title: A model of mitochondrial ATP synthase deficiencies. The role of mitochondrial carriers.

abstract: The m.8993T>G mutation of the mitochondrial MT-ATP6 gene is associated with NARP syndrome (Neuropathy, Ataxia and Retinitis Pigmentosa). The equivalent point mutation introduced in yeast Saccharomyces cerevisiae mitochondrial DNA considerably reduced the activity of ATP synthase and of cytochrome-c-oxidase preventing yeast growth on oxidative substrates. The over-expression of the mitochondrial oxodicarboxylate carrier (Odc1p) is able to rescue the growth on oxidative substrate by increasing the substrate-level phosphorylation of ADP coupled to conversion of -ketoglutarate (AKG) into succinate with an increase in Complex IV activity. Previous studies showed that equivalent point mutations in ATP synthase behave similarly and can be rescued by Odc1p overexpression and/or uncoupler of OXPHOS from ATP synthesis. In order to better understand the mechanism of ATP synthase mutation bypass, we developed a core model of mitochondrial metabolism based on AKG as respiratory substrate. We describe the different possible metabolite output and the ATP/O ratio values as a function of ATP synthase inhibition.


title: A theoretical model of mitochondrial ATP synthase deficiencies. The role of mitochondrial carriers.

category: systems biology


title: Nuclear Factor I in neurons, glia and during the formation of Muller glia-derived progenitor cells in avian, porcine and primate retinas

abstract: The regenerative potential of Muller glia (MG) is extraordinary in fish, poor in chick and terrible in mammals. In the chick model, MG readily reprogram into proliferating Muller glia-derived progenitor cells (MGPCs), but neuronal differentiation is very limited. The factors that suppress the neurogenic potential of MGPCs in the chick are slowly being revealed. Isoforms of Nuclear Factor I (NFI) are cell-intrinsic factors that limit neurogenic potential; these factors are required for the formation of MG in the developing mouse retina (Clark et al., 2019) and deletion of these factors reprograms MG into neuron-like cells in mature mouse retina (Hoang et al., 2020). Accordingly, we sought to characterize the patterns of expression NFIs in the developing, mature and damaged chick retina. In addition, we characterized patterns of expression of NFIs in the retinas of large mammals, pigs and monkeys. Using a combination of single cell RNA-sequencing (scRNA-seq) and immunolabeling we probed for patterns of expression. In embryonic chick, levels of NFIs are very low in early E5 (embryonic day 5) retinal progenitor cells (RPCs), up-regulated in E8 RPCs, further up-regulated in differentiating MG at E12 and E15. NFIs are maintained in mature resting MG, microglia and neurons. Levels of NFIs are reduced in activated MG in retinas treated with NMDA and/or insulin+FGF2, and further down-regulated in proliferating MGPCs. However, levels of NFIs in MGPCs were significantly higher than those seen in RPCs. Immunolabeling for NFIA and NFIB closely matched patterns of expression revealed in different types of retinal neurons and glia, consistent with findings from scRNA-seq. In addition, we find expression of NFIA and NFIB through progenitors in the circumferential marginal zone at the far periphery of the retina. We find similar patterns of expression for NFIs in scRNA-seq databases for pig and monkey retinas. Patterns of expression of NFIA and NFIB were validated with immunofluorescence in pig and monkey retinas wherein these factors were predominantly detected in MG and a few types of inner retinal neurons. In summary, NFIA and NFIB are prominently expressed in developing chick retina and by mature neurons and glia in the retinas of chicks, pigs and monkeys. Although levels of NFIs are decreased in chick, in MGPCs these levels remain higher than those seen in neurogenic RPCs. We propose that the neurogenic potential of MGPCs in the chick retina is suppressed by NFIs.

category: neuroscience


title: Memory-paced tapping to auditory rhythms: Effects of rate, speech and motor-engagement

abstract:
Humans have a near-automatic tendency to entrain their motor actions to rhythms in the environment. Entrainment is hypothesized to play an important role in processing naturalistic stimuli, such as speech and music, which have intrinsically rhythmic properties. Here we studied two facets of entraining ones rhythmic motor actions to an external stimulus: (1) synchronized finger tapping to auditory rhythmic stimuli, and (2) memory-paced reproduction of a previously heard rhythm. Using modifications of the Synchronization-Continuation tapping paradigm, we studied how these two rhythmic behaviours were affected by different stimulus and task features. We tested synchronization and memory-paced tapping for a broad range of rates, from sub-second to supra-second, both for isochronous tone-sequences and for rhythmic speech stimuli (counting from one to ten), which are more ecological yet less strictly isochronous. We also asked what role motor engagement plays in forming a stable internal representation for rhythms and guiding memory-paced tapping.

Results show that individuals can flexibly synchronize their motor actions to a very broad range of rhythms. However, this flexibility does not extend to memory-paced tapping, which is accurate only in a narrower range of rates, around ~1.5Hz. This pattern suggests that intrinsic rhythmic-defaults in the auditory/motor systems influence the internal representation of rhythms, in the absence of an external pace-maker. Interestingly, memory-paced tapping for speech rhythms and simple tones shared similar  optimal rates, although with reduced accuracy, suggesting that internal constraints on rhythmic entrainment may generalize to more ecological stimuli. Last, active synchronization led to more accurate memory-paced tapping vs. passive listening, which emphasizes the importance of action-perception interactions in forming stable entrainment to external rhythms.


title: Consistent coordination patterns provide near perfect behavior decoding in a comprehensive motor program for insect flight

abstract: Patterns of motor activity can be used to decode behavior state. Precise spike timing encoding is present in many motor systems, but is not frequently utilized to decode behavior or to examine how coordination is achieved across many motor units. Testing whether the same coordinated sets of muscles control different movements is difficult without a complete motor representation at the level of the currency of control - action potentials. Here, we demonstrate nearly perfect decoding of six hawk moth flight behaviors elicited in response to wide-field drifting visual stimuli about the flight axes - pitch, roll, and yaw - using a comprehensive, spike-resolved motor program and a simple linear decoding pipeline. A complex decoding scheme is not necessary, even if the functional patterns of control are nonlinear. We show that muscle covariation present in one pair of visual stimulus conditions can be used to decode behavior in a different pair of visual stimulus conditions, indicating the presence of conserved muscle coordination patterns at the level of motor neuronal timings in functionally distinct behaviors. We also demonstrate that as few as half the muscles can be used to retain decoding performance, linking coordination to redundancy in encoding, if not function, across the entire moth flight motor program.


title: PD-1 in hippocampal neurons regulates excitability, synaptic plasticity, and cognition

abstract:
Immunotherapy using monoclonal antibodies against programmed cell death protein 1 (PD-1) demonstrated improved survival in cancer patients through immune activation. Here we show that functional PD-1 is expressed in mouse and primate hippocampal neurons and PD-1 inhibition improves cognition in physiological and pathological conditions. Mice lacking the Pdcd1 gene encoding PD-1 exhibit enhanced long-term potentiation (LTP) and learning and memory. These behavioral and cellular changes can be recapitulated by selective deletion of Pdcd1 in hippocampal excitatory neurons but not in microglia. Perfusion of mouse or nonhuman primate brain slices with anti-PD-1 antibody is sufficient to increase excitability in CA1 hippocampal neurons. Conversely, re-expression of Pdcd1 in PD-1 deficient hippocampal neurons suppresses memory and LTP. Traumatic brain injury impairs learning and memory, which is improved by intraventricular administration of anti-PD-1. These findings suggest that anti-PD-1 treatment has therapeutic potential to counteract cognitive decline.

HighlightsO_LIAdult mice lacking Pdcd1 in hippocampal neurons exhibit enhanced memory and LTP
C_LIO_LIAnti-PD-1 antibody treatment increases CA1 neuron excitability in brain slices of mice and primates
C_LIO_LIRe-expression of Pdcd1 in PD-1 deficient hippocampal neurons impairs memory and LTP
C_LIO_LICognitive deficits after traumatic brain injury are improved by anti-PD-1 treatment
C_LI


title: +microstate: A MATLAB toolbox for brain microstate analysis in sensor and cortical EEG/MEG

abstract: +microstate is a MATLAB toolbox for brain functional microstate analysis. It builds upon previous EEG microstate literature and toolboxes by including algorithms for microstate analysis for other neuroimaging modalities such as sensor-space MEG and source-space data. +microstate includes codes for performing individual- and group-level brain microstate analysis in resting-state and task-based data including event-related potentials/fields. Functions are included to visualise and perform statistical analysis of microstate sequences, including novel advanced statistical approaches such as microstate-segmented functional connectivity analysis, cluster-permutation topographic ANOVAs, and{chi} 2 analysis of microstate probabilities in response to stimuli. Additionally, codes for simulating microstate sequences and their associated M/EEG data are included in the toolbox, which can be used to generate artificial data with ground truth microstates and to validate the methodology. +microstate integrates with widely used toolboxes for M/EEG processing including Fieldtrip, SPM, LORETA/sLORETA, EEGLAB, and Brainstorm to aid with accessibility, and includes wrappers for pre-existing toolboxes for brain-state estimation such as Hidden Markov modelling (HMM-MAR) and independent component analysis (FastICA) to aid with direct comparison with these techniques. In this paper, we first introduce +microstate before subsequently performing example analyses using open access datasets to demonstrate and validate the methodology. MATLAB live scripts for each of these analyses are included in +microstate, to act as a tutorial and to aid with reproduction of the results presented in this manuscript.


title: Motor memories of object dynamics are categorically organized

abstract: The ability to predict the dynamics of objects, linking applied force to motion, underlies our capacity to perform many of the tasks we carry out on a daily basis. Thus, a fundamental question is how the dynamics of the myriad objects we interact with are organized in memory. Using a custom-built three-dimensional robotic interface that allowed us to simulate objects of varying appearance and weight, we examined how participants learned the weights of sets of objects that they repeatedly lifted. We find strong support for the novel hypothesis that motor memories of object dynamics are organized categorically, in terms of families, based on covariation in their visual and mechanical properties. A striking prediction of this hypothesis, supported by our findings and not predicted by standard associative map models, is that outlier objects with weights that deviate from the family-predicted weight will never be learned despite causing repeated lifting errors.


title: Livestock as vectors of organic matter and nutrient loading in aquatic ecosystems in African savannas

abstract: Populations of large wildlife have declined in many landscapes around the world, and have been replaced or displaced by livestock. The consequences of these changes on the transfer of organic matter (OM) and nutrients from terrestrial to aquatic ecosystems are not well understood. We used behavioural data, excretion and egestion rates and C: N: P stoichiometry of dung and urine of zebu cattle, to develop a metabolism-based estimate of loading rates of OM (dung), C, N and P into the Mara River, Kenya. We also directly measured the deposition of OM and urine by cattle into the river during watering. Per head, zebu cattle excrete and/or egest 25.6 g dry matter (DM, 99.6 g wet mass; metabolism) - 27.7 g DM (direct input) of OM, 16.0-21.8 g C, 5.9-9.6 g N, and 0.3-0.5 g P per day into the river. To replace loading rates OM of an individual hippopotamus by cattle, around 100 individuals will be needed, but much less for different elements. In parts of the investigated sub-catchments loading rates by cattle were equivalent to or higher than that of the hippopotamus. The patterns of increased suspended materials and nutrients as a result of livestock activity fit into historical findings on nutrients concentrations, dissolved organic carbon and other variables in agricultural and livestock areas in the Mara River basin. Changing these patterns of OM and nutrients transport and cycling are having significant effects on the structure and functioning of both terrestrial and aquatic ecosystems.

category: ecology


title: Dicamba drift alters plant-herbivore interactions at the agro-ecological interface

abstract: Natural populations evolve in response to biotic and abiotic changes in their environment, which shape species interactions and ecosystem dynamics. Agricultural systems can introduce novel conditions via herbicide exposure to non-crop habitats in surrounding fields. While herbicide drift is known to produce a variety of toxic effects in plants, little is known about its impact on non-target wildlife species interactions. In a two-year study, we investigated the impact of herbicide drift on plant-herbivore interactions with common weed velvetleaf (Abutlion theophrasti) as the focal species. The findings reveal a significant increase in the phloem feeding silverleaf whitefly (Bermisia tabaci) abundance on the plants exposed to herbicide at drift rates of 0.5% and 1% of the field dose. Additionally, we found evidence that drift imposes correlated selection on whitefly resistance and growth rate as well as positive linear selection on herbicide resistance. We also identified a significant phenotypic tradeoff between whitefly resistance and herbicide resistance in addition to whitefly resistance and relative growth rate in the presence of dicamba drift. These findings suggest herbicide exposure to non-target communities can significantly alter herbivore populations, potentially impacting biodiversity and community dynamics of weed populations found at the agro-ecological interface.


title: Global change differentially modulates coral physiology and suggests future shifts in Caribbean reef assemblages

abstract: Global change driven by anthropogenic carbon emissions is altering ecosystems at unprecedented rates, especially coral reefs, whose symbiosis with algal endosymbionts ise particularly vulnerable to increasing ocean temperatures and altered carbonate chemistry. Here, we assess the physiological responses of the coral holobiont (animal host + algal symbiont) of three Caribbean coral species from two reef environments after exposure to simulated ocean warming (28, 31 {degrees}C), acidification (300 - 3290 atm), and the combination of stressors for 93 days. We used multidimensional analyses to assess how multiple coral holobiont physiological parameters respond to ocean acidification and warming. Our results demonstrate significantly diminishing holobiont physiology in S. siderea and P. astreoides in response to projected ocean acidification, while future warming elicited severe declines in P. strigosa. Offshore S. siderea fragments exhibited higher physiological plasticity than inshore counterparts, suggesting that this offshore population has the capacity to modulate their physiology in response to changing conditions, but at a cost to the holobiont. Plasticity of P. strigosa and P. astreoides was not clearly different between natal reef environments, however, temperature evoked a greater plastic response in both species. Interestingly, while these species exhibit unique physiological responses to ocean acidification and warming, when data from all three species are modeled together, convergent stress responses to these conditions are observed, highlighting the overall sensitivities of tropical corals to these stressors. Our results demonstrate that while ocean warming is a severe acute stressor that will have dire consequences for coral reefs globally, chronic exposure to acidification may also impact coral physiology to a greater extent than previously assumed. The variety of responses to global change we observe across species will likely manifest in altered Caribbean reef assemblages in the future.


title: Pfam domain adaptation profiles reflect plant species' evolutionary history

abstract: The increase in global temperatures predicted by climate change models presents a serious problem for agriculture because high temperatures reduce crop yields. Protein biochemistry is at the core of plant heat stress response, and understanding the interactions between protein biochemistry and temperature will be key to developing heat-tolerant crop varieties. Current experimental studies of proteome-wide plant thermostability are limited by the complexity of plant proteomes: evaluating function for thousands of proteins across a variety of temperatures is simply not feasible with existing technologies. In this paper, we use homologous prokaryote sequences to predict plant Pfam temperature adaptation and gain insights into how thermostability varies across the proteome for three species: maize, Arabidopsis, and poplar. We find that patterns of Pfam domain adaptation across organelles are consistent and highly significant between species, with cytosolic proteins having the largest range of predicted Pfam stabilities and a long tail of highly-stable ribosomal proteins. Pfam adaptation in leaf and root organs varies between species, and maize root proteins have more low-temperature Pfam domains than do Arabidopsis or poplar root proteins. Both poplar and maize populations have an excess of low-temperature mutations in Pfam domains, but only the mutations identified in poplar accessions have a negative effect on Pfam temperature adaptation overall. These Pfam domain adaptation profiles provide insight into how different plant structures adapt to their surrounding environment and can help inform breeding or protein editing strategies to produce heat-tolerant crops.

category: plant biology


title: Learning spatio-temporal properties of hippocampal place cells

abstract: Hippocampal place cells have spatio-temporal properties: they generally respond to a single spatial location of a small environment; in addition, they also display the temporal response property of theta phase precession, namely that the phase of spiking relative to the theta wave shifts from the late phase to early phase as the animal crosses the place field. Grid cells in layer II of the medial entorhinal cortex (MEC) also have spatio-temporal properties similar to hippocampal place cells, except that grid cells respond to multiple spatial locations that form a hexagonal pattern. Other non-grid spatial cells are also abundant in the entorhinal cortex (EC). Because the EC is the upstream area that projects strongly to the hippocampus, a number of EC-hippocampus models have been proposed to explain how the spatial receptive field properties of place cells emerge. However, none of these learning models have explained how the temporal response properties of hippocampal place cells emerge as a result of the EC input. A learning model is presented here based on non-negative sparse coding in which we show that the spatial and temporal properties of hippocampal place cells can be simultaneously learnt from EC input: both MEC grid cells and other EC spatial cells contribute to the spatial properties of hippocampal place cells while MEC grid cells predominantly determine the temporal response properties of hippocampal place cells.


title: Cellular resolution anatomical and molecular atlases for prenatal human brains

abstract: Increasing interest in studies of prenatal human brain development, particularly using new single-cell genomics and anatomical technologies to create cell atlases, creates a strong need for accurate and detailed anatomical reference atlases. In this study, we present two cellular-resolution digital anatomical atlases for prenatal human brain at post-conceptional weeks (PCW) 15 and 21. Both atlases were annotated on sequential Nissl-stained sections covering brain-wide structures on the basis of combined analysis of cytoarchitecture, acetylcholinesterase staining and an extensive marker gene expression dataset. This high information content dataset allowed reliable and accurate demarcation of developing cortical and subcortical structures and their subdivisions. Furthermore, using the anatomical atlases as a guide, spatial expression of 37 and 5 genes from the brains respectively at PCW 15 and 21 was annotated, illustrating reliable marker genes for many developing brain structures. Finally, the present study uncovered several novel developmental features, such as the lack of an outer subventricular zone in the hippocampal formation and entorhinal cortex, and the apparent extension of both cortical (excitatory) and subcortical (inhibitory) progenitors into the prenatal olfactory bulb. These comprehensive atlases provide useful tools for visualization, targeting, imaging and interpretation of brain structures of prenatal human brain, and for guiding and interpreting the next generation of cell census and connectome studies.


title: Mapping the subcortical connectivity of the human default mode network

abstract: The default mode network (DMN) mediates self-awareness and introspection, core components of human consciousness. Therapies to restore consciousness in patients with severe brain injuries have historically targeted subcortical sites in the brainstem, thalamus, hypothalamus, basal forebrain, and basal ganglia, with the goal of reactivating cortical DMN nodes. However, the subcortical connectivity of the DMN has not been fully mapped and optimal subcortical targets for therapeutic neuromodulation of consciousness have not been identified. In this work, we created a comprehensive map of DMN subcortical connectivity by combining high-resolution functional and structural datasets with advanced signal processing methods. We analyzed 7 Tesla resting-state functional MRI (rs-fMRI) data from 168 healthy volunteers acquired in the Human Connectome Project. The rs-fMRI blood-oxygen-level-dependent (BOLD) data were temporally synchronized across subjects using the BrainSync algorithm. Cortical and subcortical DMN nodes were jointly analyzed and identified at the group level by applying a novel Nadam-Accelerated SCAlable and Robust (NASCAR) tensor decomposition method to the synchronized dataset. The subcortical connectivity map was then overlaid on a 7 Tesla 100 micron ex vivo MRI dataset for neuroanatomic analysis using automated segmentation of nuclei within the brainstem, thalamus, hypothalamus, basal forebrain, and basal ganglia. We further compared the NASCAR subcortical connectivity map with its counterpart generated from canonical seed-based correlation analyses. The NASCAR method revealed that BOLD signal in the central lateral nucleus of the thalamus and ventral tegmental area of the midbrain is strongly correlated with that of the DMN. In an exploratory analysis, additional subcortical sites in the median and dorsal raphe, lateral hypothalamus, and caudate nuclei were correlated with the cortical DMN. We also found that the putamen and globus pallidus are negatively correlated (i.e., anti-correlated) with the DMN, providing rs-fMRI evidence for the mesocircuit hypothesis of human consciousness, whereby a striatopallidal feedback system modulates anterior forebrain function via disinhibition of the central thalamus. Seed-based analyses yielded similar subcortical DMN connectivity, but the NASCAR result showed stronger contrast and better spatial alignment with dopamine immunostaining data. The DMN subcortical connectivity map identified here advances understanding of the subcortical regions that contribute to human consciousness and can be used to inform the selection of therapeutic targets in clinical trials for patients with disorders of consciousness.


title: Electron Cryo-Tomography Structure of Axonemal Doublet Microtubule from Tetrahymena thermophila

abstract: Doublet microtubules (DMT) provide a scaffold for axoneme assembly in motile cilia. Aside from /{beta} tubulins, the DMT comprises a large number of non-tubulin proteins in the luminal wall of DMT, collectively named the microtubule inner proteins (MIPs). We used electron cryo-tomography to study axoneme DMT isolated from Tetrahymena thermophila. We present the structures of DMT at nanometer and sub-nanometer resolution. The structures confirm that MIP Rib72A/B binds to the luminal wall of the DMT by multiple DM10 domains, likely by recognizing the acetylated K40 residue of -tubulin. We found Fap115, a MIP containing multiple EF-hand domains, located at the interface of four-tubulin dimers in the lumen of the A-tubule. It functions as a "molecular staple" stabilizing both lateral and longitudinal tubulin interfaces and playing a critical role in DMT stability. Defects caused by the depletion of Fap115 propagate along the axoneme due to extensive structural changes in the DMT at and beyond the Fap115 binding site. Finally, by comparing DMT structures from Tetrahymena and Chlamydomonas, we have identified a number of conserved MIPs as well as MIPs that are unique to each organism. This conservation and diversity of the DMT structures might be linked to their specific functions. Our work provides structural insights essential for understanding the roles of MIPs during motile cilium assembly and function, as well as their relationships to human ciliopathies.

category: cell biology


title: A Lethal Genetic Incompatibility between Naturally Hybridizing Species in Mitochondrial Complex I

abstract: The evolution of reproductive barriers is the first step in the formation of new species and can help us understand the diversification of life on Earth. These reproductive barriers often take the form of "hybrid incompatibilities," where genes derived from two different species no longer interact properly. Theory predicts that hybrid incompatibilities involving multiple genes should be common and that rapidly evolving genes will be more likely to cause incompatibilities, but there has been sparse empirical data to evaluate these predictions. Here, we describe a mitonuclear incompatibility involving three genes within respiratory Complex I in naturally hybridizing swordtail fish species. Individuals with specific mismatched protein combinations fail to complete embryonic development while those heterozygous for the incompatibility have reduced function of Complex I and unbalanced representation of parental alleles in the mitochondrial proteome. We document the evolutionary history of the genes involved and localize the protein-protein interactions most likely to contribute to the incompatibility. This work thus provides the first glimpse into the genetic architecture, physiological impacts, and evolutionary origin of a multi-gene incompatibility impacting naturally hybridizing species.

category: evolutionary biology


title: Estimating dispersal rates and locating genetic ancestors with genome-wide genealogies

abstract: Spatial patterns in genetic diversity are shaped by individuals dispersing from their parents and larger-scale population movements. It has long been appreciated that these patterns of movement shape the underlying genealogies along the genome leading to geographic patterns of isolation by distance in contemporary population genetic data. However, extracting the enormous amount of information contained in genealogies along recombining sequences has, up till recently, not been computational feasible. Here we capitalize on important recent advances in gene-genealogy reconstruction and develop methods to use thousands of trees to estimate time-varying per-generation dispersal rates and to locate the genetic ancestors of a sample back through time. We take a likelihood approach in continuous space using a simple approximate model (branching Brownian motion) as our prior distribution of spatial genealogies. After testing our method with simulations we apply it to the 1001 Genomes dataset of over one thousand Arabidopsis thaliana genomes sampled across a wide geographic extent. We detect a very high dispersal rate in the recent past, especially longitudinally, and use inferred ancestor locations to visualize many examples of recent long-distance dispersal and admixture. We also use inferred ancestor locations to identify the origin and ancestry of the North American expansion and to depict alternative geographic ancestries stemming from multiple glacial refugia. Our method highlights the huge amount of information about past dispersal events and population movements contained in genome-wide genealogies.


title: A hyperthermoactive-Cas9 editing tool reveals the role of a unique arsenite methyltransferase in the arsenic resistance system of Thermus thermophilus HB27

abstract:
Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to man. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pull-down assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosyl-Lmethionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyze SAM- dependent arsenite methylation with formation of monomethylarsenite (MMAs) and dimethylarsenite (DMAs). In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9- based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus {Delta}TtarsM genome was substituted by a gene encoding a stabilized yellow fluorescent protein (sYFP) to create a sensitive genome-based bioreporter system for the detection of arsenic ions.

ImportanceWe here describe the discovery of an unknown protein by using a proteomic approach with a functionally related protein as bait. Remarkably, we successfully obtained a novel type of enzyme through the interaction with a transcription regulator, controlling the expression of this enzyme. Employing this strategy, we isolated TtArsM, the first thermophilic prokaryotic arsenite methyltransferase, as a new enzyme of the arsenic resistance mechanism in T. thermophilus HB27. The atypical arsenite binding site of TtArsM categorizes the enzyme as the first member of a new arsenite methyltransferase type, exclusively present in the Thermus genus. The enzyme methylates arsenite producing MMAs and DMAs. Furthermore, we developed an hyperthermophilic Cas9-based genome-editing tool, active up to 65{degrees}C. The tool allowed us to perform highly efficient, marker-free modifications (either gene deletion or insertion) in the T. thermophilus genome. With these modifications, we confirmed the critical role of TtArsM in the arsenite detoxification system and developed a sensitive whole cell bioreporter for arsenic ions. We anticipate that the developed tool can be easily adapted for editing the genomes of other thermophilic bacteria, significantly boosting fundamental and metabolic engineering in hyperthermophilic microorganisms.

category: biochemistry


title: CLPTM1L is a lipid scramblase involved in glycosylphosphatidylinositol biosynthesis

abstract:
Glycosylphosphatidylinositols (GPIs) are membrane anchors of many eukaryotic cell surface proteins. Biosynthesis of GPIs is initiated at the cytosolic face of the endoplasmic reticulum (ER) and the second intermediate, glucosaminyl-phosphatidylinositol (GlcN-PI), is translocated across the membrane to the lumenal face for later biosynthetic steps and attachment to proteins. The mechanism of the lumenal translocation of GlcN-PI is unclear. We report that Cleft lip and palate transmembrane protein 1-like protein (CLPTM1L), an ER membrane protein of unknown function, is a lipid scramblase involved in GPI biosynthesis. Purified CLPTM1L scrambles GlcN-PI, PI, and several other phospholipids in vitro. Knockout of CLPTM1L gene in mammalian cultured cells partially decreased GPI-anchored proteins due to impaired usage of GlcN-PI, suggesting a major role of CLPTM1L in lumenal translocation of GlcN-PI.

One-Sentence SummaryCLPTM1L translocates glucosaminyl-phosphatidylinositol across the membrane during glycosylphosphatidylinositol biosynthesis.


title: SpiderLearner: An ensemble approach to Gaussian graphical model estimation

abstract: Multivariate biological data are often modeled using networks in which nodes represent a biological variable (e.g., genes) and edges represent associations (e.g., coexpression). A Gaussian graphical model (GGM), or partial correlation network, is an undirected graphical model in which a weighted edge between two nodes represents the magnitude of their partial correlation, and the absence of an edge indicates zero partial correlation. A GGM provides a roadmap of direct dependencies between variables, providing a valuable systems-level perspective. Many methods exist for estimating GGMs; estimated GGMs are typically highly sensitive to choice of method, posing an outstanding statistical challenge. We address this challenge by developing SpiderLearner, a tool that combines a range of candidate GGM estimation methods to construct an ensemble estimate as a weighted average of results from each candidate. In simulation studies, SpiderLearner performs better than or comparably to the best of the candidate methods. We apply SpiderLearner to estimate a GGM for gene expression in a publicly available dataset of 260 ovarian cancer patients. Using the community structure of the GGM, we develop a network-based risk score which we validate in six independent datasets. The risk score requires only seven genes, each of which has important biological function. Our method is flexible, extensible, and has demonstrated potential to identify de novo biomarkers for complex diseases. An open-source implementation of our method is available at https://github.com/katehoffshutta/SpiderLearner.

category: bioinformatics


title: The Polarization Dependence of 2D IR Cross-Peaks Distinguishes Parallel-Stranded and Antiparallel-Stranded DNA G-quadruplexes

abstract: Guanine-rich nucleic acid sequences have a tendency to form four-stranded non-canonical motifs known as G-quadruplexes. These motifs may adopt a wide range of structures characterized by size, strand orientation, guanine base conformation, and fold topology. Using three K+-bound model systems, we show that vibrational coupling between guanine C6=O and ring modes varies between parallel-stranded and antiparallel-stranded G-quadruplexes, and that such structures can be distinguished by comparison of polarization dependent cross-peaks in their two-dimensional infrared (2D IR) spectra. Combined with previously defined vibrational frequency trends, this analysis reveals key features of a 30-nucleotide unimolecular variant of the Bcl-2 proximal promoter that are consistent with its reported structure. This study shows that 2D IR spectroscopy is a convenient method for analyzing G-quadruplex structures that can be applied to complex sequences where traditional high-resolution methods are limited by solubility and disorder.

category: biophysics


title: Caveolin-1 temporal modulation enhances trastuzumab and trastuzumab-drug conjugate efficacy in heterogeneous gastric cancer

abstract:
Resistance mechanisms and heterogeneity in HER2-positive gastric cancers (GC) limit trastuzumab benefit in 32% of patients, and other targeted therapies have failed in clinical trials. Using genomic data from patient tissue, patient-derived xenografts (PDXs), partially humanized biological models, and HER2-targeted imaging we identified caveolin-1 (CAV1) as a complementary biomarker in GC selection for trastuzumab therapy. In retrospective analyses of samples from patients enrolled on trastuzumab trials, the CAV1-high profile was associated with low membrane HER2 density and reduced patient survival. We found a negative correlation between CAV1 tumoral protein levels - a major protein of cholesterol-rich membrane domains - and trastuzumab-drug conjugate TDM1 tumor uptake. Finally, CAV1 depletion using knockdown or pharmacologic approaches was shown to increase HER2-directed immunoPET uptake and TDM1 efficacy in GC with incomplete HER2 membranous reactivity. In support of these findings, background statin use in patients is associated with enhanced antibody efficacy. Together, this work provides mechanistic justification and clinical evidence that require prospective investigation of HER2-targeted therapies combined with statins to delay drug resistance in GC.

SIGNIFICANCEThis study identifies how CAV1 protein expression and statin use relate to GC response in HER2-targeted imaging and therapeutic approaches. In addition, to support the synergy of CAV1 depletion with TDM1 observed in mouse models, we demonstrate that statin users had better clinical responses to antibody-based therapies in HER2+ GC.

category: cancer biology


title: Nitrogen uptake and macronutrients distribution in mango trees (cv. Keitt) under three N fertigation treatments

abstract:
We assessed the effects of N concentration in the irrigation water on nutrient uptake and distribution in leaves and fruit of mango cv. Keitt grown in a lysimeter for four years. We applied three treatments: N1 - no N fertilization (less than 2 mg/L in the tap water); N2 - 10 mg/L N; N3 - 20 mg/L N.

Deficient N conditions (N1) generated low vegetative yield, high fruit:leaf ratio, high photosynthetic activity, high leaf P and K concentrations, and high sugar content along with low acidity in the fruit. Excess N concentration (N3) induced vegetative growth, and reduced fruit yield and gas-exchange characteristics. The calculated annual nitrogen uptake heavily depended on the nitrogen supply (N1-26 g/tree; N2-196 g/tree; N3- 185 g/tree).

Fruits were the major N sink being 0.82, 0.26 and 0.05 from the total annual N supplied. The N quantities accumulated in N1 fruits during the reproductive season (May-August), were above the N quantities supplied via fertigation, suggesting that N reserve in the vegetative tissues supplied the fruits high N demand.

The finding shows the importance of adequate nitrogen supply to mango trees and the dangers of excessive fertilization.

category: physiology


title: Metabolic endotoxemia is dictated by the type of lipopolysaccharide

abstract:
Lipopolysaccharides (LPS) can promote metabolic endotoxemia, which is considered inflammatory and metabolically detrimental based on Toll-like receptor (TLR)4 agonists such as Escherichia coli-derived LPS. LPS from certain bacteria antagonize TLR4 yet contribute to endotoxemia measured by Endotoxin Units (EU). We found that E. coli LPS impaired gut barrier function and worsened glycemic control in mice, but equal doses of LPS from other bacteria did not. Matching the LPS dose from R. sphaeroides and E. coli by EU revealed that only E. coli LPS promoted dysglycemia, adipose inflammation, delayed intestinal glucose absorption, and augmented insulin and GLP-1 secretion. Metabolically beneficial endotoxemia promoted by R. sphaeroides LPS counteracted dysglycemia caused by an equal dose of E. coli LPS and promoted insulin sensitivity in obese mice. The concept of metabolic endotoxemia should be expanded beyond LPS load (EU) to include LPS characteristics, where the balance of deleterious and beneficial endotoxemia regulates host metabolism.

HighlightsO_LIType of LPS dictates gut barrier function, inflammation, insulin, GLP-1, intestinal glucose absorption and blood glucose
C_LIO_LIEndotoxin Units (EU) do not reflect how LPS influences blood glucose or hormones
C_LIO_LILPS derived from certain types of bacteria are insulin sensitizers
C_LIO_LIR. sphaeroides LPS promotes metabolically beneficial endotoxemia
C_LIO_LILPS characteristics dictate metabolically beneficial versus deleterious endotoxemia
C_LI


title: Cortical cell ensemble control of past experience-dependent memory updating

abstract: When processing current sensory inputs, animals refer to related past experiences. Current information is then incorporated into the related neural network to update previously stored memories. However, the neuronal mechanism underlying the impact of memories of prior experiences on current learning is not well understood. Here, we found that a cellular ensemble in the posterior parietal cortex (PPC) that is activated during past experience mediates an interaction between past and current information to update memory through a PPC-anterior cingulate cortex circuit in mice. Moreover, optogenetic silencing of the PPC ensemble immediately after retrieval dissociated the interaction without affecting individual memories stored in the hippocampus and amygdala. Thus, a specific subpopulation of PPC cells represents past information and instructs downstream brain regions to update previous memories.


title: Muscle effort is best minimized by the right-dominant arm in the gravity field

abstract: The central nervous system (CNS) is thought to develop motor strategies that minimize various hidden criteria, such as end-point variance or effort. A large body of literature suggests that the dominant arm is specialized for such open-loop optimization-like processes whilst the non-dominant arm is specialized for closed-loop control. Building on recent results suggesting that the brain plans arm movements that takes advantage of gravity effects to minimize muscle effort, the present study tests the hypothesized superiority of the dominant arm motor system for effort minimization. Thirty participants (22.5 {+/-} 2.1 years old; all right-handed) performed vertical arm movements between two targets (40{degrees} amplitude), in two directions (upwards and downwards) with their two arms (dominant and non-dominant). We recorded the arm kinematics and the electromyographic activity of the anterior and posterior deltoid to compare two motor signatures of the gravity-related optimization process; i.e., directional asymmetries and negative epochs on phasic muscular activity. We found that these motor signatures were still present during movements performed with the non-dominant arm, indicating that the effort-minimization process also occurs for the non-dominant motor system. However, these markers were reduced compared with movements performed with the dominant arm. This difference was especially prominent during downward movements, where the optimization of gravity effects occurs early in the movement. Assuming that the dominant arm is optimal to minimize muscle effort, as suggested by previous studies, the present results support the hypothesized superiority of the dominant arm motor system for effort-minimization.


title: Effects of sub-threshold transcutaneous auricular vagus nerve stimulation on cerebral blood flow

abstract:
BackgroundTranscutaneous auricular vagus nerve stimulation (taVNS) has shown promise as a non-invasive alternative to vagus nerve stimulation (VNS) with implantable devices, which has been used to treat drug-resistant epilepsy and treatment-resistant depression. Prior work has used functional MRI to investigate the brain response to taVNS, and more recent work has also demonstrated potential therapeutic effects of high-frequency sub-threshold taVNS in rheumatoid arthritis. However, no studies to date have measured the effects of high-frequency sub-threshold taVNS on cerebral blood flow (CBF).

Objective/HypothesisThe objective of this study was to determine whether high-frequency (20 kHz) sub-threshold taVNS induces significant changes in CBF.

MethodsArterial spin labeling (ASL) MRI scans were performed on 20 healthy subjects in a single-blind placebo-controlled repeated measures experimental design. The ASL scans were performed before and after 15 minutes of either sub-threshold taVNS treatment or a sham control.

ResultstaVNS induced significant changes in CBF in the superior posterior cerebellum that were largely localized to bilateral Crus I and Crus II. Post hoc analyses showed that the changes were driven by a treatment-related decrease in CBF.

ConclusionsFifteen minutes of high-frequency sub-threshold taVNS can induce sustained CBF decreases in the bilateral posterior cerebellum in a cohort of healthy subjects. This study lays the foundation for future studies in clinical popluations to assess whether similar effects can be observed and are related to treatment outcomes.


title: Trips and Neurotransmitters: Discovering Principled Patterns across 6,850 Hallucinogenic Experiences

abstract: Psychedelics are thought to alter states of consciousness by disrupting how the higher association cortex governs bottom-up sensory signals. Individual hallucinogenic drugs are usually studied in participants in controlled laboratory settings. Here, we have explored word usage in 6,850 free-form testimonials with 27 drugs through the prism of 40 neurotransmitter receptor subtypes, which were then mapped to 3D coordinates in the brain via their gene transcription levels from invasive tissue probes. Despite the variable subjective nature of hallucinogenic experiences, our pattern-learning approach delineated how drug-induced changes of conscious awareness (e.g., dissolving self-world boundaries or fractal distortion of visual perception) are linked to cortex-wide anatomical distributions of receptor density proxies. The dominant explanatory factor related ego-dissolution-like phenomena to a constellation of 5-HT2A, D2, KOR, and NMDA receptors, anchored especially in the brains deep hierarchy (epitomized by the associative higher-order cortex) and shallow hierarchy (epitomized by the visual cortex). Additional factors captured psychological phenomena in which emotions (5-HT2A and Imidazoline1) were in tension with auditory (SERT, 5-HT1A) or visual (5-HT2A) sensations. Each discovered receptor-experience factor spanned between a higher-level association pole and a sensory input pole, which may relate to the previously reported collapse of hierarchical order among large-scale networks. Simultaneously considering many psychoactive molecules and thousands of natural language descriptions of drug experiences our framework finds the underlying semantic structure and maps it directly to the brain. These advances could assist in unlocking their wide-ranging potential for medical treatment.


title: Co-aggregation with Apolipoprotein E modulates the function of Amyloid-β in Alzheimer's disease

abstract: Isoforms of Apolipoprotein E (ApoE) determine our risk of developing late-onset Alzheimers Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between ApoE and Amyloid-{beta} (A{beta}) remains controversial. Here, single-molecule imaging shows that in the early stages of aggregation, all isoforms of ApoE associate with A{beta} in large co-aggregates, but then fall away as fibrillation happens. Similar large co-aggregates exist in the brains of AD patients, accounting for around 50% of the mass of aggregated A{beta} detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. The cellular uptake and toxicity of these large co-aggregates are isoform-dependent, suggesting a mechanistic role for ApoE-A{beta} interactions in AD.


title: Perturb-map enables CRISPR genomics with spatial resolution and identifies regulators of tumor immune composition

abstract: The cellular architecture of a tumor, particularly immune composition, has a major impact on cancer outcome, and thus there is an interest in identifying genes that control the tumor microenvironment (TME). While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying gene functions operating extracellularly or within a tissue context. To address this, we developed an approach for spatial functional genomics called Perturb-map, which utilizes protein barcodes (Pro-Code) to enable spatial detection of barcoded cells within tissue. We show >120 Pro-Codes can be imaged within a tumor, facilitating spatial mapping of 100s of cancer clones. We applied Perturb-map to knockout dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Additionally, we paired Perturb-map and spatial transcriptomics for unbiased molecular analysis of Pro-Code/CRISPR lesions. Our studies found in Tgfbr2 knockout lesions, the TME was converted to a mucinous state and T-cells excluded, which was concomitant with increased TGF{beta} expression and pathway activation, suggesting Tgfbr2 loss on lung cancer cells enhanced suppressive effects of TGF{beta} on the TME. These studies establish Perturb-map for functional genomics within a tissue at single cell-resolution with spatial architecture preserved.

category: immunology


title: Candida auris is rendered non-viable by medium-chain fatty acids

abstract: The medium-chain fatty acids, octanoic and decanoic acid, found in coconut oil, were fungistatic and decanoic acid was fungicidal against a panel of Candida auris strains, during both planktonic and biofilm growth. The strains were from all four major geographic clades, and some were resistant to several classes of antifungal drugs. These compounds are safe, natural products and could provide a new strategy for skin decolonization and environmental decontamination.

category: microbiology


title: Correlated transcriptional responses provide insights into the synergy mechanisms of the furazolidone, vancomycin and sodium deoxycholate triple combination in Escherichia coli

abstract:
Effective therapeutic options are urgently needed to tackle antibiotic resistance. Furazolidone (FZ), vancomycin (VAN), and sodium deoxycholate (DOC) show promise as their combination can synergistically inhibit the growth of, and kill, multidrug-resistant Gram-negative bacteria that are classified as critical priority by the World Health Organization. Here, we investigated the mechanisms of action and synergy of this drug combination using a transcriptomics approach in the model bacterium Escherichia coli. We show that FZ and DOC elicit highly similar gene perturbations indicative of iron starvation, decreased respiration and metabolism, and translational stress. In contrast, VAN induced envelope stress responses, in agreement with its known role in peptidoglycan synthesis inhibition. FZ induced the SOS response consistent with its DNA damaging effects, but we demonstrate that using FZ in combination with the other two compounds enables use of lower dosages and largely mitigates its mutagenic effects. Based on the gene expression changes identified, we propose a synergy mechanism where the combined effects of FZ, VAN, and DOC amplify damage to Gram-negative bacteria while simultaneously suppressing antibiotic resistance mechanisms.

ImportanceSynergistic combinations of existing antibacterials against multidrug-resistant "superbugs" are an alternative strategy to costly and arduous development of novel antibacterial molecules. The synergistic combination of nitrofurans, vancomycin and sodium deoxycholate shows promise in inhibiting and killing multidrug-resistant Gram-negative bacteria. We examined the mechanism of action and synergy of these three antibacterials, and proposed a mechanistic basis for their synergy. Our results highlight much needed mechanistic information necessary to advance this combination as a potential therapy.


title: Trophic status and local conditions affect microbial potential for denitrification versus internal nitrogen cycling in lake sediments

abstract:
The nitrogen (N) cycle is of global importance as N is an essential element and a limiting nutrient in terrestrial and aquatic ecosystems. Excessive anthropogenic N fertilizer usage threatens sensitive downstream aquatic ecosystems. Although freshwater lake sediments remove N through various microbial transformation processes, few studies have investigated the microbial communities involved. In an integrated biogeochemical and microbiological study on a eutrophic and oligotrophic lake, we estimated N removal rates in the sediments from porewater concentration gradients. Simultaneously, the abundance of different microbial N transformation genes was investigated using metagenomics on a seasonal and spatial scale. We observed that contrasting nutrient concentrations in the sediments were reflected in distinct microbial community compositions and significant differences in the abundance of various N transformation genes. Within each lake, we observed a more pronounced spatial than seasonal variability. The eutrophic Lake Baldegg showed a higher denitrification potential with higher nosZ gene (N2O reductase) abundance and higher nirS:nirK (nitrite reductase) ratio, indicating a greater capacity for complete denitrification. Correspondingly, this lake had a higher N removal efficiency. The oligotrophic Lake Sarnen, in contrast, had a higher potential for DNRA and nitrification, and specifically a high abundance of Nitrospirae, including some capable of comammox. In general, the oligotrophic lake ecosystems had a higher microbial diversity, thus acting as an important habitat for oligotrophic microbes. Our results demonstrate that knowledge of the genomic N transformation potential is important for interpreting N process rates and understanding the limitations of the N cycle response to environmental drivers.

Importance{paragraph}Anthropogenic nitrogen (N) inputs can lead to eutrophication in aquatic systems, specifically in N limited coastal ecosystems. Lakes act as N sinks by transforming reactive N to N2 through denitrification or anammox. The N cycle in lake sediments is mediated by microbial processes and affected by environmental drivers such as the amount and quality of settling organic material or nitrate concentration. However, the microbial communities mediating the different N transformation processes and their impact on N removal in freshwater lake sediments remain largely unknown. We provide the first seasonally and spatially resolved metagenomic analysis of the N cycle in the sediments of two lakes with different trophic states. We show that the trophic state of lakes provokes other microbial communities with characteristic key players and functional potential for N transformation.


title: An osmotic laxative renders mice susceptible to prolonged Clostridioides difficile colonization and hinders clearance

abstract:
Antibiotics are a major risk factor for Clostridioides difficile infections (CDIs) because of their impact on the microbiota. However, non-antibiotic medications such as the ubiquitous osmotic laxative polyethylene glycol (PEG) 3350 also alter the microbiota. Clinicians also hypothesize that PEG helps clear C. difficile. But whether PEG impacts CDI susceptibility and clearance is unclear. To examine how PEG impacts susceptibility, we treated C57Bl/6 mice with 5-day and 1-day doses of 15% PEG in the drinking water and then challenged the mice with C. difficile 630. We used clindamycin-treated mice as a control because they consistently clear C. difficile within 10 days post-challenge. PEG treatment alone was sufficient to render mice susceptible and 5-day PEG-treated mice remained colonized for up to 30 days post-challenge. In contrast, 1-day PEG treated mice were transiently colonized, clearing C. difficile within 7 days post-challenge. To examine how PEG treatment impacts clearance, we administered a 1-day PEG treatment to clindamycin-treated, C. difficile-challenged mice. Administering PEG to mice after C. difficile challenge prolonged colonization up to 30 days post-challenge. When we trained a random forest model with community data from 5 days post-challenge, we were able to predict which mice would exhibit prolonged colonization (AUROC = 0.90). Examining the dynamics of these bacterial populations during the post-challenge period revealed patterns in the relative abundances of Bacteroides, Enterobacteriaceae, Porphyromonadaceae, Lachnospiraceae, and Akkermansia that were associated with prolonged C. difficile colonization in PEG-treated mice. Thus, the osmotic laxative, PEG, rendered mice susceptible to C. difficile colonization and hindered clearance.

ImportanceDiarrheal samples from patients taking laxatives are typically rejected for Clostridiodes difficile testing. However, there are similarities between the bacterial communities from people with diarrhea or C. difficile infections (CDI) including lower diversity compared to communities from healthy patients. This observation led us to hypothesize that diarrhea may be an indicator of C. difficile susceptibility. We explored how osmotic laxatives disrupt the microbiotas colonization resistance to C. difficile by administering a laxative to mice either before or after C. difficile challenge. Our findings suggest that osmotic laxatives disrupt colonization resistance to C. difficile, and prevent clearance among mice already colonized with C. difficile. Considering that most hospitals recommend not performing C. difficile testing on patients taking laxatives and laxatives are prescribed prior to administering fecal microbiota transplants via colonoscopy to patients with recurrent CDIs, further studies are needed to evaluate if laxatives impact microbiota colonization resistance in humans.


title: Synergistic interactions between Stachybotrys chartarum and other indoor fungal species in moisture-damaged houses

abstract: The interactions between six commonly occurring fungal species in damp or water-damaged houses in southern California were studied. These fungal species were Alternaria alternata, Aspergillus niger, Chaetomium globosum, Cladosporium herbarum, Penicillium chrysogenum and Stachybotrys chartarum. In the damp building materials, S. chartarum was found to be associated with A. niger, C. globosum, and P. chrysogenum but not with A. alternata and C. herbarum. Stachybotrys chartarum showed strong antagonistic effect against A. alternata and C. herbarum and significantly inhibited in vitro growth of A. alternata and C. herbarum but had no effect on A. niger, C. globosum, and P. chrysogenum. Two trichothecenes, produced by S. chartarum, trichodermin and trichodermol, significantly inhibited spore germination and in vitro growth of A. alternata and C. herbarum but had no effect on A. niger, C. globosum, P. chrysogenum and S. chartarum. In the damp building materials (drywall, ceiling tile, and oak woods), S. chartarum significantly inhibited the growth of A. alternata and C. herbarum and had no effect on the growth and colonization of A. niger, C. globosum, P. chrysogenum in these substrata.


title: Temporal Analyses of Postnatal Liver Development and Maturation by Single Cell Transcriptomics

abstract:
Liver is the major metabolic organ, although its postnatal development and maturation are inadequately understood. We analyzed 52,834 single cell transcriptomes and identified 31 cell types or states in mouse livers at postnatal day 1, 3, 7, 21 and 56. We observed unexpectedly high levels of hepatocyte heterogeneity in the developing liver and progressive construction of the zonated metabolic functions from pericentral to periportal hepatocytes, which was orchestrated with development of sinusoid endothelial, stellate and Kupffer cells. Trajectory and gene regulatory analyses captured 36 transcription factors, including a circadian regulator Bhlhe40, in programming liver development. Remarkably, we identified a special group of macrophages enriched at day 7 with a hybrid phenotype of macrophages and endothelial cells, which may regulate sinusoidal construction and Treg cell function. This study provides a comprehensive atlas that covers all hepatic cell types instrumental for further dissection of liver development, metabolic functions and diseases.

In BriefO_LISingle cell transcriptomics of all hepatic cell types in neonatal and adult livers
C_LIO_LIConcerted development of zonated metabolic functions in hepatocytes and NPCs
C_LIO_LITransient emergence of a distinct group of macrophages at postnatal day 7
C_LIO_LIHepatic cell-cell communications that program postnatal liver development
C_LI

category: developmental biology


title: Cylcop: An R Package for Circular-Linear Copulae with Angular Symmetry

abstract: The R package cylcop extends the copula package to allow modeling of correlated circular-linear random variables using copulae that are symmetric in the circular dimension. We present and derive several new circular-linear copulae with this property and demonstrate how they can be implemented in the cylcop package to model animal movements in discrete time. The package contains methods for estimating copulae parameters, plotting probability density and cumulative distribution functions, and simulating data.


title: Entangling snakebite dynamics: the spatiotemporal role of rainfall on snake envenoming in Colombia

abstract:
The role of climate forcing on the population dynamics of infectious diseases has typically been addressed via retrospective analyses of aggregated incidence records over whole political regions. A central question in epidemiology has been whether seasonal and interannual cycles are driven by climate variation, or instead generated by other factors such as poverty or underreporting. Here, we use process-based models to determine the role of rainfall in the dynamics of snakebite, which is one of the main neglected tropical diseases around the world. We combined space-time datasets of snakebite incidence and rainfall for Colombia in combination with stochastic epidemiological models and iterated filtering methods to show the response to rainfall forcing, specifically, modulating the encounter frequency with venomous snakes. We identified six zones with different rainfall patterns to demonstrate that the relationship between rainfall and snakebite incidence was heterogeneous. Rainfall only drives snakebite incidence in regions with marked dry seasons, where rainfall becomes the limiting resource. In addition, the encounter frequency also differs between regions, and it is higher in regions where Bothrops atrox can be found. Our results show how the heterogeneous spatial distribution of snakebite risk seasonality in the country may be related to important traits of venomous snakes natural history.

SIGNIFICANCE STATEMENTThe association between seasonal climatic variables and diseases epidemiology has helped to understand disease burden under changing environments. For example, for several tropical zoonotic diseases rainfall has been identified as a critical covariate inducing incidence seasonality. Snakebite envenoming is a disease that affects mostly economically deprived populations, and the availability of treatment is scarce. However, the role of environmental factors on snakebite is still missing in the literature. We formulated an epidemiological model to quantify the role of rainfall on snakebite seasonality throughout Colombia. We found that rainfall has a significant effect on incidence in places with a marked dry season (Caribbean and Orinoco plains), but in areas without dry season (Amazonian basin and southwestern Colombia) incidence exhibits no seasonality. This study is the first epidemiological modeling approach to snakebite and underscores the importance of rainfall as the limiting resource in this system. Thus, it is important to consider the interaction between climate forcing and venomous snakes ecology as determinants of envenomation risk.


title: Rapid sequence evolution is associated with genetic incompatibilities in the plastid Clp complex

abstract: The plastid caseinolytic protease (Clp) complex plays essential roles in maintaining protein homeostasis and comprises both plastid-encoded and nuclear-encoded subunits. Despite the Clp complex being retained across green plants with highly conserved protein sequences in most species, examples of extremely accelerated amino acid substitution rates have been identified in numerous angiosperms. The causes of these accelerations have been the subject of extensive speculation but still remain unclear. To distinguish among prevailing hypotheses and begin to understand the functional consequences of rapid sequence divergence in Clp subunits, we used plastome transformation to replace the native clpP1 gene in tobacco (Nicotiana tabacum) with counterparts from another angiosperm genus (Silene) that exhibits a wide range in rates of Clp protein sequence evolution. We found that antibiotic-mediated selection could drive a transgenic clpP1 replacement from a slowly evolving donor species (S. latifolia) to homoplasmy but that clpP1 copies from Silene species with accelerated evolutionary rates remained heteroplasmic, meaning that they could not functionally replace the essential tobacco clpP1 gene. These results suggest that observed cases of rapid Clp sequence evolution are a source of epistatic incompatibilities that must be ameliorated by coevolutionary responses between plastid and nuclear subunits.


title: Lightning Fast and Highly Sensitive Full-Length Single-cell sequencing using FLASH-Seq

abstract: In the last 10 years, single-cell RNA-sequencing (scRNA-seq) has undergone exponential growth. Emulsion droplets methods1-3, such as those commercialized by 10x Genomics, have allowed researchers to analyze tens of thousands of cells in parallel in a robust and reproducible way. However, in contrast to SMART-based full-length sequencing protocols4,5, these methods interrogate only the outer portion of the transcripts and still lack the required sensitivity for analyzing comprehensively the transcriptome of individual cells. Building upon the existing SMART-seq forerunners protocols4,5, we developed FLASH-Seq (FS), a new scRNA-seq method which displays greater sensitivity while decreasing incubation times and reducing the number of processing steps compared to its predecessors. The entire FS protocol - from lysed cells to pooled cDNA libraries - can be performed in ~4.5 hours, is automation-friendly and can be easily miniaturized to decrease costs.

category: genomics


title: Systematic in vivo interrogation identifies novel enhancers and silencers associated to Atrial Fibrillation

abstract: Cis-regulatory elements control gene expression in time and space and their disruption can lead to pathologies. Reporter assays allow the functional validation of enhancers and other regulatory elements, and such assays by means of the generation of transgenic mice provide a powerful tool to study gene regulation in development and disease. However, these experiments are time-consuming and, thus, their performance is very limited. Here, we increase the throughput of in vivo mouse reporter assays by using a piggyBac transposon-based system, and use it to decode the regulatory landscape of atrial fibrillation, a prevalent cardiac arrhythmia. We systematically interrogated ten human loci associated to atrial fibrillation in the search for regulatory elements. We found five new cardiac-specific enhancers and implicated novel genes in arrhythmia through genome editing and three-dimensional chromatin analysis by 4C-seq. Of note, functional dissection of the 7q31 locus identified a bivalent regulatory element in the second intron of the CAV1 gene differentially acting upon four genes. Our system also detected negative regulatory elements thanks to which we identified a ubiquitous silencer in the 16q22 locus that regulates ZFHX3 and can outcompete heart enhancers. Our study characterizes the function of new genetic elements that might be of relevance for the better understanding of gene regulation in cardiac arrhythmias. Thus, we have .established a new framework for the efficient dissection of the genetic contribution to common human diseases.


title: PROGRAMMED AXON DEATH EXECUTOR SARM1 IS A MULTI-FUNCTIONAL NAD(P)ASE WITH PROMINENT BASE EXCHANGE ACTIVITY, ALL REGULATED BY PHYSIOLOGICAL LEVELS OF NMN, NAD, NADP AND OTHER METABOLITES

abstract: SARM1 is an NAD glycohydrolase and TLR adapter with an essential, prodegenerative role in programmed axon death (Wallerian degeneration). It has low basal NADase activity that becomes strongly activated by NAD precursor NMN. Very high levels of NAD oppose this activation, competing for the same allosteric site on SARM1s regulatory ARM domain. Injury or diseases that deplete axons of NMNAT2, an essential enzyme converting NMN to NAD, cause SARM1 activation. The resulting NAD degradation by SARM1, combined with loss of NAD synthesis by NMNAT2, causes rapid depletion of axonal NAD. This NAD loss is widely assumed to mediate axon death and is consequently a key focus for therapeutic strategies for axonopathies. However, like other NAD(P) glycohydrolases, SARM1 has additional enzyme activities whose contributions, consequences and regulation need to be fully understood. Here, we compare the multiple actions and regulation of recombinant human SARM1 with those of two other NAD(P) glycohydrolases, human CD38 and Aplysia californica ADP ribosyl cyclase. We find that SARM1 has the highest transglycosidation (base exchange) activity of these enzymes at neutral pH and with some bases this dominates NAD(P) hydrolysis and cyclisation. Moreover, like its NADase and NADPase reactions, SARM1-mediated base exchange at neutral pH is activated by increases in the NMN:NAD ratio, which we show for the first time can act in the presence of physiological levels of both metabolites. We establish that SARM1 base exchange is the most likely physiological source of calcium mobilizing agent NaADP, and potentially of other NAD(P) analogues, which could contribute to axon and cell death. We also identify regulatory effects of free pyridine bases, of NADP and of nicotinic acid riboside (NaR) on SARM1 that represent further therapeutic opportunities. These data will help to pinpoint which of the multiple functions of SARM1 is responsible for axon degeneration and how it can be optimally targeted to block axon degeneration in disease.


title: Design of complicated all-α protein structures

abstract: A wide range of de novo protein structure designs have been achieved, but the complexity of naturally occurring protein structures is still far beyond these designs. To expand the diversity and complexity of de novo designed protein structures, we sought to develop a method for designing "difficult-to-describe"-helical protein structures composed of irregularly aligned -helices like globins. Backbone structure libraries consisting of a myriad of -helical structures with 5- or 6-helices were generated by combining 18 helix-loop-helix motifs and canonical -helices, and five distinct topologies were selected for de novo design. The designs were found to be monomeric with high thermal stability in solution and fold into the target topologies with atomic accuracy. This study demonstrated that complicated -helical proteins are created using typical building blocks. The method we developed would enable us to explore the universe of protein structures for designing novel functional proteins.


title: R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

abstract:
WEE1 inhibitors have now advanced into clinical studies as monotherapy or in combination with chemoradiotherapy in TP53, RAS, BRAF, and SETD2 mutation carriers across several tumour types, yet mechanisms of resistance are still poorly understood. Here, we further elucidate the mechanisms by which AZD1775, the most potent WEE1 inhibitor, kills cells and reveal additional genetic interactions that can result in resistance, but could be used to optimise its clinical utility.

We identified RNA Polymerase II-associated factor 1 (PAF1) complex members, CDC73, CTR9, and PAF1 as major determinants of WEE1-inhibitor sensitivity in isogenic SETD2-positive and negative cell lines. PAF1-knockdown cells resist higher doses of the WEE1 inhibitor, which we show is due to reduced DNA damage induction ({gamma}H2AX) and delayed G1 checkpoint activation, ultimately protecting cells against replicative catastrophe. Investigations into the molecular mechanisms responsible for PAF1-mediated resistance identify involvement of R-loops and subsequent activation of the cyclin-dependent kinase inhibitor p21Cip1/Waf1, which in addition to causing prolonged G1 arrest in the following cell cycle, also regulates CDK activity, therefore limiting replication.

These results provide evidence that the PAF1 complex and p21 are important regulators of proliferation under increased DNA replication stress and their expression levels might be useful biomarkers to predict clinical response to WEE1 inhibitors and other ribonucleotide reductase inhibitors.


title: Assessment of the evolutionary consequence of putative driver mutations in colorectal cancer with spatial multiomic data

abstract: Cancer genomic medicine relies on targeting driver genes. However, current catalogues of cancer drivers are mostly based on indirect measurements of mutation frequencies, positions or types, rather than their effect on clonal expansions in vivo. Moreover, non-genetic drivers are largely unknown, as are the epigenetic and transcriptomic effects of genetic drivers. Here we perform spatial computational inference on multiomic data with matched whole-genome sequencing, ATAC-seq and RNA-seq. Using 436 samples, we directly quantify the contribution, or lack thereof, of putative driver genes to subclonal expansions in vivo in 30 colorectal carcinomas (4-33 samples per patient, median=15). Although subclonal neutral evolution was widespread (13/26 cases with sufficient data), there were cases with clear evidence of subclonal selection (6/26) in which we measured epigenetic and transcriptomic differences between subclones in vivo. In 7/26 cases we could not distinguish between neutral or selective evolution with the available data. We identified expanding subclones that were not driven by known genetic alterations, and propose candidate epigenetic drivers. We identified the distinguishing patterns of genomic heterogeneity produced in fast, exponentially growing tumours (7/26) versus neoplasms growing only at the periphery (19/26), as well as identifying clonally intermixed (16/28 cases with sufficient data) versus segregated malignancies (10/28). Our model-based approach measures genetic and non-genetic subclonal selection, or lack thereof, in space and time and allows in vivo comparisons of the emergent phenotypic properties of subclones within human tumours.


title: Making Strides in Doctoral-Level Career Outcomes Reporting: Surveying the Landscape of Classification and Visualization Methodologies and Creating a Crosswalk Tool

abstract: The recent movement underscoring the importance of career taxonomies has helped usher in a new era of transparency in PhD career outcomes. The convergence of discipline-specific organizational movements, interdisciplinary collaborations, and federal initiatives have all helped to increase PhD career outcomes tracking and reporting. Transparent and publicly available PhD career outcomes are being used by institutions to attract top applicants, as prospective graduate students are factoring these in when deciding on the program and institution in which to enroll for their PhD studies. Given the increasing trend to track PhD career outcomes, the number of institutional efforts and supporting offices for these studies have increased, as has the variety of methods being used to classify and report/visualize outcomes. This report identifies and summarizes currently available PhD career taxonomy tools, resources, and visualization options to help catalyze and empower institutions to develop and publish their own PhD career outcomes. Similar fields between taxonomies were mapped to create a new crosswalk tool. This work serves as an empirical review of the career outcome tracking systems available and highlights organizations, consortia, and funding agencies that are impacting policy change toward greater transparency in PhD career outcomes reporting.

category: scientific communication and education


title: Intake, performance, and feeding behavior of Holstein and Holstein x Gyr heifers grazing intensive managed tropical grasses during the rainy season

abstract:
Holstein x Gyr and Holstein are the primary dairy breed used in tropical systems, but when rearing under pasture, feed intake, behavior, and performance might differ between them. This study aimed to evaluate the voluntary intake, nutrient digestibility, performance, and ingestive behavior of Holstein and Holstein x Gyr ([1/2] Holstein x [1/2] Gyr) heifers managed in an intermittent grazing system of Guinea grass (Panicum maximum Jacq. cv. Mombaca). The experiment was conducted during the summer season throughout four periods of 21 d. Two 8-heifers (four Holstein and four Holstein x Gyr) groups, averaging 258.6 {+/-} 24.8 kg and 157.1 {+/-} 24.99 kg body weight, were used. Each group grazed a separate set of 16 paddocks, and all heifers received a concentrate supplement daily. Heifers were weighed at the beginning and end of the experiment. Fecal, forage and concentrate samples were evaluated for their dry matter (DM), crude protein (CP), crude fat, ash, neutral detergent fiber (NDF), and indigestible NDF. Feeding behavior was evaluated through 24 h of live observation for 48 h of each experimental period. Grazing, ruminating, resting, and intake of concentrate times were recorded, and rumination criteria, bout criteria, mealtime, meal frequency, and meal duration were estimated. There was no difference in dry matter intake (DMI). The Holstein x Gyr heifers had greater NDF intake and average daily gain (ADG), and feed efficiency tended to show greater CP and NDF digestibilities. The forage DMI of Holstein x Gyr was 11.70% greater than the Holstein heifers. Holstein grazed less than Holstein x Gyr heifers in the afternoon. Ruminating time was 18.43% lower for Holstein than Holstein x Gyr heifers, and rumination criteria were greater for Holstein heifers. Holstein heifers presented more prolonged rumination bouts and resting time than Holstein x Gyr heifers. Holstein x Gyr can ingest and ruminate greater amounts of fibrous material. Holstein heifers select lower fiber material, and they need to spend more time ruminating small portions of feed. Overall, we do not recommend using young Holstein heifers in tropical pasture conditions because their ADG is low because of its lower adaptability to fibrous feed and heat stress. However, this management condition is appropriate for Holstein x Gyr heifers and results in an adequate performance.

ImplicationsThis study was the first to evaluate the performance and behavior of young Holstein x Gyr and Holsteins heifers in tropical grazing systems under the same nutritional and environmental conditions. Crossbreed and purebred heifers interacted differently with the pasture; however, without noticeable variation in grazing time. As expected, Holstein heifers performance in the tropical pasture was impaired by a reduction in intake and grazing time. The greater performance observed for Holstein x Gyr heifers was assigned to greater forage intake, rumination time, and efficient forage nutrient use, showing animals adaptability to management conditions.

category: zoology


title: NPDS Toolbox: Neural Population (De)Synchronization toolbox for Matlab

abstract: The study of synchronous or asynchronous in (stochastic) neuronal populations is an important concept both in theory and in practice in neuroscience. The NPDS toolbox provides an interactive simulation platform for exploring such processes in Matlab looking through the lens of nonlinear dynamical systems. NPDS includes two main components: neural population (de)synchronization, and neural dynamics. One can investigate distribution controls on various neural models such as HH, FHN, RH, and Thalamic. Also, it supports many numerical approaches for simulation: finite-difference, pseudo-spectral, radial basis function, and Fourier methods. In addition, this toolbox can be used for population phase shifting and clustering.


title: Differential effects of early or late exposure to prenatal maternal immune activation on mouse embryonic neurodevelopment

abstract: Exposure to maternal immune activation (MIA) in utero is a risk factor for neurodevelopmental and psychiatric disorders. MIA-induced deficits in adolescent and adult offspring have been well characterized, however, less is known about the effects of MIA-exposure on embryo development. To address this gap, we performed high-resolution ex vivo magnetic resonance imaging (MRI) to investigate the effects of early (gestational day [GD]9) and late (GD17) MIA-exposure on embryo (GD18) brain structure. We identify striking neuroanatomical changes in the embryo brain, particularly in the late exposed offspring. We further examined hippocampal neuroanatomy using electron microscopy and identified differential effects due to MIA-timing. An increase in apoptotic cell density was observed in the GD9 exposed offspring, while an increase in the density of dark neurons and glia, putative markers for increased neuroinflammation and oxidative stress, was observed in GD17 exposed offspring, particularly in females. Overall, our findings integrate imaging techniques across different scales to identify differential impact of MIA-timing on the earliest stages of neurodevelopment.


title: Regulation of MHC-I and MHC-II by CIITA in transmissible cancers

abstract: MHC-I and MHC-II molecules are critical components of antigen presentation and T cell immunity to pathogens and cancer. The two monoclonal transmissible devil facial tumours (DFT1, DFT2) exploit MHC-I pathways to overcome immunological anti-tumour and allogeneic barriers. This exploitation underpins the ongoing transmission of DFT cells across the wild Tasmanian devil population. We have previously shown that constitutive expression of NLRC5 can induce stable upregulation of MHC-I on DFT1 and DFT2 cells, but unlike IFNG-treated cells, NLRC5 does not upregulate PDL1. MHC-II expression is crucial for CD4+ T cell activation and is primarily confined to haematopoietic antigen presenting cells. Transcriptomic analysis of DFT1 and DFT2 cell lines showed that several genes of the MHC-I and MHC-II pathways were upregulated in response to constitutive overexpression of the class II transactivator (CIITA) gene. This was further supported by upregulation of MHC-I protein on DFT1 and DFT2 cells, but interestingly MHC-II protein was upregulated only on DFT1 cells. The functional significance of the MHC upregulation on DFT cells was shown using serum from devils with natural or immunotherapy-induced DFT1 regressions; binding of serum IgG was stronger in CIITA-transfected cells than wild type cells, but was less than binding to NLRC5 transfected cells. This new insight into regulation of MHC-I and MHC-II in cells that naturally overcome allogeneic barriers can inform vaccine, immunotherapy, and tissue transplant strategies for human and veterinary medicine.


title: CIITA induces expression of MHC-I and MHC-II in transmissible cancers


title: RNase HI depletion strongly potentiates cell killing by rifampicin in mycobacteria

abstract:
Multidrug resistant (MDR) tuberculosis (TB) is defined by the resistance of Mycobacterium tuberculosis, the causative organism, to the first-line antibiotics rifampicin and isoniazid. Mitigating or reversing resistance to these drugs offers a means of preserving and extending their use in TB treatment. R-loops are RNA/DNA hybrids that are formed in the genome during transcription, and can be lethal to the cell if not resolved. RNase HI is an enzyme that removes R-loops, and this activity is essential in M. tuberculosis: knockouts of rnhC, the gene encoding RNase HI, are non-viable. This essentiality supports it as a candidate target for the development of new antibiotics. In the model organism Mycolicibacterium smegmatis, RNase HI activity is provided by two RNase HI enzymes, RnhA and RnhC. We show that the partial depletion of RNase HI activity in M. smegmatis, by knocking out either of the genes encoding RnhA or RnhC, led to the accumulation of R-loops. The sensitivity of the knockout strains to the antibiotics moxifloxacin, streptomycin and rifampicin was increased, with sensitivity to the transcriptional inhibitor rifampicin strikingly increased by nearly 100-fold. We also show that R-loop accumulation accompanies partial transcriptional inhibition, suggesting a mechanistic basis for the synergy between RNase HI depletion and transcriptional inhibition. A model of how transcriptional inhibition can potentiate R-loop accumulation is presented. Finally, we identified four small molecules that inhibit recombinant RnhC activity and that also potentiated rifampicin activity in whole-cell assays against M. tuberculosis, supporting an on-target mode of action, and providing the first step in developing a new class of anti-mycobacterial drug.

ImportanceThis study validates mycobacterial RNase HI as a druggable, vulnerable candidate for a new therapeutic treatment of M. tuberculosis with a novel mode of action. RNase HI depletion shows synergistic bacterial killing with some current first- and second-line antibiotics, suggesting that RNase HI inhibitors would combine well with these regimens, and could potentially accelerate the clearance of drug-sensitive strains. RNase HI inhibitors also have the potential to reduce the effective dose of rifampicin, with the comcommitant reduction in side effects. The potentiation of rifampicin efficacy conferred by RNase HI deficiency suggests that RNase HI inhibitors may be able to mitigate against development of rifampicin resistance. The synergy may also be able to reverse rifampicin resistance, rescuing this antibiotic for therapy. The surprising finding that low levels of transcriptional inhibition potentiate R-loop formation provides a key new insight into R-loop metabolism.


title: A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR

abstract: Real-time PCR is a widely used technique for quantification of gene expression. However, commercially available kits for real-time PCR are very expensive. The ongoing coronavirus pandemic has severely hampered the economy in a number of developing countries, resulting in a reduction in available research funding. The fallout of this will result in limiting educational institutes and small enterprises from using cutting edge biological techniques such as real-time PCR. Here, we report a cost-effective approach for preparing and assembling cDNA synthesis and real-time PCR mastermixes with similar efficiencies as commercially available kits. Our results thus demonstrate an alternative to commercially available kits.

category: molecular biology


title: Ribocutter: Cas9-mediated rRNA depletion from multiplexed riboseq libraries

abstract: RNA sequencing libraries produced from fragmented RNA, especially Ribo-seq libraries, contain high proportions of reads from abundant non-coding RNAs. Here, we describe a streamlined Cas9-based protocol for removing abundant rRNA/ncRNA contaminants from Ribo-seq (or other small RNA-seq) libraries and an easy-to-use software tool, ribocutter, for designing ready-to-order sgRNA templates. Following sgRNA template design, the pool of templates is in vitro transcribed using a 1-step commercial kit, which produces enough sgRNAs for multiple treatments. A single multiplexed sequencing library is then treated with Cas9/sgRNAs, followed by a short PCR program, which can increase the fraction of useful reads by more than 3-fold. Comparison of samples before and after depletion demonstrates that Cas9 produces minimal off-target effects and preserves key features (eg. footprint length, periodicity) of Ribo-seq libraries. The method is thus highly effective, costs <{pound}0.50 per sample, and minimises non-specific depletion and technical variation between samples.


title: The J domain of sacsin disrupts intermediate filament assembly

abstract: Autosomal Recessive Spastic Ataxia of the Charlevoix Saguenay (ARSACS), is caused by loss of function mutations in the SACS gene, which encodes sacsin, a giant protein of 520 kDa. A key feature of the absence of sacsin in cells is the formation of abnormal bundles of intermediate filaments (IF) including neurofilaments (NF) in neurons and vimentin IF in fibroblasts, suggesting a role of sacsin in IF homeostasis. Sacsin contains a J domain (SacsJ) homologous to Hsp40, that can interact with Hsp70 chaperones. The SacsJ domain resolved NF bundles in cultured Sacs-/- neurons, however, its mechanism is still unclear. Here, we focused on the role of SacsJ in NF assembly. We report that the SacsJ domain directly interacts with NF proteins in vitro to disassemble NFL filaments, and to inhibit their initial assembly, in the absence of Hsp70. We generated a cell-penetrating peptide derived from this domain, SacsJ-myc-TAT, which was efficient in disassembling both vimentin IF and NF in cultured fibroblasts and Sacs+/+ motor neurons as well as NF bundles in cultured Sacs-/- motor neurons. Whereas a normal NF network was restored in Sacs-/- neurons treated with the SacsJ peptide, there was some loss of IF networks in Sacs+/+ fibroblasts or neurons. These results suggest that SacsJ is a key regulator of NF and IF networks in cells, with implications for its therapeutic use.


title: A Ca2+ wave generates a force during cell extrusion in zebrafish

abstract: When oncogenic transformed or damaged cells appear within an epithelial sheet, they are apically extruded by surrounding cells. Recently, using cultured mammalian epithelial cells and zebrafish embryonic epithelial cells, we found that a calcium (Ca2+) wave propagates from RasV12-transformed cells and laser-irradiated damaged cells to surrounding cells and promotes apical extrusion by inducing polarized movements of the surrounding cells. In mammalian cell cultures, we reported that the inositol trisphosphate (IP3) receptor, gap junctions, and the mechanosensitive Ca2+ channel TRPC1 are involved in Ca2+ wave-mediated polarized movements. However, which molecules regulate Ca2+ wave-mediated polarized movements in zebrafish and whether the Ca2+ wave can generate a force remain unknown. In this study, we aimed to answer these questions. By performing pharmacological and gene knockout experiments, we showed that a Ca2+ wave induced by the IP3 receptor and trpc1 led to formation of cryptic-lamellipodia and polarized movements of surrounding cells toward extruding cells in zebrafish. By using an in vivo force measurement method, we found that the Ca2+ wave generated approximately 1 kPa of force toward extruding cells. Our results reveal a previously unidentified molecular mechanism underlying the Ca2+ wave in zebrafish and demonstrate that the Ca2+ wave generates a force during cell extrusion.


title: The haplotype-resolved reference genome of lemon (Citrus limon L. Burm f.)

abstract:
Lemon (Citrus limon (L.) Burm. f.) is an evergreen tree belonging to the genus Citrus. The fruits are particularly prized for their organoleptic and nutraceutical properties of the juice.

Herein we report, for the first time, the release of a high-quality reference genome of the two haplotypes of lemon. The sequencing has been carried out coupling Illumina short reads and Oxford Nanopore data leading to the definition of a primary and an alternative assembly characterized by a genome size of 312.8 Mb and 324.74 Mb respectively. The analysis of the long terminal repeat (LTR) allowed the identification of 1921 regions on the primary and 1911 on the alternative assembly distributed across the nine chromosomes. Furthermore, an in-silico analysis of the microRNA genes was carried out using 246 mature miRNA and the respective pre-miRNA hairpin sequences of C. sinensis. Such analysis highlighted a high conservation between the two species with 233 mature miRNAs and 51 pre-miRNA stem-loops aligning with perfect match on the lemon genome.

In parallel, total RNA was extracted from fruit, flower, leaf and root enabling the detection of 38,205 and 37,753 predicted transcripts on primary and alternative assemblies respectively. Among those, the highest and lowest number of tissue-specific transcripts were detected in flower (2.73% and 2.71% in primary and alternative assemblies respectively) and leaf (0.7% and 0.68%) while gene ontology analysis enables a more precise characterization of the expressed genes based on their function.

The availability of a reference genome is an important prerequisite both for the set-up of high-throughput genotyping analysis and for functional genomic approaches toward the characterization of the genetic determinism of traits of agronomic interest.


title: Population model of Temnothorax albipennis as a distributed dynamical system: I. self-consistent threshold is an emergent property in combination of quorum sensing and chemical perception of limited resource

abstract: House hunting of ant, such as Temnothorax albipennis, has been shown to be a distributed dynamical system. Such a system includes agent-based algorithm [1], with agents in different roles including nest exploration, nest assessment, quorum sensing, and brood item transportation. Such an algorithm, if used properly, can be applied on artificial intelligent system, like robotic swarms. Despite of its complexity, we are focusing on the quorum sensing mechanism, which is also observed in bacteria model. In bacterial model, multiple biochemical networks co-exist within each cell, including binding of autoinducer and cognate receptors, and phosphorylation-dephosphorylation cycle. In ant hunting, we also have ant commitment to the nest, mimicking binding between autoinducer and cognate receptors. We also have assessment ant specific to one nest and information exchange between two assessment ants corresponding to different nests, which is similar process to the phosphorylation-dephosphorylation cycle in bacteria quorum sensing network. Due to the similarity between the two models, we borrow the idea from bacteria quorum sensing to clarify the definition of quorum threshold through biological plausible mechanism related to limited resource model. We further made use of the contraction analysis to explore the trade-off between decision split and decision consensus within ant population. Our work provides new generation model for understanding how ant adapt to the changing environment during quorum sensing.

category: animal behavior and cognition


title: Achieving dendritic cell subset-specific targeting in vivo by site-directed conjugation of targeting antibodies to nanocarriers

abstract:
The major challenge of nanocarrier-based anti-cancer vaccination approaches is the targeted delivery of antigens and immunostimulatory agents to cells of interest, such as specific subtypes of dendritic cells (DCs), in order to induce robust antigen-specific anti-tumor responses. An undirected cell and body distribution of nanocarriers can lead to unwanted delivery to other immune cell types like macrophages reducing the vaccine efficacy. An often-used approach to overcome this issue is the surface functionalization of nanocarriers with targeting moieties, such as antibodies, mediating cell type-specific interaction. Numerous studies could successfully prove the targeting efficiency of antibody-conjugated carrier systems in vitro, however, most of them failed when targeting DCs in vivo that is partly due to cells of the reticuloendothelial system unspecifically clearing nanocarriers from the blood stream via Fc receptor ligation.

Therefore, this study shows a surface functionalization strategy to site-specifically attach antibodies in an orientated direction onto the nanocarrier surface. Different DC-targeting antibodies, such as anti-CD11c, anti-CLEC9A, anti-DEC205 and anti-XCR1, were conjugated to the nanocarrier surface at their Fc domains. Anti-mouse CD11c antibody-conjugated nanocarriers specifically accumulated in the targeted organ (spleen) over time. Additionally, antibodies against CD11c and CLEC9A proved to specifically direct nanocarriers to the targeted DC subtype, conventional DCs type 1.

In conclusion, site-directed antibody conjugation to nanocarriers is essential in order to avoid unspecific uptake by non-target cells while achieving antibody-specific targeting of DC subsets. This novel conjugation technique paves the way for the development of antibody-functionalized nanocarriers for DC-based vaccination approaches in the field of cancer immunotherapy.

category: bioengineering