsis leaves contains small RNAs (sRNAs). To investigate whether these sRNAs are encapsulated inside extracellular vesicles (EVs), we treated EVs isolated from Arabidopsis leaves with the protease trypsin and RNase A, which should degrade RNAs located outside EVs but not those located inside. These analyses revealed that apoplastic RNAs are mostly located outside EVs and are associated with proteins. Further analyses of these extracellular RNAs (exRNAs) revealed that they comprise both sRNAs and long non-coding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the post-transcriptional modification N6-methyladenine (m6A). Consistent with this, we identified a putative m6A-binding protein in apoplastic wash fluid, GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7), as well as the small RNA-binding protein ARGONAUTE2 (AGO2). These two proteins co-immunoprecipitated with each other, and with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of apoplastic wash fluid, suggesting that these proteins contribute to the secretion and/or stabilization of exRNAs. We propose that these extravesicular RNAs mediate host-induced gene silencing, rather than RNA inside EVs.

One-sentence summaryThe apoplast of Arabidopsis leaves contains diverse small and long-noncoding RNAs, including circular RNAs, that are bound to protein complexes and are located outside extracellular vesicles.


title: Characterization of Heat Responsive microRNAs and Phased Small Interfering RNAs in Reproductive Development of Flax

abstract: Plants will face increased heat stress due to rising global temperatures. Heat stress affects plant reproductive development and decreases productivity; however, the underlying molecular mechanisms of these processes are poorly characterized. Plant small RNAs (sRNAs) have important regulatory roles in plant reproductive development following abiotic stress responses. We generated sRNA transcriptomes of three reproductive bud stages at three different time points to identify sRNA-mediated pathways responsive to heat stress in flax. With added sRNA transcriptomes of vegetative tissues, we comprehensively annotated miRNA and phasiRNA-encoding genes (PHAS) in flax. We identified 173 miRNA genes, of which 42 are novel. Our analysis revealed that 141 miRNA genes were differentially expressed between tissue types while 18 miRNA genes were differentially expressed in reproductive tissues following heat stress, including members of miR2118/482 and miR2275 families, known triggers of reproductive phasiRNAs. Furthermore, we identified 68 21-PHAS flax loci from protein coding and non-coding regions, four 24-PHAS loci triggered by miR2275, and 658 24-PHAS-like loci with unknown triggers, derived mostly from non-coding regions. The reproductive phasiRNAs are mostly downregulated in response to heat stress. Overall, we found that several previously unreported miRNAs and phasiRNAs are responsive to heat stress in flax reproductive tissues.


title: Enzymes degraded under high light maintain proteostasis by transcriptional regulation in Arabidopsis

abstract: Photo-inhibitory high light stress in Arabidopsis leads to increases in markers of protein degradation and transcriptional upregulation of proteases and proteolytic machinery, but proteostasis is largely maintained. We find significant increases in the in vivo degradation rate for specific molecular chaperones, nitrate reductase, glyceraldehyde-3 phosphate dehydrogenase, and phosphoglycerate kinase and other plastid, mitochondrial, peroxisomal, and cytosolic enzymes involved in redox shuttles. Coupled analysis of protein degradation rates, mRNA levels, and protein abundance reveal that 57% of the nuclear-encoded enzymes with higher degradation rates also had high light-induced transcriptional responses to maintain proteostasis. In contrast, plastid-encoded proteins with enhanced degradation rates showed decreased transcript abundances and must maintain protein abundance by other processes. This analysis reveals a light-induced transcriptional program for nuclear-encoded genes, beyond the regulation of PSII D1 subunit and the function of PSII, to replace key protein degradation targets in plants and ensure proteostasis under high light stress.


title: Mechanistic Model of Replication Fork Progression in Yeast

abstract: Replication fork progression complex plays an essential role during DNA replication. It travels along with the DNA with a particular speed called replication fork speed. Faithful duplication of the genome requires strict control over replication fork speed. Both acceleration and pausing mechanisms of the replication fork complex are regulated at the molecular level. Based on the experimental evidence, DNA replicates faster in normal cells than cancer cells, whereas cancer cells duplicate themselves more quickly than normal cells. Then in principle, accelerating the replication fork complex in cancer cells beyond a specific threshold speed limit can cause DNA damage and plausibly kill them. A modular mathematical model is proposed to explain the dynamics of replication fork control during DNA replication using the underlying molecular mechanisms in yeast which can extend to the mammalian system in the future.


title: Reconstruction of distinct vertebrate gastrulation modes via modulation of key cell behaviours in the chick embryo

abstract: The morphology of gastrulation driving the internalisation of the mesoderm and endoderm differs dramatically among vertebrate species. It ranges from involution of epithelial sheets of cells through a circular blastopore in amphibians to ingression of mesenchymal cells through a primitive streak in amniotes. By targeting signalling pathways controlling critical cell behaviours in the chick embryo, we generated crescent- and ring-shaped mesendoderm territories in which cells can or cannot ingress. These alterations subvert the formation of the chick primitive streak into the gastrulation modes seen in amphibians, reptiles and teleost fish. Our experimental manipulations are supported by a theoretical framework linking cellular behaviors to self-organized multi-cellular flows in the accompanying paper. All together, this suggests that the evolution of gastrulation movements are largely determined by the shape of and cell behaviours in the mesendoderm territory across different species, and controlled by a relatively small number of signalling pathways.


title: Stress-dependent dynamic and reversible formation of cytoskeleton-like filaments and gel-transition by tardigrade tolerance proteins.

abstract: Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective proteins. Cytoplasmic-abundant heat-soluble (CAHS) proteins are uniquely invented in the lineage of eutardigrades, a major class of the phylum Tardigrada and are essential for their anhydrobiotic survival. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins that reversibly form condensates upon dehydration-like stress. Through comprehensive analysis, we identified 336 such proteins, collectively dubbed "Desolvataion-induced ReversiblY condensing Proteins (DRYPs)". Unexpectedly, we rediscovered CAHS proteins as highly enriched in DRYPs, 3 of which were major components of DRYPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro. CAHS filamentation increases cell stiffness to resist deformation and improves resistance to dehydration-like stress. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeletal proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and also contribute to the exceptional physical stability in a dehydrated state.


title: Stress-dependent cell stiffening by tardigrade tolerance proteins through reversible formation of cytoskeleton-like filamentous network and gel-transition


title: A Bidirectional Switch in the Shank3 Phosphorylation State Biases Synapses toward Up or Down Scaling

abstract: Homeostatic synaptic plasticity requires widespread remodeling of synaptic signaling and scaffolding networks, but the role of posttranslational modifications in this process has not been systematically studied. Using deepscale, quantitative analysis of the phosphoproteome in mouse neocortical neurons, we found wide-spread and temporally complex changes during synaptic scaling up and down. We observed 424 bidirectionally modulated phosphosites that were strongly enriched for synapse-associated proteins, including S1539 in the ASD-associated synaptic scaffold protein Shank3. Using a parallel proteomic analysis performed on Shank3 isolated from rat neocortical neurons by immunoaffinity, we identified two sites that were hypo-phosphorylated during scaling up and hyper-phosphorylated during scaling down: one (rat S1615) that corresponded to S1539 in mouse, and a second highly conserved site, rat S1586. The phosphorylation status of these sites modified the synaptic localization of Shank3 during scaling protocols, and dephosphorylation of these sites via PP2A activity was essential for the maintenance of synaptic scaling up. Finally, phosphomimetic mutations at these sites prevented scaling up but not down, while phosphodeficient mutations prevented scaling down but not up. Thus, an activity-dependent switch between hypo- and hyperphosphorylation at S1586/ S1615 of Shank3 enables scaling up or down, respectively. Collectively our data show that activity-dependent phosphoproteome dynamics are important for the functional reconfiguration of synaptic scaffolds, and can bias synapses toward upward or downward homeostatic plasticity.


title: Pre-clinical validation of an RNA-based precision oncology platform for patient-therapy alignment in a diverse set of human malignancies resistant to standard treatments

abstract: Predicting tumor sensitivity to antineoplastics remains an elusive challenge, with no methods demonstrating predictive power. Joint analysis of tumors--from patients with distinct malignancies who had progressed on multiple lines of therapy--and drug perturbation transcriptional profiles predicted sensitivity to 28 of 350 drugs, 26 of which (93%) were confirmed in low-passage, patient-derived xenograft (PDX) models. Drugs were prioritized based on their ability to either invert the activity of individual Master Regulator proteins, with available high-affinity inhibitors, or of the modules they comprise (Tumor-Checkpoints), based on de novo mechanism of action analysis. Of 138 PDX mice enrolled in 16 single and 18 multi-protein treatment arms, a disease control rate (DCR) of 68% and 91 %, and an objective response rate (ORR) of 12% and 17%, were achieved respectively, compared to 6% and 0% in the negative controls arm, with multi-protein drugs achieving significantly more durable responses. Thus, these approaches may effectively complement and expand current precision oncology approaches, as also illustrated by a case study.


title: The cerebellum initiates severe tonic-clonic seizures by altering neuronal activity in the ventral posteromedial nucleus (VPM) of the thalamus

abstract: Thalamo-cortical networks are central to seizures, yet its unclear how these circuits initiate the seizures. Here, we test the hypothesis that a facial region of the thalamus, the VPM, is a source of convulsive, tonic-clonic seizures. We devised an in vivo optogenetic mouse model to elicit tonic-clonic seizures by driving convergent input to the VPM. With viral tracing, we show dense cerebellar and cerebral cortical afferent input to the VPM. Lidocaine microinfusions into the cerebellar nuclei selectively block seizure initiation. We perform single-unit electrophysiology recordings during awake, convulsive seizures to define the local activity of thalamic neurons before, during, and after seizure onset. We find highly dynamic activity with biphasic properties, raising the possibility that heterogenous activity patterns promote seizures. These data reveal the VPM as a source of tonic-clonic seizures, with cerebellar input providing the predominant signals.


title: The cerebellum contributes to tonic-clonic seizures by altering neuronal activity in the ventral posteromedial nucleus (VPM) of the thalamus


title: Biased credit assignment in motivational learning biases arises through prefrontal influences on striatal learning

abstract: Actions are biased by the outcomes they can produce: Humans are more likely to show action under reward prospect, but hold back under punishment prospect. Such motivational biases derive not only from biased response selection, but also from biased learning: humans tend to attribute rewards to their own actions, but are reluctant to attribute punishments to having held back. The neural origin of these biases is unclear; in particular, it remains open whether motivational biases arise solely from an evolutionarily old, subcortical architecture or also due to younger, cortical influences. Simultaneous EEG-fMRI allowed us to track which regions encoded biased prediction errors in which order. Biased prediction errors occurred in cortical regions (ACC, vmPFC, PCC) before subcortical regions (striatum). These results highlight that biased learning is not a mere feature of the basal ganglia, but arises through prefrontal cortical contributions, revealing motivational biases to be a potentially flexible, sophisticated mechanism.


title: The first transchromosomic rat model with human chromosome 21 shows robust Down syndrome features

abstract: Progress in earlier detection and symptom management has increased life expectancy and quality of life in people with Down syndrome (DS). However, no drug has been approved to help individuals with DS live independently and fully. Although rat models could support more robust physiological, behavioral, and toxicology analysis than mouse models during preclinical validation, no DS rat model is available due to technical challenges. We developed the first transchromosomic rat model of DS, TcHSA21rat, which contains a freely segregating, EGFP-inserted, human chromosome 21 (HSA21) with >93% of its protein coding genes. RNA-Seq of neonatal forebrains demonstrates that TcHSA21rat not only expresses HSA21 genes but also has an imbalance in global gene expression. Using EGFP as a marker for trisomic cells, flow cytometry analyses of peripheral blood cells from 361 adult TcHSA21rat animals show that 81% of animals retain HSA21 in >80% of cells, the criterion for a "Down syndrome karyotype" in people. TcHSA21rat exhibits learning and memory deficits and shows increased anxiety and hyperactivity. TcHSA21rat recapitulates well-characterized DS brain morphology, including smaller brain volume and reduced cerebellar size. In addition, the rat model shows reduced cerebellar foliation, a prominent feature of DS that is not observed in DS mouse models. Moreover, TcHSA21rat exhibits anomalies in craniofacial morphology, heart development, husbandry, and stature. TcHSA21rat is a robust DS animal model that can facilitate DS basic research and provide a unique tool for preclinical validation to accelerate DS drug development.


title: Pain is so close to pleasure: the same dopamine neurons can mediate approach and avoidance in Drosophila

abstract: In mammals, dopamine is considered a central neuromodulator involved in all kinds of rewarding experiences ( common currency hypothesis). In insects, the role of do-paminergic neurons in aversive stimuli was discovered before dopaminergic neurons were found to also be involved in processing appetitive stimuli. Here, we screened about 50 transgenic Drosophila lines, representing different subpopulations of dopa-minergic neurons for their ability to sustain approach or avoidance behavior, when activated optogenetically in four different operant self-stimulation paradigms. None of the lines sustain consistent behavioral valence in all experiments. Individual lines sustain approach in one experiment and avoidance in another. One line mediated strong avoidance early in the experiment and weak approach in later stages. The evidence presented here appears to contradict a  common currency dopamine function in flies. Instead, different dopaminergic neurons convey valence in a context-dependent and flexible manner, reflecting the genetic heterogeneity of the dopaminergic neuronal population.


title: Inter-subunit Crosstalk via PDZ Synergistically Governs Allosteric Activation of Proapoptotic HtrA2

abstract: Mitochondrial serine protease - High temperature requirement A2 (HtrA2), is associated with various diseases including neurodegenerative disorders and cancer. Despite availability of structural details, the reports on HtrA2s mechanistic regulation that varies with the type of activation signals still remain non-concordant. To expound the role of regulatory PDZ domains in promoting synergistic coordination between HtrA2 subunits, we generated heterotrimeric HtrA2 variants comprising different numbers of PDZs and/or active-site mutations. Sequential deletion of PDZs from the trimeric ensemble significantly affected its residual activity in a way that proffered a hypothesis advocating intermolecular allosteric crosstalk via PDZ domains in trimeric HtrA2. Furthermore, structural and computational snapshots affirmed the role of PDZs in secondary structural element formation and coordinated reorganization of the N-terminal region and regulatory loops. Therefore, apart from providing cues for devising structure-guided therapeutic strategies, this study establishes a physiologically relevant working model of complex allosteric regulation through a multifaceted trans-mediated cooperatively-shared energy landscape.


title: A force-balance model for centrosome positioning and spindle elongation during Interphase and Anaphase B

abstract: A computational model in one dimension is proposed to position a single centrosome using astral microtubules (MTs) interacting with the cell cortex. The mechanism exploits mutually antagonistic pulling and pushing forces arising from the astral MTs binding to cortical dynein motors in the actin-rich cell cortex and their buckling while growing against the cell cortex, respectively. The underlying mechanism is extended to account for the elongation and positioning of the bipolar spindle during mitotic anaphase B. Besides astral MTs, the model for bipolar spindle involves interpolar microtubules (IPMTs). The composite model can predict spindle elongation and position under various circumstances. The outcome reveals that the bipolar spindle elongation, weakened by decreasing overlap between the antiparallel IPMTs in the spindle mid-zone, is recovered by the astral MTs. The one-dimensional models are extended in two dimensions to include the effect of cortical sliding of the astral MTs for studying the dynamics of the interphase centrosome and the anaphase B spindles in elongated cells. The results reveal that the dynamics in two dimensions stay qualitatively similar to the one dimension.


title: Cryo-EM Structure of the Pol Polyprotein Provides Insights into HIV Maturation

abstract: Many retroviral proteins are initially translated from unspliced full-length RNA as polyprotein precursors that are subsequently processed by the viral protease (PR) to yield the mature forms. In HIV-1, the enzymes, PR, reverse transcriptase (RT), and integrase (IN), are produced as part of the Gag-Pol polyprotein. While structures of the mature proteins have aided our understanding of catalytic mechanisms and the design of antiretroviral drugs, knowledge of the architecture and functional implications of the immature forms prior to PR-mediated cleavage is limited. We developed a system to produce and purify the HIV-1 Pol polyprotein intermediate precursor and determined its high-resolution cryo-EM structure. The RT portion of the polyprotein has an architecture similar to the mature RT p66/p51 heterodimer, and dimerization of the RT portion draws together two PR monomers to activate proteolytic processing. HIV-1 thus may leverage the dimerization interfaces in Pol to regulate the assembly and maturation of the polyprotein precursors.


title: Machine learning-based modulation of Ca2+-binding affinity in EFhand proteins and comparative structural insights into site-specific cooperative binding

abstract: Ca2+-binding proteins are present in almost all living organisms and different types display different levels of binding affinities for the cation. Here, we applied two new scoring schemes enabling the user to manipulate the binding affinities of such proteins. We specifically designed a unique EF-hand loop capable of binding calcium with high affinity by altering five residues of the loop based on the scoring scheme. We worked on the N-terminal domain of Entamoeba histolytica calcium-binding protein1 (NtEhCaBP1), and used site-directed mutagenesis to incorporate the designed loop sequence into the second EF hand motif of this protein. The binding isotherms calculated using ITC calorimetry showed a [~]500-fold greater association constant (Ka) for the mutant. The crystal structure of the mutant was also determined, and displayed more compact Ca2+-coordination spheres in both of its EF loops than did the structure of the wildtype protein, consistent with the greater calcium-binding affinities of the mutant. The NtEhCaBP1 mutant was also shown to form a hexamer rather than just a trimer, and this hexamer formation was attributed to the position of the last helix of the mutant having been changed as a result of the strong calcium coordination. Further dynamic correlation analysis revealed that the mutation in the second EF loop changed the entire residue network of the monomer, resulting in a stronger coordination of Ca2+ even in other EF hand loop.


title: {micro}-Theraphotoxin-Pn3a inhibition of Cav3.3 channels reveals a novel isoform-selective drug binding site

abstract: Low voltage-activated calcium currents are mediated by T-type calcium channels CaV3.1, CaV3.2, and CaV3.3, which modulate a variety of physiological processes including sleep, cardiac pace-making, pain, and epilepsy. CaV3 isoforms biophysical properties, overlapping expression and lack of subtype-selective pharmacology hinder the determination of their specific physiological roles in health and disease. Notably, CaV3.3s contribution to normal and pathophysiological function has remained largely unexplored. We have identified Pn3a as the first subtype-selective spider venom peptide inhibitor of CaV3.3, with >100-fold lower potency against the other T-type isoforms. Pn3a modifies CaV3.3 gating through a depolarizing shift in the voltage dependence of activation thus decreasing CaV3.3-mediated currents in the normal range of activation potentials. Paddle chimeras of KV1.7 channels bearing voltage sensor sequences from all four CaV3.3 domains revealed preferential binding of Pn3a to the S3-S4 region of domain II (CaV3.3DII). This novel T-type channel pharmacological site was explored through computational docking simulations of Pn3a into all T-type channel isoforms highlighting it as subtype-specific pharmacophore with therapeutic potential. This research expands our understanding of T-type calcium channel pharmacology and supports the suitability of Pn3a as a molecular tool in the study of the physiological roles of CaV3.3 channels.

category: pharmacology and toxicology


title: Ultrastructural analysis of neuroimplant-parenchyma interfaces uncover remarkable neuroregeneration along-with barriers that limit the implant electrophysiological functions

abstract: Despite increasing use of in-vivo multielectrode array (MEA) implants for basic research and medical applications, the critical structural interfaces formed between the implants and the brain parenchyma, remain elusive. Prevailing view assumes that formation of multicellular inflammatory encapsulating-scar around the implants (the foreign body response) degrades the implant electrophysiological functions. Using gold mushroom shaped microelectrodes (gMEs) based perforated polyimide MEA platforms (PPMPs) that in contrast to standard probes can be thin sectioned along with the interfacing parenchyma; we examined here for the first time the interfaces formed between brains parenchyma and implanted 3D vertical microelectrode platforms at the ultrastructural level. Our study demonstrates remarkable regenerative processes including neuritogenesis, axon myelination, synapse formation and capillaries regrowth in contact and around the implant. In parallel, we document that individual microglia adhere tightly and engulf the gMEs. Modeling of the formed microglia-electrode junctions suggest that this configuration suffice to account for the low and deteriorating recording qualities of in vivo MEA implants. These observations help define the anticipated hurdles to adapting the advantageous 3D in-vitro vertical-electrode technologies to in-vivo settings, and suggest that improving the recording qualities and durability of planar or 3D in-vivo electrode implants will require developing approaches to eliminate the insulating microglia junctions.


title: Neutrophil reverse migration from liver fuels neutrophilic inflammation to tissue injury in Nonalcoholic Steatohepatitis.

abstract: Inflammation is a hallmark in the progression of nonalcoholic-fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH). Patients with NAFLD are characterized by a chronic low-grade systemic metabolic inflammation (i.e., metainflammation), which contributes to exacerbated however dysfunctional immune response. Neutrophils play an important pathological role in NAFLD progression to NASH; however, how NASH and associated chronic systemic inflammation impact overall the neutrophil response to injury is completely unexplored. Here, we investigated how neutrophil response to tissue injury is altered by the presence of NASH. We used a diet-induced NASH zebrafish model combined with tailfin transection in transgenic zebrafish larvae to study neutrophilic inflammation. Live non-invasive confocal microscopy was used to investigate neutrophil recruitment to tailfin injury through time. Photoconvertion of neutrophils at the liver area followed by time-lapse microscopy was performed to evaluate migration of neutrophils from liver to tailfin injury. Metformin and Pentoxifylline were used to pharmacologically reduce NASH and liver inflammation. We found that larvae with NASH display systemic inflammation and increased myelopoiesis. NASH larvae display a dysfunctional and exacerbated neutrophil response to tailfin injury, characterized by increased neutrophil recruitment, and delayed resolution of inflammation. Interestingly, we showed that neutrophils undergo reverse migration from the NASH liver to the wounded tailfin area. Finally, pharmacological treatment of NASH with Pentoxifylline and Metformin significantly reduced systemic chronic inflammation and the exacerbated recruitment of neutrophils to tissue injury. Taken together, our findings suggest that NASH exacerbates neutrophilic inflammation probably via neutrophil priming at the liver, which can further undergo reverse migration and respond to secondary inflammatory triggers such as tissue injury. Reverse migration of primed neutrophils from the liver might be an important mechanism that fuels the exacerbated neutrophil response observed in NASH conditions and associated metainflammation contributing to poor prognosis and increasing death in patients with metabolic syndrome.


title: Cockroaches adjust body and appendages to traverse cluttered large obstacles

abstract: To traverse complex natural terrain, animals often transition between locomotor modes. It is well known that locomotor transitions can be induced by switching in neural control circuits or be driven by a need to minimize metabolic energetic cost. Recent work discovered that locomotor transitions in complex 3-D terrain cluttered with large obstacles can also emerge from physical interaction with the environment controlled by the nervous system. To traverse cluttered, stiff grass-like beams, the discoid cockroach often transitions from using a strenuous pitch mode to push across to using a less strenuous roll mode to maneuver through the gaps, during which a potential energy barrier must be overcome. Although previous robotic physical modeling demonstrated that kinetic energy fluctuation from body oscillation generated by leg propulsion can help overcome the barrier and facilitate this transition, the animal was observed to transition even when the barrier still exceeds kinetic energy fluctuation. Here, we further studied whether and how the cockroach makes active adjustments to facilitate this locomotor transition to traverse cluttered beams. We observed that the animal flexed its head and abdomen, reduced hind leg sprawl, and used both hind legs differentially during the pitch-to-roll transition, which were absent when running on a flat ground. Using a refined potential energy landscape with additional degrees of freedom modeling these adjustments, we found that head flexion did not substantially reduce the transition barrier, whereas the leg sprawl reduction did so dramatically. We discussed likely functions of the observed adjustments and suggested future directions.

category: animal behavior and cognition