title: Accurate de novo identification of biosynthetic gene clusters with GECCO

abstract: Biosynthetic gene clusters (BGCs) are enticing targets for (meta)genomic mining efforts, as they may encode novel, specialized metabolites with potential uses in medicine and biotechnology. Here, we describe GECCO (GEne Cluster prediction with COnditional random fields; https://gecco.embl.de), a high-precision, scalable method for identifying novel BGCs in (meta)genomic data using conditional random fields (CRFs). Based on an extensive evaluation of de novo BGC prediction, we found GECCO to be more accurate and over 3x faster than a state-of-the-art deep learning approach. When applied to over 12,000 genomes, GECCO identified nearly twice as many BGCs compared to a rule-based approach, while achieving higher accuracy than other machine learning approaches. Introspection of the GECCO CRF revealed that its predictions rely on protein domains with both known and novel associations to secondary metabolism. The method developed here represents a scalable, interpretable machine learning approach, which can identify BGCs de novo with high precision.

category: bioinformatics


title: Tree Diet: Reducing the Treewidth to Unlock FPT Algorithms in RNA Bioinformatics

abstract:
Hard graph problems are ubiquitous in Bioinformatics, inspiring the design of specialized Fixed-Parameter Tractable algorithms, many of which rely on a combination of tree-decomposition and dynamic programming. The time/space complexities of such approaches hinge critically on low values for the treewidth tw of the input graph. In order to extend their scope of applicability, we introduce the TO_SCPLOWREEC_SCPLOW-DO_SCPLOWIETC_SCPLOW problem, i.e. the removal of a minimal set of edges such that a given tree-decomposition can be slimmed down to a prescribed treewidth tw'. Our rationale is that the time gained thanks to a smaller treewidth in a parameterized algorithm compensates the extra post-processing needed to take deleted edges into account.

Our core result is an FPT dynamic programming algorithm for TO_SCPLOWREEC_SCPLOW-DO_SCPLOWIETC_SCPLOW, using 2O(tw)n time and space. We complement this result with parameterized complexity lower-bounds for stronger variants (e.g., NP-hardness when tw' or tw - tw' is constant). We propose a prototype implementation for our approach which we apply on difficult instances of selected RNA-based problems: RNA design, sequence-structure alignment, and search of pseudoknotted RNAs in genomes, revealing very encouraging results. This work paves the way for a wider adoption of tree-decomposition-based algorithms in Bioinformatics.


title: Asynchronous glutamate exocytosis is enhanced in low release probability synapses and is widely dispersed across the active zone

abstract: The balance between fast synchronous and delayed asynchronous release of neurotransmitters has a major role in defining computational properties of neuronal synapses and regulation of neuronal network activity. However, how it is tuned at the single synapse level remains poorly understood. Here, using the fluorescent glutamate sensor SF-iGluSnFR, we image quantal vesicular release in tens to hundreds of individual synaptic outputs (presynaptic boutons) from single pyramidal cells in culture with 4 millisecond temporal resolution, and localise vesicular release sites with ~ 75 nm spatial resolution. We find that the ratio between synchronous and asynchronous synaptic vesicle exocytosis varies extensively among presynaptic boutons supplied by the same axon, and that asynchronous release fraction is elevated in parallel with short-term facilitation at synapses with low release probability. We further demonstrate that asynchronous exocytosis sites are more widely distributed within the presynaptic release area than synchronous sites. These findings are consistent with a model in which functional presynaptic properties are regulated via a synapsespecific adjustment of the coupling distance between presynaptic Ca2+ channels and releaseready synaptic vesicles. Together our results reveal a universal relationship between the two major functional properties of synapses - the timing and the overall probability of neurotransmitter release.

category: neuroscience


title: Phonological and temporal regularities lead to differential ERP effects in self- and externally generated speech

abstract: Some theories of predictive processing propose reduced sensory and neural responses to anticipated events. Support comes from M/EEG studies, showing reduced auditory N1 and P2 responses to self-compared to externally generated events, or when stimulus properties are more predictable (e.g. prototypical). The current study examined the sensitivity of N1 and P2 responses to statistical regularities of speech. We employed a motor-to-auditory paradigm comparing ERP responses to externally and self-generated pseudowords, varying in phonotactic probability and syllable stress. We expected to see N1 and P2 suppression for self-generated stimuli, with greater suppression effect for more predictable features such as high phonotactic probability and first syllable stress in pseudowords. We observe an interaction between phonotactic probability and condition on the N1 amplitude, with an enhanced effect of phonotactic probability in processing self-generated stimuli. However, the directionality of this effect was reversed compared to what was expected, namely a larger N1 amplitude for high probability items, possibly indicating a perceptual bias toward the more predictable item. We further observed an effect of syllable stress on the P2 amplitude, with greater amplitudes in response to first syllable stress items. The current results suggest that phonotactic probability plays an important role in processing self-generated speech, supporting feedforward models involved in speech production.


title: Splice-specific lyn knockout mice reveal a dominant function of LynB in preventing autoimmunity

abstract: The unique roles of the Src-family kinases LynA and LynB in immune activating and inhibitory signaling have eluded definition. Here we report that LynB, the shorter splice product of lyn, carries the dominant immunosuppressive function. We used CRISPR/Cas9 gene editing to constrain lyn splicing and expression to a single product: LynAKO or LynBKO mice. While activities of both isoforms regulate homeostatic Lyn expression, only LynB protects against autoimmune disease. LynBKO monocytes and dendritic cells are TLR4-hyper-responsive, and TLR4 expression increases with age in LynBKO myeloid and B cells. These changes are accompanied by the development of an inflammatory disease that resembles human systemic lupus erythematosus (SLE). The interplay between LynB and TLR4 likely underlies the autoimmunity risk associated with LYN hypomorph and TLR4 hypermorph alleles.

category: immunology


title: Engineering a customizable antibacterial T6SS-based platform in Vibrio natriegens

abstract: Bacterial pathogens are a major risk to human, animal, and plant health. To counteract the spread of antibiotic resistance, alternative antibacterial strategies are urgently needed. Here, we constructed a proof-of-concept customizable, modular, and inducible antibacterial toxin delivery platform. By engineering a type VI secretion system (T6SS) that is controlled by an externally induced on/off switch, we transformed the safe bacterium, Vibrio natriegens, into an effective antibacterial weapon. Furthermore, we demonstrated that the delivered effector repertoire, and thus the toxicity range of this platform, can be easily manipulated and tested. We believe that this platform can serve as a foundation for novel antibacterial bio-treatments, as well as a unique tool to study antibacterial toxins.

category: microbiology


title: Imaging Nanoscale Nuclear Structures with Expansion Microscopy

abstract:
Commonly applied super-resolution light microscopies have provided insight into subcellular processes at the nanoscale. However, imaging depth, speed, throughput and cost remain significant challenges, reducing the numbers of three-dimensional, nanoscale processes that can be investigated and the number of laboratories able to undertake such analysis. Expansion microscopy solves many of these limitations but its application to imaging nuclear processes has been constrained by concerns of unequal nuclear expansion.

Here we demonstrate the conditions for isotropic expansion of the nucleus. Using DNA damage response proteins, BRCA1, 53BP1 and RAD51 as exemplars we quantitatively describe the three-dimensional nanoscale organisation of over 50,000 DNA damage response structures. We demonstrate the ability to assess chromatin regulated events and show the simultaneous assessment of four elements. This study thus provides the means by which expansion microscopy can contribute to the investigation of nanoscale nuclear processes.

category: molecular biology


title: Unbiased and UMI-informed sequencing of cell-free miRNAs at single-nucleotide resolution

abstract: Terminal nucleotidyl transferases are enzymes that add non-templated nucleotides to RNA molecules. In the case of microRNAs, this process was shown to be functionally relevant for their maturation process and generation of isomiRs with non-canonical mRNA targets. Deconvolution of these posttranscriptional modifications is challenging in particular for extracellular miRNAs that are considered as a target for minimally-invasive diagnostics. Massively parallel RNA sequencing is the only method that can truthfully reveal isomiR diversity in biological samples and determine relative quantities. Improvements aside, current small RNA sequencing strategies remain imprecise. We developed IsoSeek that diverges from these methods by making use of randomized 5- and 3-adapters combined with a 10N unique molecular identifier (UMI). Using synthetic miRNA and isomiR spike-in sets and testing depletion and RNA competition strategies in 7 sequencing rounds of >100 samples, we rigorously optimized and validated the technical accuracy of the IsoSeek method. In genetically-altered HEK293, we characterized the terminal uridylase (TUT4/TUT7) dependent miRNA uridylome and discovered extensive uridylation of disease-associated miRNAs. Notably, 3-uridylated isomiR profiles of plasma extracellular vesicles (EVs) rely on UMI-correction. Thus, IsoSeek advances our knowledge of cell-free miRNAs and supports development into non-invasive biomarkers.

category: genomics


title: A high-continuity and annotated tomato reference genome

abstract: Genetic and functional genomics studies require a high-quality genome assembly. Tomato (Solanum lycopersicum), an important horticultural crop, is an ideal model species for the study of fruit development. Here, we assembled an updated reference genome of S. lycopersicum cv. Heinz 1706 that was 799.09 Mb in length, containing 34,384 predicted protein-coding genes and 65.66% repetitive sequences. By comparing the genomes of S. lycopersicum and S. pimpinellifolium LA2093, we found a large number of genomic fragments probably associated with human selection, which may have had crucial roles in the domestication of tomato. Our results offer opportunities for understanding the evolution of the tomato genome and will facilitate the study of genetic mechanisms in tomato biology. Information for the assembled genome SLT1.0 was deposited both into the Genome Warehouse (GWH) database (https://bigd.big.ac.cn/gwh/) in the BIG Data Center under Accession Number GWHBAUD00000000.


title: Delta oscillations coordinate intra-cerebellar and cerebello-hippocampal network dynamics during sleep

abstract:
During sleep, the widespread coordination of neuronal oscillations across both cortical and subcortical brain regions is thought to support various physiological functions, including memory consolidation. However, how sleep-related activity within the brains largest sensorimotor structure, the cerebellum, is multiplexed with well described sleep-related mechanisms in regions such as the hippocampus remains unknown.

To address this gap in knowledge, we simultaneously recorded from the dorsal hippocampus and three distinct regions of the cerebellum (Crus I, lobule VI and lobules II/III) during natural murine sleep. We found that LFP oscillations are coordinated between the two structures in a sleep-stage specific manner. Particularly during non-REM sleep, prominent delta frequency coherence was observed between lobule VI and hippocampus. We additionally observed that non-REM associated hippocampal sharp wave ripple activity can drive discrete LFP modulation in all the recorded cerebellar regions, with the shortest latency effects observed in lobule VI.

We also describe discrete phasic sharp potentials, synchronized across cerebellar regions, which were strongly phase locked to the peak of ongoing cerebellar delta oscillations and found in greatest numbers during REM. These phasic sharp potentials recorded within the cerebellar cortex were found to be phase-locked to the trough of the hippocampal theta oscillation, further suggesting cross-structural coordination. During REM, cerebellar delta oscillation phase significantly modulated hippocampal theta frequency, and this effect was greatest when phasic sharp potentials were most abundant. Within all three cerebellar regions, prominent LFP oscillations were observed at both low (delta, <4 Hz) and very high frequencies (~ 250 Hz) during non-REM and REM sleep. Intra-cerebellar cross-frequency analysis revealed that delta frequency oscillations strongly modulate those in the very high frequency range.

Together, these results reveal multiple candidate physiological mechanisms to support  offline, bi-directional interaction within distributed cerebello-hippocampal networks. In particular, we describe a prominent cerebellar delta oscillation, which appears to act as a temporal coordinator of cerebellar activity at both local (intra-cerebellar) and distributed (cerebello-hippocampal) network levels.


title: Semantic object-scene inconsistencies affect eye movements, but not in the way predicted by contextualized meaning maps

abstract: Semantic information is important in eye-movement control. An important semantic influence on gaze guidance relates to object-scene relationships: objects that are semantically inconsistent with the scene attract more fixations than consistent objects. One interpretation of this effect is that fixations are driven towards inconsistent objects because they are semantically more informative. We tested this explanation using contextualized meaning maps, a method that is based on crowd-sourced ratings to quantify the spatial distribution of context-sensitive  meaning in images. In Experiment 1, we compared gaze data and contextualized meaning maps for images, in which objects-scene consistency was manipulated. Observers fixated more on inconsistent vs. consistent objects. However, contextualized meaning maps did not assigned higher meaning to image regions that contained semantic inconsistencies. In Experiment 2, a large number of raters evaluated the meaningfulness of a set of carefully selected image-regions. The results suggest that the same scene locations were experienced as slightly less meaningful when they contained inconsistent compared to consistent objects. In summary, we demonstrated that - in the context of our rating task - semantically inconsistent objects are experienced as less meaningful than their consistent counterparts, and that contextualized meaning maps do not capture prototypical influences of image meaning on gaze guidance.


title: Forest structure and degradation drive canopy gap sizes across the Brazilian Amazon

abstract:
Canopy gaps are openings in the forest canopy resulting from branch fall and tree mortality events. Light reaches the lower layers of the canopy through these gaps, enabling understory trees to grow and maintaining the high heterogeneity and biodiversity of tropical forests. The size-frequency distribution of canopy gaps follows a power-law distribution, and the slope of this power-law () is a key indicator of forest structure and dynamics.

We detected canopy gaps using a unique LiDAR data set consisting of 650 transects randomly distributed across the Brazilian Amazon Forest providing an unprecedented perspective on forest structural variation over 2500 km2 of forest. We then investigated how  varied with forest structure, elevation, soil fertility, water deficit, wind gust speed and lightning intensity.

We found that human modified forests had more large gaps than intact forests. Within the intact forests we observed a large-scale Northwest to Southeast pattern in  (more large gaps in the Southeast), which aligns with recent work on tree mortality rates. The two most important variables in predicting  were median canopy height and maximum modeled height, which had opposite effects on the number of large gaps. Forests with higher median canopy height contain fewer large gaps but the presence of very tall trees was associated with more large gaps. Environmental variables were of secondary importance in our model, with larger gaps occurring in drier forests with high soil fertility, wind speed, and lightning intensity.

The distribution of large gaps in the forest canopy varies substantially over the Brazilian Amazon as a result of canopy structure and mortality rates. We mapped this variation and found more large gaps in human modified forests, forests on fertile soils and those exposed to higher wind, lightning and drought stress. Increasing extreme weather events due to climate change may therefore increase the number of large gaps in currently intact forests, causing them to resemble human modified forests.

category: ecology


title: Mobile antimicrobial resistance genes in probiotics

abstract: Even though people around the world tend to consume probiotic products for their beneficial health effects on a daily basis, recently, concerns were outlined regarding the uptake and potential intestinal colonisation of the bacteria that they transfer. These bacteria are capable of executing horizontal gene transfer (HGT) which facilitates the movement of various genes, including antimicrobial resistance genes (ARGs), among the donor and recipient bacterial populations. Within our study, 47 shotgun sequencing datasets deriving from various probiotic samples (isolated strains and metagenomes) were bioinformatically analysed. We detected more than 70 ARGs, out of which rpoB mutants conferring resistance to rifampicin, tet(W/N/W) and potentially extended-spectrum beta-lactamase (ESBL) coding TEM-116 were the most common. Numerous ARGs were associated with integrated mobile genetic elements, plasmids or phages promoting the HGT. Our findings raise clinical and public health concerns as the consumption of probiotic products may lead to the transfer of ARGs to human gut bacteria.


title: Semi-supervised identification of SARS-CoV-2 molecular targets

abstract: SARS-CoV-2 genomic sequencing efforts have scaled dramatically to address the current global pandemic and aid public health. In this work, we analyzed a corpus of 66,000 SARS-CoV-2 genome sequences. We developed a novel semi-supervised pipeline for automated gene, protein, and functional domain annotation of SARS-CoV-2 genomes that differentiates itself by not relying on use of a single reference genome and by overcoming atypical genome traits. Using this method, we identified the comprehensive set of known proteins with 98.5% set membership accuracy and 99.1% accuracy in length prediction compared to proteome references including Replicase polyprotein 1ab (with its transcriptional slippage site). Compared to other published tools such as Prokka (base) and VAPiD, we yielded an 6.4- and 1.8-fold increase in protein annotations. Our method generated 13,000,000 molecular target sequences-- some conserved across time and geography while others represent emerging variants. We observed 3,362 non-redundant sequences per protein on average within this corpus and describe key D614G and N501Y variants spatiotemporally. For spike glycoprotein domains, we achieved greater than 97.9% sequence identity to references and characterized Receptor Binding Domain variants. Here, we comprehensively present the molecular targets to refine biomedical interventions for SARS-CoV-2 with a scalable high-accuracy method to analyze newly sequenced infections.


title: LiPyphilic: A Python toolkit for the analysis of lipid membrane simulations

abstract:
Molecular dynamics simulations are now widely used to study emergent phenomena in lipid membranes with complex compositions. Here, we present LiPyphilic - a fast, fully tested, and easy to install Python package for analysing such simulations. Analysis tools in LiPyphilic include the identification of cholesterol flip-flop events, the classification of local lipid environments, and the degree of interleaflet registration. LiPyphilic is both force field and resolution agnostic, and thanks to the powerful atom selection language of MDAnalysis it can handle membranes with highly complex compositions. LiPyphilic also offers two on-the-fly trajectory transformations to i) fix membranes split across periodic boundaries and ii) perform nojump coordinate unwrapping. Our implementation of nojump unwrapping accounts for fluctuations in box volume under the NPT ensemble -- an issue that most current implementations have overlooked. The full documentation of LiPyphilic, including installation instructions, is available at https://lipyphilic.readthedocs.io/en/latest.

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category: biophysics


title: Discrete mechanical model of lamellipodial actin network implements molecular clutch mechanism and generates arcs and microspikes

abstract: Mechanical forces, actin filament turnover, and adhesion to the extracellular environment regulate lamellipodial protrusions. Computational and mathematical models at the continuum level have been used to investigate the molecular clutch mechanism, calculating the stress profile through the lamellipodium and around focal adhesions. However, the forces and deformations of individual actin filaments have not been considered while interactions between actin networks and actin bundles is not easily accounted with such methods. We develop a filament-level model of a lamellipodial actin network undergoing retrograde flow using 3D Brownian dynamics. Retrograde flow is promoted in simulations by pushing forces from the leading edge (due to actin polymerization), pulling forces (due to molecular motors), and opposed by viscous drag in cytoplasm and focal adhesions. Simulated networks have densities similar to measurements in prior electron micrographs. Connectivity between individual actin segments is maintained by permanent and dynamic crosslinkers. Remodeling of the network occurs via the addition of single actin filaments near the leading edge and via filament bond severing. We investigated how several parameters affect the stress distribution, network deformation and retrograde flow speed. The model captures the decrease in retrograde flow upon increase of focal adhesion strength. The stress profile changes from compression to extension across the leading edge, with regions of filament bending around focal adhesions. The model reproduces the observed reduction in retrograde flow speed upon exposure to cytochalasin D, which halts actin polymerization. Changes in crosslinker concentration and dynamics, as well as in the orientation pattern of newly added filaments demonstrate the models ability to generate bundles of filaments perpendicular (actin arcs) or parallel (microspikes) to the protruding direction.


title: Modelling of synaptic interactions between two brainstem half-centre oscillators that coordinate breathing and swallowing

abstract:
AO_SCPLOWBSTRACTC_SCPLOWRespiration and swallowing are vital orofacial motor behaviours that require the coordination of the activity of two brainstem central pattern generators (r-CPG, sw-CPG). Here, we use computational modelling to further elucidate the neural substrate for breathing-swallowing coordination. We progressively construct several computational models of the breathing-swallowing circuit, starting from two interacting half-centre oscillators for each CPG. The models are based exclusively on neuronal nodes with spike-frequency adaptation, having a parsimonious description of intrinsic properties. These basic models undergo a stepwise integration of synaptic connectivity between central sensory relay, sw- and r-CPG neuron populations to match experimental data obtained in a perfused brainstem preparation. In the model, stimulation of the superior laryngeal nerve (SLN, 10s) reliably triggers sequential swallowing with concomitant glottal closure and suppression of inspiratory activity, consistent with the motor pattern in experimental data. Short SLN stimulation (100ms) evokes single swallows and respiratory phase resetting yielding similar experimental and computational phase response curves. Subsequent phase space analysis of model dynamics provides further understanding of SLN-mediated respiratory phase resetting. Consistent with experiments, numerical circuit-busting simulations show that deletion of ponto-medullary synaptic interactions triggers apneusis and eliminates glottal closure during sequential swallowing. Additionally, systematic variations of the synaptic strengths of distinct network connections predict vulnerable network connections that can mediate clinically relevant breathing-swallowing disorders observed in the elderly and patients with neurodegenerative disease. Thus, the present model provides novel insights that can guide future experiments and the development of efficient treatments for prevalent breathing-swallowing disorders.

KO_SCPLOWEYC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWPOINTSC_SCPLOWO_LIThe coordination of breathing and swallowing depends on synaptic interactions between two functionally distinct central pattern generators (CPGs) in the dorsal and ventral brainstem.
C_LIO_LIWe model both CPGs as half-centre oscillators with spike-frequency adaptation to identify the minimal connectivity sufficient to mediate physiologic breathing-swallowing interactions.
C_LIO_LIThe resultant computational model(s) can generate sequential swallowing patterns including concomitant glottal closure during simulated 10s stimulation of the superior laryngeal nerve (SLN) consistent with experimental data.
C_LIO_LIIn silico, short (100 ms) SLN stimulation triggers a single swallow which modulates the respiratory cycle duration consistent with experimental recordings.
C_LIO_LIBy varying the synaptic connectivity strengths between the two CPGs and the sensory relay neurons, and by inhibiting specific nodes of the network, the model predicts vulnerable network connections that may mediate clinically relevant breathing-swallowing disorders.
C_LI

category: physiology


title: Biological complexity facilitates tuning of the neuronal parameter space

abstract:
The electrical and computational properties of neurons in our brains are determined by a rich repertoire of membrane-bound ion channels and elaborate dendritic trees. However, the precise reason for this inherent complexity remains unknown. Here, we generated large stochastic populations of biophysically realistic hippocampal granule cell models comparing those with all 15 ion channels to their reduced but functional counterparts containing only 5 ion channels. Strikingly, valid parameter combinations in the full models were more frequent and more stable in the face of perturbations. Scaling up the numbers of ion channels artificially in the reduced models recovered these advantages confirming the key contribution of the actual number of ion channels. We conclude that the diversity of ion channels allows a neuron to achieve a target excitability through random channel expression with increased robustness and higher flexibility.

Significance statementOver the course of billions of years, evolution has led to a wide variety of biological systems. The emergence of the more complex among these seems surprising in the light of the high demands on searching viable solutions in a correspondingly high-dimensional parameter space. In realistic neuron models with their inherently complex ion channel composition, we find a surprisingly large number of viable solutions when selecting parameters randomly. This effect is strongly reduced in models with less ion channel types but is recovered when inserting additional artificial ion channels. Because concepts from probability theory provide a plausible explanation for such an improved arrangement of valid model parameters, we propose that this generalises to evolutionary selection in other complex biological systems.

In briefStudying ion channel diversity in neuronal models we show how robust biological systems may evolve not despite but through their complexity.

HighlightsO_LI15 channel model of hippocampal granule cells (GCs) reduces to 5 ion channels without loss of spiking behaviour.
C_LIO_LIBut knocking out ion channels can be compensated only in the full model.
C_LIO_LIRandom sampling leads to[~] 6% solutions in full but only[~] 1% in reduced model.
C_LIO_LILaw of large numbers generalises our observations to other complex biological systems.
C_LI


title: Protective heterologous T cell immunity in COVID-19 induced by MMR and Tdap vaccine antigens

abstract: T cells are critical for control of viral infection and effective vaccination. We investigated whether prior Measles-Mumps-Rubella (MMR) or Tetanus-Diphtheria-pertussis (Tdap) vaccination elicit cross-reactive T cells that mitigate COVID-19. Using co-cultures of antigen presenting cells (APC) loaded with antigens and autologous T cells, we found a high correlation between responses to SARS-CoV-2 (Spike-S1 and Nucleocapsid) and MMR and Tdap vaccine proteins in both SARS-CoV-2 infected individuals and individuals immunized with mRNA-based SARS-CoV-2 vaccines. The overlapping T cell population contained effector memory T cells (TEMRA) previously implicated in anti-viral immunity and their activation required APC-derived IL-15. TCR- and scRNA-sequencing detected cross-reactive clones with TEMRA features among the cells recognizing SARS-CoV-2, MMR and Tdap epitopes. A propensity-weighted analysis of 73,582 COVID-19 patients revealed that severe disease outcomes (hospitalization and transfer to intensive care unit or death) were reduced in MMR or Tdap vaccinated individuals by 38-32% and 23-20% respectively. In summary, SARS-CoV-2 re-activates memory T cells generated by Tdap and MMR vaccines, which may reduce disease severity.


title: Highly functional Cellular Immunity in SARS-CoV-2 Non-Seroconvertors is associated with immune protection

abstract:
The role of T cells in the control of SARS-CoV-2 infection has been underestimated in favor of neutralizing antibodies. However, cellular immunity is essential for long-term viral control and protection from disease severity. To understand T-cell immunity in the absence of antibody generation we focused on a group of SARS-CoV-2 Non-Seroconvertors (NSC) recovered from infection. We performed an immune comparative analysis of SARS-CoV-2 infected individuals stratified by the absence or presence of seroconversion and disease severity. We report high levels of total naive and low effector CD8+ T cells in NSC. Moreover, polyfunctional Nucleocapsid (NP)-specific CD8+ T-cell responses, as well as reduced levels of T-cell activation monitored by PD-1 and activation-induced markers, were distinctive immunological traits in NSC. Longitudinal data support the stability of the NSC phenotype over three months. Our results implicate highly functional SARS-CoV-2 Spike and NP T-cell responses with low immune activation in protection from disease severity in the absence of seroconversion.

SUMMARYTo understand SARS-CoV-2 specific T-cell immunity in the absence of seroconversion, we characterized immunological features of Non-Seroconvertors recovered from infection. Highly functional specific T-cell responses and low immune activation were determinants of immune protection from severe disease.


title: Tropism, susceptibility, infectivity, and cytokine releases of differentiated human tonsillar epithelial cells by different Influenza viruses

abstract:
Human tonsil epithelium cells (HTEC) are a heterogeneous group of actively differentiating cells comprising stratified squamous epithelial and reticulated crypt cells with abundant keratin expression. We hypothesized that the tonsils are a primary site for influenza infection and sustained viral replication. Primary HTEC were grown using an air-liquid culture and infected apically with different influenza viruses (IVs) to measure viral growth kinetics. These cultures were highly differentiated, with subpopulations of heterogenous surface stratified squamous cells rich with both cilia and microvilli; these cells contained more 2,6-linked sialic acids, those preferentially bound by human IVs, than 2,3-linked avian like sialic acids. The stratified squamous cells were interrupted by patches of reticular epithelial cells rich in 2,3-linked sialic acids. The HTEC were permissive for influenza A and B virus replication. Following infection, a subset of cells, mostly ciliated cells, underwent apoptosis while others remained intact despite being positive for IV nucleoprotein. H3N2 virus antigen colocalized with non-ciliated cells while H1N1 virus antigen was mostly associated with ciliated cells. Exposure of HTECs to IVs triggers an early proinflammatory response that fluctuates between viruses. The H3N2 IV induces an early response that persists, whereas pH1N1 induces a primarily late response in HTECs. Our results implicated HTEC as a site for IV replication. The HTEC differentiated system provides a valuable in vitro model for studying cellular tropism, infectivity, cytokine responses and the pathogenesis of IVs.

IMPORTANCETo develop an effective intervention against influenza, it is important to identify host factors affecting transmission, pathogenesis, and immune response. Tonsils are lymphoepithelial organs characterized by infiltration of B and T lymphocytes into the squamous epithelium of tonsillar crypts, beneath which germinal centers play key roles in antigen processing and immune response. The heterogenicity of HTECs as well as the sialic acid distributions supports the replication of IVs and may play a role in IV adaptation. Furthermore, Tonsillectomy is a surgical procedure in which tonsils are fully removed from the human throat and may contribute to the diverse outcomes among infected individuals.


title: A novel highly potent inhibitor of TMPRSS2-like proteases blocks SARS-CoV-2 variants of concern and is broadly protective against infection and mortality in mice

abstract: The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced against emerging variants of concern (VOCs) 1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against VOCs 3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs), such as TMPRSS2, whose essential role in the virus lifecycle is responsible for the cleavage and priming of the viral spike protein 5-7. Here, we identify and characterize a small-molecule compound, N-0385, as the most potent inhibitor of TMPRSS2 reported to date. N-0385 exhibited low nanomolar potency and a selectivity index of >106 at inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids 8. Importantly, N-0385 acted as a broad-spectrum coronavirus inhibitor of two SARS-CoV-2 VOCs, B.1.1.7 and B.1.351. Strikingly, single daily intranasal administration of N-0385 early in infection significantly improved weight loss and clinical outcomes, and yielded 100% survival in the severe K18-human ACE2 transgenic mouse model of SARS-CoV-2 disease. This demonstrates that TTSP-mediated proteolytic maturation of spike is critical for SARS-CoV-2 infection in vivo and suggests that N-0385 provides a novel effective early treatment option against COVID-19 and emerging SARS-CoV-2 VOCs.


title: A New Assay Capturing Chromosome Fusions Shows a Protection Trade-off at Telomeres and NHEJ Vulnerability to Low Density Ionising Radiation

abstract: Chromosome fusions threaten genome integrity and promote cancer by engaging catastrophic mutational processes, namely chromosome breakage-fusion-bridge cycles and chromothripsis. Chromosome fusions are frequent in cells incurring telomere dysfunctions or those exposed to DNA breakage. Their occurrence and therefore their contribution to genome instability in unchallenged cells is unknown. To address this issue, we constructed a genetic assay able to capture and quantify rare chromosome fusions in budding yeast. This chromosome fusion capture assay (CFC) relies on the controlled inactivation of one centromere to rescue unstable dicentric chromosome fusions. It is sensitive enough to quantify the basal rate of end-to-end chromosome fusions occurring in wild-type cells. These fusions depend on canonical nonhomologous end-joining (NHEJ). Our results show that chromosome end protection results from a trade-off at telomeres between positive effectors (Rif2, Sir4, telomerase) and a negative effector partially antagonizing them (Rif1). The CFC assay also captures NHEJ-dependent chromosome fusions induced by ionising radiation. It provides evidence for chromosomal rearrangements stemming from a single photon-matter interaction.


title: Immunolocalization studies of vimentin and ACE2 on the surface of cells exposed to SARS-CoV-2 Spike proteins

abstract: The Spike protein from SARS-CoV-2 mediates docking of the virus onto cells and contributes to viral invasion. Several cellular receptors are involved in SARS-CoV-2 Spike docking at the cell surface, including ACE2 and neuropilin. The intermediate filament protein vimentin has been reported to be present at the surface of certain cells and act as a co-receptor for several viruses; furthermore, its potential involvement in interactions with Spike proteins has been proposed. Here we have explored the binding of Spike protein constructs to several cell types using low-temperature immunofluorescence approaches in live cells, to minimize internalization. Incubation of cells with tagged Spike S or Spike S1 subunit led to discrete dotted patterns at the cell surface, which showed scarce colocalization with a lipid raft marker, but consistent coincidence with ACE2. Under our conditions, vimentin immunoreactivity appeared as spots or patches unevenly distributed at the surface of diverse cell types. Remarkably, several observations including potential antibody internalization and adherence to cells of vimentin-positive structures present in the extracellular medium exposed the complexity of vimentin cell surface immunoreactivity, which requires careful assessment. Notably, overall colocalization of Spike and vimentin signals markedly varied with the cell type and the immunodetection sequence. In turn, vimentin-positive spots moderately colocalized with ACE2; however, a particular enrichment was detected at elongated structures positive for acetylated tubulin, consistent with primary cilia, which also showed Spike binding. Thus, these results suggest that vimentin-ACE2 interaction could occur at selective locations near the cell surface, including ciliated structures, which can act as platforms for SARS-CoV-2 docking.

category: cell biology


title: The length scale of multivalent interactions is evolutionarily conserved in fungal and vertebrate phase-separating proteins

abstract: One key feature of proteins that form liquid droplets by phase separation inside a cell is the presence of multiple sites - multivalency - that mediate interactions with other proteins. We know little about the variation of multivalency on evolutionary time scales. Here, we investigated the long-term evolution ([~]600 million years) of multivalency in fungal mRNA decapping subunit 2 protein (Dcp2), and in the FET protein family. We found that multivalency varies substantially among the orthologs of these proteins. However, evolution has maintained the length scale at which sequence motifs that enable protein-protein interactions occur. That is, the total number of such motifs per hundred amino acids is higher and less variable than expected by neutral evolution. To help explain this evolutionary conservation, we developed a conformation classifier using machine-learning algorithms. This classifier demonstrates that disordered segments in Dcp2 and FET proteins tend to adopt compact conformations, which is necessary for phase separation. Thus, the evolutionary conservation we detected may help proteins preserve the ability to undergo phase separation. Altogether, our study reveals that the length scale of multivalent interactions is an evolutionarily conserved feature of two classes of phase-separating proteins in fungi and vertebrates.

category: evolutionary biology


title: Phasing Alleles Improves Network Inference with Allopolyploids

abstract: Accurately reconstructing the reticulate histories of polyploids remains a central challenge for understanding plant evolution. Although phylogenetic networks can provide insights into relationships among polyploid lineages, inferring networks may be hampered by the complexities of homology determination in polyploid taxa. We use simulations to show that phasing alleles from allopolyploid individuals can improve inference of phylogenetic networks under the multispecies coalescent. Phased allelic data can also improve divergence time estimates for networks, which is helpful for evaluating allopolyploid speciation hypotheses and proposing mechanisms of speciation. To achieve these outcomes, we present a novel pipeline that leverages a recently developed phasing algorithm to reliably phase alleles from polyploids. This pipeline is especially appropriate for target enrichment data, where depth of coverage is typically high enough to phase entire loci. We provide an empirical example in the North American Dryopteris fern complex that demonstrates how phasing can help reveal the mode of polyploidization and improve network inference. We establish that our pipeline (PATE: Phased Alleles from Target Enrichment data) is capable of recovering a high proportion of phased loci from both diploids and polyploids, and that these data improve network estimates compared to using haplotype consensus assemblies. This approach is shown to be especially effective in reticulate complexes where there are multiple hybridization events. The pipeline is available at: https://github.com/gtiley/Phasing.


title: Gene by environment interaction mouse model reveals a functional role for 5-hydroxymethylcytosine in neurodevelopmental disorders

abstract: Mouse knockouts of Cntnap2 exhibit altered neurodevelopmental behavior and a genome-wide disruption of 5-hydroxymethylcytosine (5hmC). Here we examined whether adult Cntnap2 heterozygous mice (Cntnap2+/-, lacking behavioral or neuropathological abnormalities) subjected to a prenatal stress would have disruptions in brain 5hmC levels and exhibit altered behaviors similar to the knockout mice. Adult prenatally stressed Cntnap2+/- female mice showed repetitive behaviors and altered sociability, similar to the homozygote phenotype. Genomic profiling revealed disruptions in hippocampal and striatal 5hmC levels that were correlated to altered transcript levels of genes linked to these phenotypes (e.g., Reln, Dst, Trio and Epha5). Chromatin-immunoprecipitation coupled with high-throughput sequencing and hippocampal nuclear lysate pull-down data indicated that 5hmC abundance alters the binding of the transcription factor CLOCK near the promoters of differentially expressed genes (e.g., Palld, Gigyf1, and Fry), providing a mechanistic role for 5hmC (disruption of transcription factor binding) in gene regulation of developmentally important genes.

category: genetics


title: The Automated Systematic Search Deduplicator (ASySD): a rapid, open-source, interoperable tool to remove duplicate citations in biomedical systematic reviews.

abstract:
BackgroundResearchers who perform systematic searches across multiple databases often identify duplicate publications. Identifying such duplicates ("deduplication") can be extremely time-consuming, but failure to remove these citations can, in the worst instance, lead to the wrongful inclusion of duplicate data. Many existing tools are not sensitive enough, lack interoperability with other tools, are not freely accessible, or are difficult to use without programming knowledge. Here, we report the performance of our Automated Systematic Search Deduplicator (ASySD), a novel tool to perform automated deduplication of systematic searches for biomedical reviews.

MethodsWe evaluated ASySDs performance on 5 unseen biomedical systematic search datasets of various sizes (1,845 - 79,880 citations), which had been deduplicated by human reviewers. We compared the performance of ASySD with Endnotes automated deduplication option and with the Systematic Review Accelerator Deduplication Module (SRA-DM).

ResultsASySD identified more duplicates than either SRA-DM or Endnote, with a sensitivity in different datasets of 0.95 to 0.99. The false-positive rate was comparable to human performance, with a specificity of 0.94-0.99. The tool took less than 1 hour to deduplicate all datasets.

ConclusionsFor duplicate removal in biomedical systematic reviews, ASySD is a highly sensitive, reliable, and time-saving tool. It is open source and freely available online as both an R package and a user-friendly web application.


title: Transcriptome analysis of heterogeneity in mouse model of metastatic breast cancer

abstract: Cancer metastasis is a complex process involving the spread of malignant cells from a primary tumor to distal organs. Understanding this cascade at a mechanistic level could provide critical new insights into the disease and potentially reveal new avenues for treatment. Transcriptome profiling of spontaneous cancer models is an attractive method to examine the dynamic changes accompanying tumor cell spread. However, such studies are often complicated by the underlying heterogeneity of the cell types involved. Here we performed a comprehensive analysis of tumor heterogeneity and organ tropism during breast cancer metastasis. We created a suite of organ-derived metastatic cell lines from the MMTV-PyMT mouse model. Bulk sequencing analyses uncovered tissue-specific genes across the different metastatic and primary tumor samples. We further investigated intratumoral heterogeneity by performing single-cell RNA-seq. These data revealed transcriptomes that may contribute to sub-clonal evolution during cancer progression. Collectively, the organ-derived cell lines provide a platform for comprehensive studies of metastatic breast cancer progression.

category: cancer biology


title: AZBA: A 3D Adult Zebrafish Brain Atlas for the Digital Age

abstract: Zebrafish have made significant contributions to our understanding of the vertebrate brain and the neural basis of behavior, earning a place as one of the most widely used model organisms in neuroscience. Their appeal arises from the marriage of low cost, early life transparency, and ease of genetic manipulation with a behavioral repertoire that becomes more sophisticated as animals transition from larvae to adults. To further enhance the use of adult zebrafish, we created the first fully segmented three-dimensional digital adult zebrafish brain atlas (AZBA). AZBA was built by combining tissue clearing, light-sheet fluorescence microscopy, and three-dimensional image registration of nuclear and antibody stains. These images were used to guide segmentation of the atlas into over 200 neuroanatomical regions comprising the entirety of the adult zebrafish brain. As an open source, online azba.wayne.edu), updatable digital resource, AZBA will significantly enhance the use of adult zebrafish in furthering our understanding of vertebrate brain function in both health and disease.


title: Impaired bidirectional communication between interneurons and oligodendrocyte precursor cells affects cognitive behavior

abstract: Cortical neural circuits are complex but very precise networks of balanced excitation and inhibition (E/I). Yet, the molecular and cellular mechanisms that form the E/I balance are just beginning to emerge. Here, using conditional GABAB receptor-deficient mice we identified a GABA/TNF-related cytokine (TNFSF12)-mediated bidirectional communication pathway between Parvalbumin-positive (PV+) fast spiking interneurons and oligodendrocyte precursor cells (OPCs) that determines the density and function of interneurons in the developing medial prefrontal cortex (mPFC). Interruption of the GABAergic signaling to OPCs resulted in reduced myelination and hypoactivity of interneurons, strong changes of cortical network activities and impaired cognitive behavior. In conclusion, glial transmitter receptors are pivotal elements in finetuning distinct brain functions.


title: Contingent-behavior assay shows zebrafish preference for alcohol is biphasic

abstract: Alcohol use disorders are complex, multifactorial phenomena with a large footprint within the global burden of diseases. Here, we report the development of an accessible, two-choice self-administration zebrafish assay (SAZA) to study the neurobiology of addiction. Using this assay, we first demonstrated that, while zebrafish avoid higher concentrations of alcohol, they are attracted to low concentrations. Pre-exposure to alcohol did not change this relative preference, but acute exposure to an alcohol deterrent approved for human use decreased alcohol self-administration. A pigment mutant used in whole-brain imaging studies displayed a similar relative alcohol preference profile, however, mutants in CCSER1, a gene associated with alcohol dependence in human genetic studies, showed a reversal in relative preference. The presence of a biphasic response (hormesis) in zebrafish validated a key aspect of vertebrate responses to alcohol. SAZA adds a new dimension for discovering novel alcohol deterrents, and studying the neurogenetics of addiction using the zebrafish.


title: Contingent-behavior assay to study the neurogenetics of addiction shows zebrafish preference for alcohol is biphasic


title: Circadian Control of Heparan Sulfate Levels Times Phagocytosis of Amyloid Beta Aggregates

abstract:
Alzheimers Disease (AD) is a neuroinflammatory disease characterized partly by the inability to clear, and subsequent build-up, of amyloid-beta (A{beta}). A{beta} clearance is regulated by several pathways and has a circadian component. However, the mechanism underlying the circadian clearance of A{beta} has not been defined. Myeloid-based phagocytosis, a key mechanism in the metabolism of A{beta}, is circadianly-regulated, presenting a potential mechanism for the circadian clearance of A{beta}. In this work, we revealed that the phagocytosis of A{beta}42 undergoes a daily oscillation that is dependent on the circadian clock. We found the circadian timing of global heparan sulfate proteoglycan (HSPG) biosynthesis was the molecular timer for the clock-controlled phagocytosis of A{beta} and that both HSPG binding and A{beta}42 aggregation were essential for this oscillation. These data highlight that circadian regulation in immune cells may play a role in the intricate relationship between the circadian clock and AD.

Graphical Abstract

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title: Pivotal Roles for Ribonucleases in Streptococcus pneumoniae Pathogenesis

abstract:
RNases perform indispensable functions in regulating gene expression in many bacterial pathogens by processing and/or degrading RNAs. Despite the pivotal role of RNases in regulating bacterial virulence factors, the functions of RNases have not yet been studied in the major human respiratory pathogen Streptococcus pneumoniae (pneumococcus). Here, we sought to determine the impact of two conserved RNases, the endoribonuclease RNase Y and exoribonuclease polynucleotide phosphorylase (PNPase), on the physiology and virulence of S. pneumoniae serotype 2 strain D39. We report that RNase Y and PNPase are essential for pneumococcal pathogenesis as both deletion mutants showed strong attenuation of virulence in murine models of invasive pneumonia. Genome-wide transcriptomic analysis revealed that nearly 200 mRNA transcripts were significantly up-regulated, whereas the abundance of several pneumococcal sRNAs, including the Ccn (CiaR Controlled Noncoding RNA) sRNAs, were altered in the {Delta}rny mutant relative to the wild-type strain. Additionally, lack of RNase Y resulted in pleiotropic phenotypes that included defects in pneumococcal cell morphology and growth in vitro. In contrast, {Delta}pnp mutants showed no growth defect in vitro, but differentially expressed a total of 40 transcripts including the tryptophan biosynthesis operon genes and numerous 5-cis-acting regulatory RNAs, a majority of which were previously shown to impact pneumococcal disease progression in mice using the serotype 4 strain TIGR4. Altogether our data suggest that RNase Y exerts a global impact on pneumococcal physiology, while PNPase-mediates virulence phenotypes, likely through sRNA regulation.

IMPORTANCEStreptococcus pneumoniae is a notorious human pathogen that adapts to conditions in distinct host tissues and responds to host cell interactions by adjusting gene expression. Ribonucleases (RNases) are key players that modulate gene expression by mediating the turnover of regulatory and protein-coding transcripts. Here, we characterized two highly conserved RNases, RNase Y and PNPase, and evaluated their impact on the S. pneumoniae transcriptome for the first time. We show that PNPase influences the levels of a narrow set of mRNAs, but a large number of regulatory RNAs primarily implicated in virulence control, whereas RNase Y has a more sweeping effect on gene expression, altering levels of transcripts involved in diverse cellular processes including cell division, metabolism, stress response, and virulence. This study further reveals that RNase Y regulates expression of genes governing competence by mediating the turnover of CiaR-controlled-noncoding (Ccn) sRNAs.


title: Sex-biased response to and brain cell infection by SARS-CoV-2 in a highly susceptible human ACE2 transgenic model

abstract: The COVID-19 pandemic is caused by SARS-CoV-2 infection. Human angiotensin-converting enzyme II (hACE2) has been identified as the receptor enabling SARS-CoV-2 host entry. To establish a mouse model for COVID-19, we generated transgenic mouse lines using the (HS4)2-pCAG-hACE2-HA-(HS4)2 transgene cassette, which expresses HA-tagged hACE2 under control of the CAG promoter and is flanked by HS4 insulators. Expression levels of the hACE2 transgene are respectively higher in lung, brain and kidney of our CAG-hACE2 transgenic mice and relatively lower in duodenum, heart and liver. The CAG-hACE2 mice are highly susceptibility to SARS-CoV-2 infection, with 100 PFU of SARS-CoV-2 being sufficient to induce 87.5% mortality at 9 days post-infection and resulting in a sole (female) survivor. Mortality was 100% at the higher titer of 1000 PFU. At lower viral titers, we also found that female mice exposed to SARS-CoV-2 infection suffered much less weight loss than male mice, implying sex-biased responses to SARS-CoV-2 infection. We subjected neuronal cultures to SARS-CoV-2 pseudovirus infection to ascertain the susceptibilities of neurons and astrocytes. Moreover, we observed that expression of SARS-CoV-2 Spike protein alters the synaptic responses of cultured neurons. Our transgenic mice may serve as a model for severe COVID-19 and sex-biased responses to SARS-CoV-2 infection, aiding in the development of vaccines and therapeutic treatments for this disease.


title: A temperate Siphoviridae bacteriophage isolate from Siberian tiger enhances the virulence of Methicillin-resistant Staphylococcus aureus through distinct mechanisms

abstract:
The emergence and worldwide spread of Methicillin-resistant Staphylococcus aureus (MRSA) poses a threat to human health. While bacteriophages are recognized as an effective alternative to treat infections caused by drug resistant pathogens, some bacteriophages in particular the temperate bacteriophage may also influence the virulence of the host bacteria in distinct ways. In this study, we isolated a bacteriophage vB_Saus_PHB21 from an epidermal sample of Siberian tiger (Panthera tigris altaica) using a MRSA strain SA14 as the indicator. Our following laboratory tests and whole genome sequencing analyses revealed that vB_Saus_PHB21 was a temperate bacteriophage belonging to the Siphoviridae family, and this bacteriophage did not contain any virulence genes. However, the integration of PHB21 genome into the host MRSA increased the bacterial capacities of cell adhesion, cell invasion, anti-phagocytosis and biofilm formation. Challenge of the lysogenic strain (SA14+) caused severer mortalities in both Galleria mellonella and mouse models. Mice challenged with SA14+ showed more serious organ lesions and produced higher inflammatory cytokines (IL-8, IFN-{gamma} and TNF-) compared to those challenged with SA14. In mechanism, we found the integration of PHB21 genome caused the upregulated expression of many genes encoding products involved in bacterial biofilm formation, adherence and invasion to host cells, anti-phagocytosis, and virulence. This study may provide novel knowledge of "bacteria-phage-interactions" in MRSA.

IMPORTANCEThe interaction between bacteriophage and bacteria is like a "double-edged sword": phages can either kill bacteria, or they may contribute to the bacterial fitness and virulence. In general, phages have positive impacts on bacterial fitness and virulence mainly because they carry antimicrobial resistance genes (ARGs) and/or virulence factors encoding genes (VFGs) and they can spread these harmful genes to the host bacteria. However, we found those phages which do not harbor ARGs and/or VFGs may also enhance the bacterial fitness and virulence. In addition, we also found the integration of phage genomes may lead to the upregulated expression of virulence associated genes in bacteria. Our study may provide new insights to redefine the relationship between phage and bacteria, and the results may also remind a cautious way to set phage-therapy for bacterial infections, before which the safety of a phage intends to be used should be fully evaluated.


title: High arsenic levels increase activity rather than diversity or abundance of arsenic metabolism genes in paddy soils

abstract:
Arsenic (As) metabolism genes are generally present in soils but their diversity, relative abundance, and transcriptional activity in response to different As concentrations remain unclear, limiting our understanding of the microbial activities that control the fate of an important environmental pollutant. To address this issue, we applied metagenomics and metatranscriptomics to paddy soils showing a gradient of As concentrations to investigate As resistance genes (ars) including arsR, acr3, arsB, arsC, arsM, arsI, arsP, and arsH as well as energy-generating As respiratory oxidation (aioA) and reduction (arrA) genes. Somewhat unexpectedly, the relative DNA abundances and diversity of ars, aioA, and arrA genes were not significantly different between low and high ([~]10 vs [~]100 mg kg-1) As soils. By comparison to available metagenomes from other soils, geographic distance rather than As levels drove the different compositions of microbial communities. Arsenic significantly increased ars genes abundance only when its concentration was higher than 410 mg kg-1. In contrast, between low and high As soils, metatranscriptomics revealed a significant increase in transcription of ars and aioA genes, which are induced by arsenite, the dominant As species in paddy soils, but not arrA genes, which are induced by arsenate. These patterns appeared to be community-wide as opposed to taxon-specific. Collectively, our findings advance understanding of how microbes respond to high As levels and the diversity of As metabolism genes in paddy soils and indicated that future studies of As metabolism in soil, or other environments, should include the function (transcriptome) level.

IMPORTANCEArsenic (As) is a toxic metalloid pervasively present in the environment. Microorganisms have evolved the capacity to metabolize As, and As metabolism genes are ubiquitously present in the environment even in the absence of high concentrations of As. However, these previous studies were carried out at the DNA level and thus, the activity of the As metabolism genes detected remains essentially speculative. Here, we show that the high As levels in paddy soils increased the transcriptional activity rather than the relative DNA abundance and diversity of As metabolism genes. These findings advance our understanding of how microbes respond to and cope with high As levels, and have implications for better monitoring and managing an important toxic metalloid in agricultural soils and possibly other ecosystems.


title: Increased mitochondrial activity upon CatSper channel activation is required for sperm capacitation.

abstract:
To fertilize an oocyte, sperm must become hyperactive. However, whether they obtain ATP for hyperactivated motility via glycolysis or mitochondrial respiration is unclear. Here, high-resolution respirometry, flow cytometry, and confocal microscopy experiments revealed that mitochondrial respiration and membrane potential increased during mouse sperm capacitation. Treatment with inhibitors of mitochondrial respiration prevented sperm from hyperactivating and fertilizing an oocyte. Mitochondrial respiration was impaired in sperm from mice lacking the calcium channel CatSper. We developed a method to image mitochondrial calcium in sperm and found that CatSper activation led to increased mitochondrial calcium concentration. Finally, treating sperm with an inhibitor of mitochondrial calcium import impaired mitochondrial function and sperm hyperactivation. Together, our results uncover a new role of sperm mitochondria and reveal a new pathway connecting calcium influx through CatSper to mitochondrial activity and the sperm hyperactivation required to fertilize an oocyte.

SummaryThe source of ATP for sperm hyperactivation is unclear. Ferreira et al. show that mitochondrial activity increases during, and is required for, hyperactivation and fertilization ability. Increased mitochondrial activity depends on calcium influx through the channel CatSper.


title: Multivalent interactions make adherens junction-cytoskeletal linkage robust during morphogenesis

abstract: Embryogenesis requires cells to change shape and move without disrupting epithelial integrity. This requires robust, responsive linkage between adherens junctions and the actomyosin cytoskeleton. Using Drosophila morphogenesis we define molecular mechanisms mediating junction-cytoskeletal linkage and explore the role of mechanosensing. We focus on the junction-cytoskeletal linker Canoe, a multidomain protein. We engineered the canoe locus to define how its domains mediate its mechanism of action. To our surprise, the PDZ and FAB domains, which we thought connected junctions and F-actin, are not required for viability or mechanosensitive recruitment to junctions under tension. The FAB domain stabilizes junctions experiencing elevated force, but in its absence most cells recover, suggesting redundant interactions. In contrast, the Rap1-binding RA domains are critical for all Cno functions and for enrichment at junctions under tension. This supports a model in which junctional robustness derives from a large protein network assembled via multivalent interactions, with proteins at network nodes and some node connections more critical than others.


title: Reorganization of molecular networks associated with DNA methylation and changes in the rearing environments of the house wren (Troglodytes aedon)

abstract: Environmental change, such as increased rates of urbanization, can induce shifts in phenotypic plasticity with some individuals adapting to city life while others are displaced. A key trait that can facilitate adaptation is the degree at which animals respond to stress. This stress response has a heritable component and exhibits intra- and inter-individual variation. However, the mechanisms behind this variability and whether they might be responsible for adaptation to different environments are not known. Variation in DNA methylation can be a potential mechanism that mediates environmental effects on the stress response. We used an inter- and intra-environmental cross-foster experiment to analyze the contribution of DNA methylation to early-life phenotypic variation. We found that at hatching, urban house wren (Troglodytes aedon) offspring had increased methylation as compared to their rural counterparts, and observed plasticity in methylation as offspring aged, indicating developmental effects of the rearing environment on methylation. Differential methylation analyses showed that cellular respiration genes were differentially expressed at hatching and behavioral and metabolism genes were differentially expressed at fledgling. Lastly, hyper-methylation of a single gene (CNTNAP2) is associated with increased glucocorticoid levels. These differential methylation patterns linked to a specific physiological phenotype suggest that DNA methylation may be a mechanism by which individuals adapt to novel environments. Characterizing genetic and environmental influences on methylation is critical for understanding the role of epigenetic mechanisms in evolutionary adaptation.


title: Time to fixation in changing environments

abstract: Although a large number of experimental and theoretical studies have been carried out in a constant environment, as natural environments vary in time, it is important to ask if and how these results are affected by a changing environment. Here, we study the properties of the conditional fixation time of a mutant in a finite, randomly mating diploid population which is evolving in a periodically changing environment. In a static environment, as the conditional mean fixation time of a co-dominant beneficial mutant is equal to that of a deleterious mutant with the same magnitude of selection coefficient, similar patterns for beneficial and deleterious sweeps may result. We find that this symmetry breaks even when the environment is changing slowly. Furthermore, for intermediate dominance, the conditional mean fixation time of a beneficial mutant in a slowly changing environment depends weakly on the dominance coefficient and is close to the corresponding results in the static environment; however, the fixation time for a deleterious mutant under moderate selection with a slowly varying selection coefficient differs substantially from that in the constant environment when the mutant is recessive. Our results thus suggest that the variability patterns and levels for beneficial sweeps are mildly affected by temporally varying environment but changing environment is likely to strongly impact those due to recessive deleterious sweeps.


title: The Hypoxia Response Pathway Promotes PEP Carboxykinase Expression And Gluconeogenesis

abstract: Actively dividing cells, including some cancers, rely on aerobic glycolysis rather than oxidative phosphorylation to generate energy, a phenomenon termed "the Warburg effect1." Constitutive activation of the Hypoxia Inducible Factor (HIF-1), a transcription factor known for mediating an adaptive response to oxygen deprivation (hypoxia), is a hallmark of the Warburg effect2. HIF-1 is thought to promote glycolysis and suppress oxidative phosphorylation. Here, we show instead that HIF-1 can promote gluconeogenesis. Using a multiomics approach, we determined the genomic, transcriptomic, and metabolomic landscapes regulated by constitutively active HIF-1 in C. elegans. We performed RNA-seq and ChIP-seq under aerobic conditions in mutants lacking EGL-9, a key negative regulator of HIF-1, and then integrated these approaches to identify over a hundred genes directly and functionally upregulated by HIF-1. We show that HIF-1 directly promotes the expression of PCK-1, a PEP carboxykinase that is a rate-limiting mediator of gluconeogenesis3. This activation of PCK-1 by HIF-1 promotes survival in response to both oxidative and hypoxic stress. Our work is the first to identify functional direct targets of HIF-1 in vivo, and it describes the first complete metabolome induced by constitutive HIF-1 activation in any organism.


title: Comprehensive PAM prediction for CRISPR-Cas systems reveals evidence for spacer sharing, preferred strand targeting and conserved links with CRISPR repeats

abstract: The adaptive CRISPR-Cas immune system stores sequences from past invaders as spacers in CRISPR arrays and thereby provides direct evidence that links invaders to hosts. Mapping CRISPR spacers has revealed many aspects of CRISPR biology, including target requirements such as the protospacer adjacent motif (PAM). However, studies have so far been limited by a low number of mapped spacers in the database. By using vast metagenomic sequence databases, we mapped one third ([~]70,000) of more than 200,000 unique CRISPR spacers from a variety of microbes, and derived a catalog of more than one hundred unique PAM sequences associated with specific CRISPR subtypes. These PAMs were further used to correctly assign the orientation of CRISPR arrays, revealing conserved patterns between the last nucleotides of the CRISPR repeat and PAM. From the curated CRISPR arrays dataset we could also deduce CRISPR subtype specific preferences for targeting either template or coding strand of open reading frames. While some DNA-targeting systems (e.g. Type I-E and Type II systems) prefer the template strand and avoid mRNA, other DNA- and RNA-targeting systems (i.e. Type I-A, I-B and Type III systems) prefer the coding strand and mRNA. In addition, we found large scale evidence that both CRISPR adaptation machinery and CRISPR arrays are shared between different CRISPR-Cas systems. This could lead to simultaneous DNA- and RNA targeting of invaders, which may be effective at combating mobile genetic invaders.


title: Wikidata and the bibliography of life

abstract: Biological taxonomy rests on a long tail of publications spanning nearly three centuries. Not only is this literature vital to resolving disputes about taxonomy and nomenclature, for many species it represents a key source - indeed sometimes the only source - of information about that species. Unlike other disciplines such as biomedicine, the taxonomic community lacks a centralised, curated literature database (the "bibliography of life"). This paper argues that Wikidata can be that database as it has flexible and sophisticated models of bibliographic information, and an active community of people and programs ("bots") adding, editing, and curating that information. The paper also describes a tool to visualise and explore bibliography information in Wikidata and how it links to both taxa and taxonomists.


title: Comparative meta-proteomic analysis for the identification of novel plasmodesmata proteins and regulatory cues.

abstract: A major route for cell-to-cell signaling is via cell wall-embedded pores termed plasmodesmata (PD) forming the symplasm. PD regulate many aspects of plant development and responses to the environment however, our understanding of what factors affect their structure and permeability is limited. In this paper, a meta-analysis is presented as a tool for the identification of conditions affecting PD transport and in silico generation of PD proteomes for species of interest. The custom-built pipeline searches the whole genome for protein structural features and conserved domains identified on experimental proteomes and use it to predict PD candidates in 22 compatible plant species. Using the in silico proteome and microarray analysis, interactions between PD genes and conditions affecting PD function are identified. High salinity and osmotic stress affect a significant number of PD candidate genes and we provide evidence that these conditions regulate symplasmic transport of GFP. Using the pipeline, the in silico PD proteome for Medicago truncatula was generated, as an example of a plant in which experimental data is not available. The identification of a candidate receptor like protein was experimentally validated in M. truncatula transgenic roots expressing fluorescently tagged protein fusion. Together the results highlight the power of our newly designed tool in the identification of new factors and proteins influencing PD in diverse plant species.

category: plant biology


title: An interpretable connectivity-based decoding model for classification of chronic marijuana use

abstract:
BackgroundPsychiatric neuroimaging typically proceeds with one of two approaches: encoding models, which aim to model neural mechanisms, or decoding models, which aim to predict behavioral or clinical characteristics from brain imaging data. In this study, we seek to combine these aims by developing interpretable decoding models that offer both accurate prediction and novel neural insights. We demonstrate the effectiveness of this combined approach in a case study of chronic marijuana use.

MethodsChronic marijuana (MJ) users (n=195) and non-using healthy controls (n=128) completed a cue-elicited craving task during functional magnetic resonance imaging. Linear machine learning methods were used to classify individuals into chronic MJ users and non-users based on task-evoked, whole-brain functional connectivity. We then used graph theoretic analyses to identify  predictive functional connectivities among brain regions that contributed most substantially to the classification of chronic marijuana use.

ResultsWe obtained high (~80% out-of-sample) accuracy across four different classification models, demonstrating that task-evoked, whole-brain functional connectivity can successfully differentiate chronic marijuana users from non-users. Subsequent network analyses revealed key predictive regions (e.g., anterior cingulate cortex, dorsolateral prefrontal cortex, and precuneus) that are often implicated in neuroimaging studies of substance use disorders, as well as some key exceptions. We also identified a core set of networks of brain regions that contributed to successful classification, comprised of many of the same predictive regions.

ConclusionsOur dual aims of accurate prediction and interpretability were successful, producing a predictive model that also provides interpretability at the neural level. This novel approach may complement other predictive-exploratory approaches for a more complete understanding of neural mechanisms in drug use and other neuropsychiatric disorders.


title: Impaired T-cell and antibody immunity after COVID-19 infection in chronically immunosuppressed transplant recipients

abstract: Assessment of T-cell immunity to the COVID-19 coronavirus requires reliable assays and is of great interest, given the uncertain longevity of the antibody response. Some recent reports have used immunodominant spike (S) antigenic peptides and anti-CD28 co-stimulation in varying combinations to assess T-cell immunity to SARS-CoV-2. These assays may cause T-cell hyperstimulation and could overestimate antiviral immunity in chronically immunosuppressed transplant recipients, who are predisposed to infections and vaccination failures. Here, we evaluate CD154-expressing T-cells induced by unselected S antigenic peptides in 204 subjects-103 COVID-19 patients and 101 healthy unexposed subjects. Subjects included 72 transplanted and 130 non-transplanted subjects. S-reactive CD154+T-cells co-express and can thus substitute for IFN{gamma} (n=3). Assay reproducibility in a variety of conditions was acceptable with coefficient of variation of 2-10.6%. S-reactive CD154+T-cell frequencies were a) higher in 42 healthy unexposed transplant recipients who were sampled pre-pandemic, compared with 59 healthy non-transplanted subjects (p=0.02), b) lower in Tr COVID-19 patients compared with healthy transplant patients (p<0.0001), c) lower in Tr patients with severe COVID-19 (p<0.0001), or COVID-19 requiring hospitalization (p<0.05), compared with healthy Tr recipients. S-reactive T-cells were not significantly different between the various COVID-19 disease categories in NT recipients. Among transplant recipients with COVID-19, cytomegalovirus co-infection occurred in 34%; further, CMV-specific T-cells (p<0.001) and incidence of anti-receptor-binding-domain IgG (p=0.011) were lower compared with non-transplanted COVID-19 patients. Healthy unexposed transplant recipients exhibit pre-existing T-cell immunity to SARS-CoV-2. COVID-19 infection leads to impaired T-cell and antibody responses to SARS-CoV-2 and increased risk of CMV co-infection in transplant recipients.


title: CCR2-dependent monocyte-derived cells restrict SARS-CoV-2 infection

abstract: SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19) and current evidence suggests severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts viral burden in the lung. We find that a recently developed mouse-adapted MA-SARS-CoV-2 strain, as well as the emerging B. 1.351 variant, trigger an inflammatory response in the lung characterized by expression of pro-inflammatory cytokines and interferon-stimulated genes. scRNA-seq analysis of lung homogenates identified a hyper-inflammatory monocyte profile. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes infiltration of classical monocytes into the lung and expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.


title: ORAI1 establishes resistance to SARS-CoV-2 infection by regulating tonic type I interferon signaling

abstract: ORAI1 and STIM1 are the critical mediators of store-operated Ca2+ entry by acting as the pore subunit and an endoplasmic reticulum-resident signaling molecule, respectively. In addition to Ca2+ signaling, STIM1 is also involved in regulation of a cytosolic nucleic acid sensing pathway. Using ORAI1 and STIM1 knockout cells, we examined their contribution to the host response to SARS-CoV-2 infection. STIM1 knockout cells showed strong resistance to SARS-CoV-2 infection due to enhanced type I interferon response. On the contrary, ORAI1 knockout cells showed high susceptibility to SARS-CoV-2 infection as judged by increased expression of viral proteins and a high viral load. Mechanistically, ORAI1 knockout cells showed reduced homeostatic cytoplasmic Ca2+ concentration and severe impairment in tonic interferon signaling. Transcriptome analysis showed downregulation of multiple cellular defense mechanisms, including antiviral signaling pathways in ORAI1 knockout cells, which are likely due to reduced expression of the Ca2+-dependent transcription factors of the activator protein 1 (AP-1) family and MEF2C. Our results identify a novel role of ORAI1-mediated Ca2+ signaling in regulating the baseline type I interferon level, which is a determinant of host resistance to SARS-CoV-2 infection.


title: Mouse Adapted SARS-CoV-2 protects animals from lethal SARS-CoV challenge

abstract: The emergence of SARS-CoV-2 has resulted in a worldwide pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of COVID-19 disease have been hampered by the lack of robust mouse models. To overcome this barrier, we utilized a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARSCoV-2 variants, this model recapitulates critical elements of human infection including viral replication in the lung, immune cell infiltration, and significant in vivo disease. Importantly, mouse-adaptation of SARS-CoV-2 does not impair replication in human airway cells and maintains antigenicity similar to human SARS-CoV-2 strains. Utilizing this model, we demonstrate that SARS-CoV-2 infected mice are protected from lethal challenge with the original SARS-CoV, suggesting immunity from heterologous CoV strains. Together, the results highlight the utility of this mouse model for further study of SARS-CoV-2 infection and disease.


title: DUX4 induces a homogeneous sequence of molecular changes, culminating in the activation of a stem-cell-like transcriptional network and induction of apoptosis in somatic cells

abstract: Facioscapulohumeral muscular dystrophy (FSHD) is a muscle degenerative disease that disproportionally affects the muscles of the face, shoulder girdle and upper arms. FSHD is caused by the misexpression of Double Homeobox 4 (DUX4), a transcription factor that is normally expressed during early embryonic development. Ectopic expression of DUX4 in somatic cells is cytotoxic and leads to rapid apoptosis. To elucidate the mechanism by which DUX4 induces apoptosis, we determined the temporal transcriptional changes induced by Dux4 at the single-cell level. We observed that induction of DUX4 expression induces a non-random, consecutive sequence of transcriptional changes. DUX4 homogenously induces the activation of a stem cell signature and activates a network of transcription factors that is typically expressed during early embryogenesis and in pluripotent stem cells. Ultimately, these transcriptional changes trigger the induction of apoptosis, suggesting that the induction of this early stemness program is incompatible with a somatic cell program. Our findings shed new light on the timing and dynamic of DUX4-mediated transcriptional reprogramming and may help elucidate why the DUX4 stemness program is required during early embryogenesis, but incompatible with somatic cell viability.


title: Evolution of enzyme levels in metabolic pathways: A theoretical approach

abstract: The central role of metabolism in cell functioning and adaptation has given rise to count-less studies on the evolution of enzyme-coding genes and network topology. However, very few studies have addressed the question of how enzyme concentrations change in response to positive selective pressure on the flux, considered a proxy of fitness. In particular, the way cellular constraints, such as resource limitations and co-regulation, affect the adaptive landscape of a pathway under selection has never been analyzed theoretically. To fill this gap, we developed a model of the evolution of enzyme concentrations that combines metabolic control theory and an adaptive dynamics approach, and integrates possible dependencies between enzyme concentrations. We determined the evolutionary equilibria of enzyme concentrations and their range of neutral variation, and showed that they differ with the properties of the enzymes, the constraints applied to the system and the initial enzyme concentrations. Simulations of long-term evolution confirmed all analytical and numerical predictions, even though we relaxed the simplifying assumptions used in the analytical treatment.


title: Single gene initiates evolution of epithelial architecture and function

abstract: Epithelial monolayers are a hallmark of the architecture of metazoan tissues: they provide stability, serve as barriers, and fold into organs. Epithelial cells vary in shape, ranging from flat and spread out to tall and slim. Dynamic epithelial shape changes have been explored in the context of tissue folding, where local cytoskeletal modulations cause epithelial bending and folding. Comparatively little is known about how entire tissues are transformed from a short to tall architecture. Here we show that shape regulation in epithelia can be governed by the activity of a single gene. We use a comparative approach in distantly related flies to experimentally decode the developmental program that directs the formation of columnar epithelia in the blastoderm and thus determines the physiological features of the resulting epithelium. We uncover an evolutionary novel, membrane-associated protein that emerged in flies and triggered a new development program, the cuboidal-to-columnar transformation of epithelial tissues. slow-as-molasses (slam) encodes a Dia/F-actin regulator that exploits an intrinsic morphological plasticity of cells to transform tissues. Our findings demonstrate that a single, newly emerged factor that amplifies its activity in epithelia provides the basis for adaptation and initiates the evolution of novel developmental programs.


title: TOPLESS promotes plant immunity by repressing auxin signaling and is targeted by the fungal effector Naked1

abstract: In plants, the antagonism between growth and defense is hardwired by hormonal signaling. Perception of Pathogen Associated Molecular Patterns (PAMPs) from invading microorganisms inhibits auxin signaling and growth. Conversely, pathogens manipulate auxin signaling to promote disease, but how this hormone inhibits immunity is not fully understood. Ustilago maydis is a maize pathogen that induces auxin signaling in its host. We characterized an U. maydis effector protein, Naked1 (Nkd1), that is translocated into the host nucleus. Through its native EAR motif, Nkd1 binds to the transcriptional co-repressors TOPLESS/TOPLESS related (TPL/TPRs) and prevents the recruitment of transcriptional repressors involved in hormonal signaling, leading to de-repression of auxin and jasmonate signaling. Moderate up-regulation of auxin signaling inhibits an early defense response, PAMP-triggered ROS burst. Thus, our findings establish a clear mechanism for auxin-induced pathogen susceptibility. Manipulation of TPL by Nkd1 is evolutionary well balanced as engineered Nkd1 variants with increased expression or increased EAR-mediated TPL/TPR binding trigger typical salicylic acid mediated defense reactions, leading to pathogen resistance.


title: Histone marks are drivers of the splicing changes necessary for an epithelial-to-mesenchymal transition

abstract: Cell differentiation and reprogramming depend on coordinated changes in specific alternative splicing events. How these cell type-specific splicing patterns are dynamically modified in response to a stimulus remains elusive. Taking advantage of the epithelial-to-mesenchymal transition (EMT), a reversible cell reprogramming intimately involved in cancer cell invasiveness and metastasis, we found a strong correlation between changes in the alternative splicing of key exons for EMT, such as at the Fgfr2 and Cnntd1 loci, and changes in the enrichment levels of specific histone modifications, namely H3K27ac and H3K27me3. Localised CRISPR epigenome editing of these exon-specific histone marks was sufficient to induce changes in splicing capable of recapitulating important aspects of EMT, such as a motile and invasive cell phenotype. Whereas, impairment of the changes in H3K27 marks observed during EMT, using histone deacetylase inhibitors, repressed inclusion of the mesenchymal isoform despite an EMT induction, supporting a driving effect for H3K27 modifications in establishing the new cell type-specific splicing patterns necessary for EMT cell reprogramming. Finally, H3K27 marks were shown to impact splicing by modulating recruitment of the splicing factor PTB to its RNA binding sites, suggesting a direct link between chromatin modifications and the splicing machinery. Taken together, these results prove the causal role of H3K27 marks in driving the dynamic splicing changes necessary for induction of important aspects of EMT. They also prove that chromatin-mediated splicing changes are sufficient to impact the cells phenotype, which expands the cells toolkit to adapt and respond to diverse stimuli, such as EMT induction.


title: Vacuole-specific lipid release for tracking intracellular lipid metabolism and transport in Saccharomyces cerevisiae

abstract: Lipid metabolism is spatiotemporally regulated within cells, yet intervention into lipid functions at subcellular resolution remains difficult. Here we report a method that enables site-specific release of sphingolipids and cholesterol inside the vacuole in Saccharomyces cerevisiae. Using this approach, we monitored real-time sphingolipid metabolic flux out of the vacuole by mass spectrometry and found that the ER-vacuole tethering protein Mdm1 facilitated the metabolism of sphingoid bases into ceramides. In addition, we showed that cholesterol, once delivered into yeast using our method, could restore cell proliferation induced by ergosterol deprivation, overcoming the previously described sterol-uptake barrier under aerobic conditions. Together, these data define a new way to study intracellular lipid metabolism and transport from the vacuole in yeast.


title: Multi-view confocal microscopy enables multiple organ and whole organism live-imaging

abstract: Understanding how development is coordinated in multiple tissues and gives rise to fully functional organs or whole organisms necessitates microscopy tools. Over the last decade numerous advances have been made in live-imaging, enabling high resolution imaging of whole organisms at cellular resolution. Yet, these advances mainly rely on mounting the specimen in agarose or aqueous solutions, precluding imaging of organisms whose oxygen uptake depends on ventilation. Here, we implemented a multi-view multi-scale microscopy strategy based on confocal spinning disk microscopy, called Multi-View confocal microScopy (MuViScopy). MuViScopy enables live-imaging of multiple organs with cellular resolution using sample rotation and confocal imaging without the need of sample embedding. We illustrate the capacity of MuViScopy by live-imaging Drosophila melanogaster pupal development throughout metamorphosis, highlighting how internal organs are formed and multiple organ development is coordinated. We foresee that MuViScopy will open the path to better understand developmental processes at the whole organism scale in living systems that necessitates gas exchange by ventilation.

category: developmental biology


title: Haploinsufficiency of the essential gene RpS12 causes defects in erythropoiesis and hematopoietic stem cell maintenance

abstract: Ribosomal protein (Rp) gene haploinsufficiency can result in Diamond-Blackfan Anemia (DBA), characterized by defective erythropoiesis and skeletal defects. Some mouse Rp mutations recapitulate DBA phenotypes, although others lack erythropoietic or skeletal defects. We generated a conditional knockout mouse to partially delete RpS12, which results in homozygous embryonic lethality. Rps12+/- mice have growth and morphological defects, pancytopenia and impaired erythropoiesis. A striking reduction in hematopoietic stem cells (HSCs) and progenitors in the bone marrow (BM) was associated with decreased ability to repopulate the blood system after competitive and non-competitive BM transplantation. The mutants exhibited loss of HSC quiescence, which was associated with ERK and MTOR activation and increased global translation in HSC and progenitors. Thus, RpS12 has a very strong requirement in maintaining HSC quiescence and function, in addition to erythropoiesis that is affected in DBA patients.


title: The role of elections as drivers of tropical deforestation

abstract: Tropical forests support immense biodiversity and provide essential ecosystem services for billions of people. Despite this value, tropical deforestation continues at a high rate. Emerging evidence suggests that elections can play an important role in shaping deforestation, for instance by incentivising politicians to allow increased utilisation of tropical forests in return for political support and votes. Nevertheless, the role of elections as a driver of deforestation has not been comprehensively tested at broad geographic scales. Here, we created an annual database from 2001 to 2018 on political elections and forest loss for 55 tropical nations and modelled the effect of elections on deforestation. In total, 1.5 million km2 of forest was lost during this time period, and the rate of deforestation increased in 37 (67%) of the analysed countries. Deforestation was significantly lower in years with presidential or lower chamber elections compared to non-election years, which is in contrast to previous local-scale studies. Moreover, deforestation was significantly higher in presidential or lower chamber elections that are competitive (i.e. when the opposition can participate in elections and has a legitimate chance to gain governmental power) compared to uncompetitive elections. Our results document a pervasive loss of tropical forests and suggest that competitive elections are potential drivers of deforestation. We recommned that organisations monitoring election transparency and fairness should also monitor environmental impacts such as forest loss, habitat destruction and resource exploitation. This would benefit the tracking of potential illegal vote buying with natural resources.


title: Same role but different actors: genetic regulation of post-translational modification of two distinct proteins

abstract: Post-translational modifications (PTMs) diversify protein functions and dynamically coordinate their signalling networks, influencing most aspects of cell physiology. Nevertheless, their genetic regulation or influence on complex traits is not fully understood. Here, we compare for the first time the genetic regulation of the same PTM of two proteins - glycosylation of transferrin and immunoglobulin G (IgG). By performing genome-wide association analysis of transferrin glycosylation, we identified 10 significantly associated loci, all novel. Comparing these with IgG glycosylation-associated genes, we note protein-specific associations with genes encoding glycosylation enzymes (transferrin - MGAT5, ST3GAL4, B3GAT1; IgG - MGAT3, ST6GAL1) as well as shared associations (FUT6, FUT8). Colocalisation analyses of the latter suggest that different causal variants in the FUT genes regulate fucosylation of the two proteins. We propose that they affect the binding of different transcription factors in different tissues, with fucosylation of IgG being regulated by IKZF1 in B-cells and of transferrin by HNF1A in liver.


title: Telomere-to-telomere genome assembly of Phaeodactylum tricornutum

abstract: Phaeodactylum tricornutum is a marine diatom with a growing genetic toolbox available and is being used in many synthetic biology applications. While most of the genome has been assembled, the currently available genome assembly is not a completed telomere-to-telomere assembly. Here, we used Oxford Nanopore long reads to build a telomere-to-telomere genome for Phaeodactylum tricornutum. We developed a graph-based approach to extract all unique telomeres, and used this information to manually correct assembly errors. In total, we found 25 nuclear chromosomes that comprise all previously assembled fragments, in addition to the chloroplast and mitochondrial genomes. We found that chromosome 19 has filtered long-read coverage and a quality estimate that suggests significantly less haplotype sequence variation than the other chromosomes. This work improves upon the previous genome assembly and provides new opportunities for genetic engineering of this species, including creating designer synthetic chromosomes.


title: Characterization of the ABC methionine transporter from Neisseria meningitidis reveals that MetQ is a lipoprotein

abstract: NmMetQ is a substrate binding protein (SBP) from Neisseria meningitidis that has been identified as a surface-exposed candidate antigen for meningococcal vaccines. However, this location for NmMetQ challenges the prevailing view that SBPs in Gram-negative bacteria are localized to the periplasmic space to promote interaction with their cognate ABC transporter embedded in the bacterial inner membrane. To address the roles of NmMetQ, we characterized NmMetQ with and without its cognate ABC transporter (NmMetNI). Here, we show that NmMetQ is a lipoprotein (lipo-NmMetQ) that binds multiple methionine analogs and stimulates the ATPase activity of NmMetNI. Using single-particle electron cryo-microscopy, we determined the structures of NmMetNI in the absence and presence of lipo-NmMetQ. Based on our data, we propose that NmMetQ tethers to membranes via a lipid anchor and has dual function/topology, playing a role in NmMetNI-mediated transport at the inner-membrane in addition to moonlighting functions on the bacterial surface.

category: biochemistry