title: Identification and drug-induced reversion of molecular signatures of Alzheimers disease onset and progression in AppNL-G-F, AppNL-F and 3xTg-AD mouse models

abstract:
Alzheimers disease (AD) is the most common form of dementia. Over fifty years of intense research have revealed many key elements of the biology of this neurodegenerative disorder. However, our understanding of the molecular bases of the disease is still incomplete, and the medical treatments available for AD are mainly symptomatic and hardly effective. Indeed, the robustness of biological systems has revealed that the modulation of a single target is unlikely to yield the desired outcome and we should therefore move from gene-centric to systemic therapeutic strategies. Here we present the complete characterization of three murine models of AD at different stages of the disease (i.e. onset, progression and advanced). To identify genotype-to-phenotype relationships, we combine the cognitive assessment of these mice with histological analyses and full transcriptional and protein quantification profiling of the hippocampus. Comparison of the gene and protein expression trends observed in AD progression and physiological aging revealed certain commonalities, such as the upregulation of microglial and inflammation markers. However, although AD models show accelerated aging, other factors specifically associated with A{beta} pathology are involved. Despite the clear correlation between mRNA and protein levels of the dysregulated genes, we discovered a few proteins whose abundance increases with AD progression, while the corresponding transcript levels remain stable. Indeed, we show that at least two of these proteins, namely lfit3 and Syt11, co-localize with A{beta} plaques in the brain. Finally, we derived specific A{beta}-related molecular AD signatures and looked for drugs able to globally revert them. We found two NSAIDs (dexketoprofen and etodolac) and two anti-hypertensives (penbutolol and bendroflumethiazide) that overturn the cognitive impairment in AD mice while reducing A{beta} plaques in the hippocampus and partially restoring the physiological levels of AD signature genes to wild-type levels.

TeaserThe comprehensive characterization of three AD mouse models reveals disease signatures that we used to identify approved drugs able to modify the etiology of the pathology and overturn cognitive impairment.


title: Cyto/myeloarchitectural changes of cortical gray matter and superficial white matter in early neurodevelopment: Multimodal MRI study of preterm neonates

abstract: The developing cerebral cortex undergoes rapid microstructural and morphological changes throughout the third trimester. Recently, increased attention has been focused on the identification of imaging features that represent the underlying cortical cyto/myeloarchitecture driving intracortical myelination and the maturation of cortical gray matter (GM) and its adjacent superficial white matter (sWM). However, the characterization and spatiotemporal pattern of complex cyto/myeloarchitectural changes in this critical time period remain incompletely understood. Using 92 MRI scans from 78 preterm neonates (baseline: n{square}={square}78, postmenstrual age=33.1{+/-}1.8 weeks; follow-up: n=14, 37.3{+/-}1.3), the current study leveraged combined T1/T2 intensity ratio and diffusion tensor imaging (DTI) measurements, including fractional anisotropy (FA) and mean diffusivity (MD), to characterize the cyto/myeloarchitectural architecture of cortical GM and its adjacent sWM in preterm neonates. DTI metrics during these weeks showed an overall linear developmental trajectory: FA decreased along with time in GM but increased in sWM; MD decreased in both GM and sWM. In contrast, T1/T2 measurements showed a distinctive parabolic developmental trajectory, revealing additional cyto/myeloarchitectural signature inferred. Furthermore, the spatiotemporal courses of T1/T2 ratio and DTI parameters were found to be regionally heterogeneous across the cerebral cortex, suggesting these imaging features specific relationship to regional cyto/myeloarchitectural maturation: faster T1/T2 ratio changes were found in the central, ventral, and temporal regions of GM and sWM, faster FA increases in anterior sWM areas, and faster MD decreases in GM and sWM central and cingulate areas. Taken together, our results may offer an explanation of the novel pattern of cyto/myeloarchitectural processes observed throughout the third trimester, including dendritic arborization, synaptogenesis, glial proliferation, as well as radial glial cell organization and apoptosis. Finally, T1/T2 ratio and DTI measurements were significantly associated with 1 year outcome scores of language and cognitive performance as well as perinatal clinical conditions, including intraventricular hemorrhage and chronic lung disease, demonstrating their potential as imaging biomarkers characterizing microstructural deviation in atypical neurodevelopment. Ultimately, with combined properties of cortical T1/T2 and DTI measurements, this study provides unique insights into the cellular processes and associated developmental mechanisms during the critical development of the third trimester.

category: neuroscience


title: Spinal sensory neurons project onto hindbrain to stabilize posture and enhance locomotor speed

abstract:
In the spinal cord, cerebrospinal fluid-contacting neurons (CSF-cNs) are GABAergic interoceptive sensory neurons that detect spinal curvature via a functional coupling with the Reissner fiber. This mechanosensory system has recently been found involved in spine morphogenesis and postural control but the underlying mechanisms are not fully understood. In zebrafish, CSF-cNs project an ascending and ipsilateral axon reaching two to six segments away. Rostralmost CSF-cNs send their axons ipsilaterally into the hindbrain, a brain region containing motor nuclei and reticulospinal neurons (RSNs), which send descending motor commands to spinal circuits. Until now, the synaptic connectivity of CSF-cNs has only been investigated in the spinal cord where they synapse onto motor neurons and premotor excitatory interneurons. The identity of CSF-cN targets in the hindbrain and the behavioral relevance of these sensory projections from spinal cord to hindbrain are unknown. Here, we provide anatomical and molecular evidence that rostralmost CSF-cNs synapse onto the axons of large RSNs including the Mauthner cells and early born chx10+ neurons. Functional anatomy and optogenetic-assisted mapping reveal that rostral CSF-cNs also synapse onto the soma and dendrites of cranial motor neurons innervating hypobranchial muscles. During acousto-vestibular evoked escape responses, ablation of rostralmost CSF-cNs results in a weaker escape response with a decreased C-bend amplitude, lower speed and a deficient postural control. Our study demonstrates that spinal sensory feedback enhances speed and stabilizes posture, and reveals a novel spinal gating mechanism acting on the output of descending commands sent from the hindbrain to the spinal cord.

eTOCCerebrospinal fluid-contacting neurons are mechanosensory cells that detect spinal curvature. Wu et al. show here that rostralmost CSF-cNs synapse in the hindbrain onto cranial motor neurons and the descending axons of reticulospinal neurons, and enhance speed and power as well as postural control during active locomotion.

HighlightsO_LICerebrospinal fluid-contacting neurons (CSF-cNs) in rostral spinal cord form inhibitory synapses onto cranial motor neurons
C_LIO_LIRostral CSF-cNs synapse onto descending axons of reticulospinal neurons
C_LIO_LICSF-cN sensory feedback in the rostral spinal cord enhance speed and power of locomotion
C_LIO_LIRostral CSF-cNs projecting to the hindbrain contribute to postural control
C_LI


title: Tumor-reactivity of d2 negative gdT cells and the role of the gdT cell receptor

abstract: {beta}T cells engineered to express a defined {gamma}{delta}TCR (TEG) to attack cancer cells have shown great promise when using a {gamma}9{delta}2TCR to redirect {beta}T cells (1-9). Reports by us (1-9) and recent reports by others (10, 11) support the key role of the {gamma}9{delta}2TCR in cancer recognition. We further emphasized the crucial role of the {delta}TCR chain and that differences in CDR3 sequences of the {delta}TCR chain modulates functional avidity of TEGs (2, 8). We and others demonstrated that also {delta}2 negative {gamma}{delta}TCRs are able to redirect {beta}T cells towards different tumor cell lines (12-15). However, some studies suggest that {delta}2 negative {gamma}{delta}TCRs play a minor role in the tumor recognition by {delta}2 negative {gamma}{delta}T cells (16, 17). In addition for both modes of action for tumor-recognition, {delta}2 negative {gamma}{delta}TCR-dependent and -independent, it has been suggested that CMV infection is not only a major driver of {delta}2 negative {gamma}{delta}T cell expansion (18) but also induces tumor-cross reactive {delta}2 negative {gamma}{delta}T cells (19-21). Therefore, we aimed to systematically explore frequencies of tumor reactive {delta}2 negative {gamma}{delta}T cells in naive repertoires (cord blood) and patients with or without CMV infection and examined the potential role the parental {delta}2 negative {gamma}{delta}TCR in anti-tumor reactivity of selected clones. We observed that approximately 30% of all tested clones were tumor-reactive, though no differences were observed between different sources. Surprisingly, none of the so far tested {gamma}{delta}TCR did mediate strong anti-tumor reactivity of the parental clones. Though numbers of tested TCR sequences are still low, our data imply that tumor-reactivity of {delta}2 negative {gamma}{delta}T cells is frequently not mediated by the {delta}2 negative {gamma}{delta}TCR alone.

category: immunology


title: Whats under the Christmas tree? Soil acidification alters fir tree rhizosphere bacterial and eukaryotic communities, their interactions, and functional traits

abstract:
pH has been identified as a master regulator of the soil environment, controlling the solubility and availability of nutrients. As such, soil pH exerts a strong influence on indigenous microbial communities. In this study we describe a soil acidification experiment and the resulting effects on the rhizosphere communities of fir trees on a Christmas tree plantation. The acidification treatment reduced the pH of bulk soil by [~]1.4 pH units and was associated with reduced Ca, Mg, and organic matter content. Similarly, root chemistry differed due to soil acidification with roots in acidified soils showing significantly higher Al, Mn, and Zn content and reduced levels of B and Ca. 16S rRNA and 18S rRNA gene sequencing was pursued to characterize the bacterial/archaeal and eukaryotic communities in the rhizosphere soils. The acidification treatment induced dramatic and significant changes in the microbial populations, with thousands of 16S RNA gene sequence variants and hundreds of 18S rRNA gene variants being significantly different in relative abundance between the treatments. Additionally, co-occurrence networks showed that bacterial and eukaryotic interactions, network topology, and hub taxa were significantly different when constructed from the control and acidified soil rRNA gene amplicon libraries. Finally, metagenome sequencing showed that the taxonomic shifts in the community resulted in alterations to the functional traits of the dominant community members. Several biochemical pathways related to sulfur and nitrogen cycling distinguished the metagenomes generated from the control and acidified soils, demonstrating the myriad of effects soils acidification induces to rhizosphere microbes.

IMPORTANCESoil pH has been identified as the property that exerts the largest influence on soil microbial populations. We employed a soil acidification experiment to investigate the effect of lowering soil pH on the bacterial and eukaryotic populations in the rhizosphere of Christmas trees. Acidification of the soils drove alterations of fir tree root chemistry and large shifts in the taxonomic and functional composition of the communities, involving pathways in sulfur and nitrogen cycling. These data demonstrate that soil pH influences are manifest across all organisms inhabiting the soil, from the host plant to the microorganisms inhabiting the rhizosphere soils. Thus, pH is an important factor that needs to be considered when investigating soil and plant health, the status of the soil microbiome, and terrestrial nutrient cycling.

category: microbiology


title: The Fragile X Mental Retardation Protein protects the lung from xenobiotic stress by facilitating the Integrated Stress Response

abstract: Stress response pathways protect the lung from the damaging effects of environmental toxicants. Here we investigate the role of the Fragile X Mental Retardation Protein (FMRP), a multifunctional protein implicated in stress responses, in the lung. We report that FMRP is expressed in murine and human lungs, in the airways and more broadly. Analysis of airway stress responses in mice and in a murine cell line ex vivo, using the well-established Naphthalene (Nap) injury model, reveals that FMRP-deficient cells exhibit increased expression of markers of oxidative and genotoxic stress and increased cell death. We find that FMRP-deficient cells fail to actuate the Integrated Stress Response Pathway (ISR) and upregulate the transcription factor ATF4. Knockdown of ATF4 expression phenocopies the loss of FMRP. We extend our analysis of the role of FMRP to human bronchial BEAS-2B cells, using a 9, 10-Phenanthrenequinone air pollutant model, to find that FMRP-deficient BEAS-2B also fail to actuate the ISR and exhibit greater susceptibility. Taken together, our data suggest that FMRP has a conserved role in protecting the airways by facilitating the ISR.

category: cell biology


title: Investigating temperature effects on coastal microbial populations and trophic interactions with 16S and 18S rRNA metabarcoding

abstract: Temperature is a universal driver of microbial life, with rising sea surface temperatures expected to differentially influence the physiology, biodiversity, and distribution of bacteria and plankton. The impact of ocean warming on microbial interactions remains unclear, despite the importance of these relationships for ecosystem functioning. We employed weekly to monthly 18S and 16S rRNA gene amplicon metabarcoding over a full year (33 d) in a subtropical estuary, investigating microbial population dynamics and network interactions with respect to a temperature gradient (9-31{degrees}C). Certain microbes (e.g., Acidimicrobiia, Nitrososphaeria, and Syndiniales) increased in relative abundance with rising temperatures (Spearman {rho} > 0.69), whereas other groups (e.g., Alpha- and Gammaproteobacteria, Bacillariophyta, and Dinophyceae) slightly decreased, became saturated, or remained stable. With network analysis, we observed an increase in 18S- 18S interactions in warm (23-31{degrees}C) vs. cold (<23{degrees}C) temperatures, largely involving Syndiniales, Bacillariophyta, and Dinophyceae ASVs. Bacteria ASVs were more connected to other microbes (higher degree and centrality) and became more prominent in the cold network, highlighted by well-established cross-domain relationships (e.g., diatom-bacteria) and positive interactions among bacteria (e.g., SAR11 and Rhodobacterales). These efforts highlight the types of interactions that may be more common under changing temperatures, with implications for modeling biogeochemistry and assessing ecosystem health.

category: ecology


title: A Phytosociological Classification Of The Peruvian Vegetation

abstract: AbstractThe present work is an update of the syntaxonomic scheme of Peru, with the results obtained to present. We currently recognize 218 phytosociological associations, of which 11 are novelties: Ageratino azangaroensis-Polylepidetum incarum ass. nov., Alchemillo pinnatae-Stipetum hans-meyeri ass. nov., Barnadesio dombeyanae-Polylepidetum racemosae ass. nov., Eclipto prostratae-Echinochloetum crus-pavonis ass. nov., Geoffroeetum decorticantis ass. nov., Junellio arequipensis-Proustietum cuneifoliae ass. nov., Lemnetum aequinoctialis ass. nov., Mutisio cochabambensis-Polylepidetum incarum ass. nov., Poo glaberrimae-Calamagrostietum eminentis ass. nov., Sarcocornietum andinae ass. nov., and Schino mollis-Prosopidetum calderensis ass. nov. These 218 associations belong to 34 phytosociological classes, four of which are described here for the first time: Limoselletea australis cl. nov., Mimulo glabrati-Plantaginetea australis cl. nov., Tillandsietea latifolio-landbeckii cl. nov., Woodsio montevidensis-Cheilanthetea pruinatae cl. nov. In addition, 5 new orders (Alternanthero pungentis-Cynodontetalia dactylonis ord. nov., Coreopsietalia fasciculatae ord. nov., Echinopsio schoenii-Proustetalia cuneifoliae ord. nov., Mimulo glabrati-Plantaginetalia australis ord. nov., Tillandsietalia latifolio-landbeckii ord. nov.), and 7 new alliances (Azollo filiculoidis-Lemnion gibbae all. nov., Ditricho submerse-Isoetion lechleri all. nov., Echinopsio schoenii-Proustion cuneifoliae all. nov., Haageocerion acranthi all. nov., Lepidio chichicarae-Cynodontion dactylonis all. nov., Schoenoplection tatorae all. nov., and Tillandsion purpureo-latifoliae all. nov.) are also described.


title: The mayfly subimago explained. The regulation of metamorphosis in Ephemeroptera

abstract: In the Paleozoic era, more than 400 million years ago, insects continued molting after forming functional wings. Today, however, all flying insects stop molting after metamorphosis when they become fully winged. The only exception is the mayflies (Ephemeroptera), which molt in the subimago, a flying intermediate stage between the nymph and the adult. However, the identity and homology of the subimago remains underexplored. Debate remains regarding whether this stage represents a modified nymph, an adult, or a pupa like that of butterflies. Another relevant question is why do mayflies maintain the subimago stage despite the risk of molting fragile membranous wings. These questions have intrigued numerous authors but nonetheless, clear answers have not yet been found. However, by combining morphological studies, hormonal treatments, and molecular analysis in the mayfly species Cloeon dipterum, we found new answers to these old questions. We observed that treatment with a juvenile hormone analog in the last nymphal instar stimulated the expression of Kr-h1 gene and reduced that of E93, which suppress and trigger metamorphosis, respectively. Consequently, the subimago is not formed in these treated mayflies. This indicates that metamorphosis is determined prior to the formation of the subimago, which must therefore be considered an instar of the adult stage. We also observed that the forelegs dramatically grow between the last nymphal instar, the subimago, and the adult. This necessary growth is spread over the last two stages, which could explain, at least in part, the adaptive sense of the subimago.

category: evolutionary biology


title: Meiotic Cas9 expression mediates genotype conversion in the male and female mouse germline

abstract: Highly efficient genotype conversion systems have potential to facilitate the study of complex genetic traits using laboratory mice and to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of genotype conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double strand DNA break is feasible in the female mouse germline. In the male germline, however, all double strand breaks were instead repaired by end joining mechanisms to form an  insertion/deletion (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate genotype conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient double strand DNA break formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of genotype conversion and further increase the rate of  super-Mendelian inheritance from both male and female mice.

category: genetics


title: Identification of anthracnose (Colletotrichum lentis) race 1 resistance loci in lentil by integrating linkage mapping and a genome-wide association study

abstract: Anthracnose, caused by Colletotrichum lentis, is a devastating disease of lentil in Western Canada. Growing resistant lentil cultivars is the most cost-effective and environmentally friendly approach to prevent seed yield losses that can exceed 70%. To identify loci conferring resistance to anthracnose race 1 in lentil, biparental quantitative trait loci (QTL) mapping of two recombinant inbred line (RIL) populations was integrated with a genome-wide association study (GWAS) using 200 diverse lentil accessions from a lentil diversity panel (LDP). A major-effect QTL (qAnt1.Lc-3) conferring resistance to race 1 was mapped to lentil chromosome 3 and co-located on the lentil physical map for both RIL populations. Clusters of candidate nucleotide binding-leucine-rich repeats (NB-LRR) and other defense-related genes were uncovered within the QTL region. A GWAS detected 14 significant SNP markers associated with race 1 resistance on chromosomes 3, 4, 5, and 6. The most significant GWAS SNPs on chromosome 3 supported qAnt1.Lc-3 and delineated a region of 1.6 Mb containing candidate resistance genes. The identified SNP markers can be directly applied in marker-assisted selection to accelerate the introgression of race 1 resistance in lentil breeding.

category: genomics


title: Beyond single discrete responses: An integrative and multidimensional analysis of behavioral dynamics assisted by Machine Learning

abstract: Behavioral systems, understanding it as an emergent system comprising the environment and organism subsystems, include spatial dynamics as a primary dimension in natural settings. Nevertheless, under the standard approaches, the experimental analysis of behavior is based on the single response paradigm and the temporal distribution of discrete responses. Thus, the continuous analysis of spatial behavioral dynamics has been a scarcely studied field. The technological advancements in computer vision have opened new methodological perspectives for the continuous sensing of spatial behavior. With the application of such advancements, recent studies suggest that there are multiple features embedded in the spatial dynamics of behavior, such as entropy, and that they are affected by programmed stimuli (e.g., schedules of reinforcement), at least, as much as features related to discrete responses. Despite the progress, the characterization of behavioral systems is still segmented, and integrated data analysis and representations between discrete responses and continuous spatial behavior are exiguous in the Experimental Analysis of Behavior. Machine Learning advancements, such as t-SNE, variable ranking, provide invaluable tools to crystallize an integrated approach for analyzing and representing multidimensional behavioral data. Under this rationale, the present work: 1) proposes a multidisciplinary approach for the integrative and multilevel analysis of behavioral systems, 2) provides sensitive behavioral measures based on spatial dynamics and helpful data representations to study behavioral systems, and 3) reveals behavioral aspects usually ignored under the standard approaches in the experimental analysis of behavior. To exemplify and evaluate our approach, the spatial dynamics embedded in phenomena relevant to behavioral science, namely water-seeking behavior, and motivational operations, are examined, showing aspects of behavioral systems hidden until now.

category: animal behavior and cognition


title: The Burden Borne by Protein Methyltransferases: Rates and Equilibria of Nonenzymatic Methylation of Amino Acid Side-Chains by SAM in Water

abstract: SAM is a powerful methylating agent, with a methyl group transfer potential matching the phosphoryl group transfer potential of ATP. SAM-dependent N-methyltransferases have evolved to catalyze the modification of specific lysine residues in histones and transcription factors, in addition to generating epinephrine, N-methylnicotinamide, and a quaternary amine (betaine) that is used to maintain osmotic pressure in plants and halophilic bacteria. To assess the catalytic power of these enzymes and their potential susceptibility to transition state and multisubstrate analogue inhibitors, we determined the rates and positions of equilibrium of methyl transfer from the trimethylsulfonium ion to model amines in the absence of a catalyst. Unlike the methyl group transfer potential of SAM, which becomes more negative with increasing pH throughout the normal pH range, equilibrium constants for the hydrolytic demethylation of secondary, tertiary and quaternary amines are found to be insensitive to changing pH and resemble to each other in magnitude, with an average {Delta}G value of [~] -0.7 kcal/mol at pH 7. Thus, each of the three steps in the mono- di- and trimethylation of lysine by SAM is accompanied by a free energy change of -7.5 kcal/mol in neutral solution. Arrhenius analysis of the uncatalyzed reactions shows that the unprotonated form of glycine attacks the trimethylsulfonium ion (TMS++) with a second order rates constant of 1.8 x 10-7 M-1 s-1 at 25 {degrees}C ({Delta}H{ddagger} = 22 kcal/mol and T{Delta}S{ddagger} = -6 kcal/mol). Comparable values are observed for the methylation of secondary and tertiary amines, with k25 = 1.1 x 10-7 M-1 s-1 for sarcosine and 4.3 x 10-8 M-1 s-1 for dimethylglycine. The nonenzymatic methylation of imidazole and methionine by TMS++, benchmarks for the methylation of histidine and methionine residues by SETD3, exhibit k25 values of 3.3 x 10-9 and 1.2 x 10-9 M-1 s-1 respectively. Lysine methylation by SAM, although slow under physiological conditions (t1/2 7 weeks at 25 {degrees}C), is accelerated 1.1 x 1012 -fold at the active site of a SET domain methyltransferase.

category: biochemistry


title: GdDO3NI allows imaging of hypoxia after brain injury

abstract:
PurposeIn this study, we use the hypoxia targeting agent (GdDO3NI, a nitroimidazole-based T1 MRI contrast agent) for imaging hypoxia in the injured brain after experimental traumatic brain injury (TBI) using magnetic resonance imaging (MRI), and validate the results with immunohistochemistry (IHC) using pimonidazole.

MethodsTBI induced mice (controlled cortical impact model) were imaged at 7T using a T2 weighted fast spin-echo sequence to estimate the extent of the injury. The mice were then were intravenously injected with either conventional T1 agent (gadoteridol) or GdDO3NI at 0.3 mmol/kg dose (n=5 for each cohort) along with pimonidazole (60 mg/kg). Mice were imaged pre- and post-contrast using a T1-weighted spin-echo sequence for three hours. Regions of interests were drawn on the brain injury region, the contralateral brain as well as on the cheek muscle region for comparison of contrast kinetics. Brains were harvested immediately post imaging for immunohistochemical analysis.

ResultsGdDO3NI is retained in the injury region for up to 3 hours post-injection (p< 0.05 compared to gadoteridol) while it rapidly clears out of the muscle region. On the other hand, conventional MRI contrast agent gadoteridol clears out of both the injury region and muscle rapidly, although with a relatively more delayed wash out in the injury region. Minimal contrast enhancement was seen for both agents in the contralateral hemisphere. Pimonidazole staining confirms the presence of hypoxia in both gadoteridol and GdDO3NI cohorts, and the later cohort shows good agreement with MRI contrast enhancement.

ConclusionGdDO3NI was successfully shown to visualize hypoxia in the brain post-TBI using T1-wt MRI.

category: bioengineering


title: Structural analysis of the OC43 coronavirus 2'-O-RNA methyltransferase

abstract:
The OC43 coronavirus is a human pathogen that usually causes only the common cold. One of its key enzymes, similar to other coronaviruses, is the 2'-O-RNA methyltransferase (MTase) that is essential for viral RNA stability and expression. Here, we report the crystal structure of the 2'-O-RNA MTase in a complex with the pan-methyltransferase inhibitor sinefungin solved at 2.2 [A] resolution. The structure revealed an overall fold consistent with the fold observed in other coronaviral MTases. The major differences are in the conformation of the C-terminus of the nsp16 subunit and an additional helix in the N-terminus of the nsp10 subunits. The structural analysis also revealed very high conservation of the SAM binding pocket suggesting that the SAM pocket is a suitable spot for the design of antivirals effective against all human coronaviruses.

ImportanceSome coronaviruses are dangerous pathogens while some cause only common colds. The reasons are not understood although the spike proteins probably play an important role. However, to understand the coronaviral biology in sufficient detail we need to compare the key enzymes from different coronaviruses. We solved the crystal structure of 2'-O-RNA methyltransferase of the OC43 coronavirus, a virus that usually causes mild colds. The structure revealed some differences in the overall fold but also revealed that the SAM binding site is conserved suggesting that development of antivirals against multiple coronaviruses is feasible.

category: biophysics


title: Characterizing Locus Specific Chromatin Structure and Dynamics with Correlative Conventional and Super Resolution imaging in living cells

abstract: The dynamic rearrangement of chromatin is critical for gene regulation, but mapping both the spatial organization of chromatin and its dynamics remains a challenge. Many structural conformations are too small to be resolved via conventional fluorescence microscopy and the long acquisition time of super-resolution PALM imaging precludes the structural characterization of chromatin below the optical diffraction limit in living cells due to chromatin motion. Here we develop a correlative conventional fluorescence and PALM imaging approach to quantitatively map time-averaged chromatin structure and dynamics below the optical diffraction limit in living cells. By assigning localizations to a locus as it moves, we reliably discriminate between bound and searching dCas9 molecules, whose mobility overlap. Our approach accounts for changes in DNA mobility and relates local chromatin motion to larger scale domain movement. In our experimental system, we show that compacted telomeres have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Correlative conventional and PALM imaging therefore improves the ability to analyze the mobility and time-averaged nanoscopic structural features of locus specific chromatin with single molecule precision and yields unprecedented insights across length and time scales.


title: Case study of a rhizosphere microbiome assay on a bamboo rhizome with excessive shoots

abstract: Young Moso bamboo shoots are a very popular seasonal food. Bamboo is an important source of income for farmers and the value for cultivation has recently been estimated to $30,000 per hectare. A rare and valuable phenomenon has recently appeared where dozens of adjacent buds within a single Moso bamboo rhizome have grown into shoots. Due to its rarity, this phenomenon, which is of practical importance for the production of edible shoots, has not been scientifically studied. We report the occurrence of a rhizome with 18 shoots, of which the microbiome were analyzed, using rhizomes having one or no shoots as controls. The community of prokaryotes, but not fungi, correlated with the shoot numbers. Burkholderia was the most abundant genus, which negatively correlated with rhizome shoot number, while Clostridia and Ktedonobacteria positively correlated with many shoots. Two Burkholderia strains were isolated and their plant-growth promoting activity was tested. The isolated Burkholderia strains attenuated the growth of bamboo seedlings. Analysis of collected events of enhanced shoot production in China showed no evidence that enhanced shoot development was heritable. Overall, our data provides a firsthand study on excessive shoot development of bamboo.

category: plant biology


title: Neural correlates of metacognition across the adult lifespan

abstract: Metacognitive accuracy describes the degree of overlap between the subjective perception of ones decision accuracy (i.e., confidence) and objectively observed performance. With older age, the need for accurate metacognitive evaluation increases; however, error detection rates typically decrease. We investigated the effect of ageing on metacognitive accuracy using event-related potentials (ERPs) reflecting error detection and confidence: the error/correct negativity (Ne/c) and the error/correct positivity (Pe/c). Sixty-five healthy adults (20 to 76 years) completed a complex perceptual task and provided confidence ratings. We found that metacognitive accuracy declined with age beyond the expected decline in task performance, while the adaptive adjustment of behaviour was well preserved. Pe/c amplitudes varied by confidence rating, but they did not mirror the reduction in metacognitive accuracy. Ne/c amplitudes decreased with age except for high confidence correct responses. The results suggest that age-related difficulties in metacognitive evaluation could be related to an impaired integration of decision accuracy and confidence information processing. Ultimately, training the metacognitive evaluation of fundamental decisions in older adults might constitute a promising endeavour.


title: Stable brain PET metabolic networks using a multiple sampling scheme

abstract: The human brains interregional communication is vital for its proper functioning. A promising direction for investigating how these regions communicate relies on the assumption that the brain is a complex network. In this context, images derived from positron emission tomography (PET) have been proposed as a potential source for understanding brain networks. However, such networks are often assembled via direct computation of inter-subject correlations, neglecting variabilities between subjects and jeopardizing the construction of group representative networks. Here, we used [18F]FDG-PET data from 1027 individuals at different syndromal stages (352 CU, 621 MCI and 234 AD) to develop a novel method for constructing stable (i.e. resilient to spurious data points) metabolic brain networks. Our multiple sampling (MS) scheme generates brain networks with higher stability when compared to the conventional approach. In addition, the proposed method is robust to imbalanced datasets and requires 50% fewer subjects to achieve stability than the conventional approach. Our method has the potential to considerably boost PET data reutilization and advance our understating of human brain network patterns in health and disease.


title: An optimal population code for global motion estimation in local direction-selective cells

abstract: Nervous systems allocate computational resources to match stimulus statistics. However, the physical information that needs to be processed depends on the animals own behavior. For example, visual motion patterns induced by self-motion provide essential information for navigation. How behavioral constraints affect neural processing is not known. Here we show that, at the population level, local direction-selective T4/T5 neurons in Drosophila represent optic flow fields generated by self-motion, reminiscent to a population code in retinal ganglion cells in vertebrates. Whereas in vertebrates four different cell types encode different optic flow fields, the four uniformly tuned T4/T5 subtypes described previously represent a local snapshot. As a population, six T4/T5 subtypes encode different axes of self-motion. This representation might serve to efficiently encode more complex flow fields generated during flight. Thus, a population code for optic flow appears to be a general coding principle of visual systems, but matching the animals individual ethological constraints.


title: GABAergic neuronal IL-4R mediates T cell effect on memory

abstract: Mechanisms governing how immune cells and their derived molecules impact homeostatic brain function are still poorly understood. Here, we elucidate neuronal mechanisms underlying effects of T cells on synaptic function and episodic memory. Depletion of CD4 T cells led to memory deficits and impaired long term potentiation. Severe combined immune-deficient (SCID) mice exhibited amnesia, which was reversible by repopulation with T cells from wild-type but not from IL-4-knockout mice. This rescue was mediated via IL-4 receptors (IL-4R) expressed on neurons. Exploration of snRNAseq of neurons participating in memory processing provided insights into synaptic organization and plasticity-associated pathways and genes regulated by immune cells and molecules. IL-4R knockout in inhibitory neurons impaired contextual fear memory, suggesting participation of an IL-4-associated switch in regulating synaptic function and promoting contextual fear memory. These findings provide insights into neuroimmune interactions at the transcriptional and functional levels in neurons.


title: Antibody elicited by HIV-1 immunogen vaccination in macaques displaces Env fusion peptide and destroys a neutralizing epitope

abstract: HIV-1 vaccine design aims to develop an immunogen that elicits broadly neutralizing antibodies against a desired epitope, while eliminating responses to off-target regions of HIV-1 Env. Here we report isolation and characterization of Ab1245, an off-target antibody against the Env gp120-gp41 interface, from V3-glycan patch immunogen-primed and boosted macaques. A 3.7[A] cryo-EM structure of an Ab1245-Env complex reveals one Ab1245 Fab binding asymmetrically to Env trimer at the gp120-gp41 interface using its long CDRH3 to mimic regions of gp41. The mimicry includes positioning of a CDRH3 methionine into the gp41 tryptophan clasp, resulting in displacement of the fusion peptide and fusion peptide-proximal region. Despite fusion peptide displacement, Ab1245 is non-neutralizing even at high concentrations, implying that only two fusion peptides per trimer are required for viral-host membrane fusion. These structural analyses facilitate immunogen design to prevent elicitation of Ab1245-like antibodies that block neutralizing antibodies against the fusion peptide.


title: Strong genetic structure and limited connectivity among populations of Clark's Anemonefish (Amphiprion clarkii) in the centre of marine biodiversity

abstract: Populations of anemonefish species often show signs of local isolation due limited dispersal potential and oceanographic conditions. Additionally, anthropogenic pressure, such as overharvesting and coral reef exploitation causes reduced population size, eventually leading to local extinction. The understanding of the genetic population structure, as well as the influence of both historical and current connectivity, is required to design effective marine protected area (MPA) networks. In this study, the genetic structure of Clarks Anemonefish (Amphiprion clarkii) populations of the Indo-Malay Archipelago (IMA) is assessed through mitochondrial control region (mtCR) sequences and nuclear microsatellites. Results provided evidence of a significant genetic structure (mtCR: {Phi}st = 0.42039, {Phi}ct = 0.63852; microsatellites: Fst = 0.01449, Fct = 0.05199). Genetic breaks were identified among Western (Padang Karimunjawa), Central (Sulawesi, Borneo, Bali, Komodo, Timor), and Eastern (Biak) IMA populations, which matches with patterns obtained for congeneric and other coral reef taxa. Due to the restricted connectivity among these three regions, it is suggested to consider them as separate management areas in the design of MPA networks.

category: molecular biology


title: Dissecting the roles of MBD2 isoforms in regulating NuRD complex function during cellular differentiation

abstract: The Nucleosome Remodelling and Deacetylation (NuRD) complex is a crucial regulator of cellular differentiation. Two members of the Methyl-CpG-binding domain (MBD) protein family, MBD2 and MBD3, are known to be integral, but mutually exclusive subunits of the NuRD complex. Several MBD2 and MBD3 isoforms are present in mammalian cells, resulting in distinct MBD-NuRD complexes. If these different complexes serve distinct biochemical and/or functional activities during differentiation is not completely understood. Based on the essential role of MBD3 in lineage commitment, we systematically investigated a diverse set of MBD3 and MBD2 variants for their potential to rescue the differentiation block observed in mouse embryonic stem cells (ESCs) lacking MBD3. Our study reveals that while MBD3 is indeed crucial for ESC differentiation to neuronal cells, this function is independent of its MBD domain or binding to methylated DNA. While MBD3 isoforms are highly redundant, we identify that MBD2 isoforms vary in their potential to fully rescue the absence of MBD3 during lineage commitment. Full-length MBD2a only partially rescues the differentiation block; MBD2b, which lacks the N-terminal GR-rich repeat, fully rescues the differentiation block in MBD3 KO ES cells, and cells expressing the testis-specific isoform MBD2t that lacks the coiled-coil domain required for NuRD interactions are not able to generate any differentiated cells. In case of MBD2a, we further show that removing the m-CpG DNA binding capacity or the GR-rich repeat renders the protein fully redundant to MBD3, highlighting the requirements for these domains in diversifying NuRD complex function. In sum, our results highlight a partial redundancy of MBD2 and MBD3 during cellular differentiation and point to specific functions of distinct MBD2 isoforms and specific domains within the NuRD complex.


title: Genome sequencing and morphometric analysis of alpine bumblebees experiencing climate change reveal ongoing phenotypic shifts and a lack of population structure

abstract: Over the last six decades, populations of the bumblebees Bombus sylvicola and Bombus balteatus in Colorado have experienced decreases in tongue length, a trait important for plant-pollinator mutualisms. It has been hypothesized that this observation reflects selection resulting from shifts in floral composition under climate change. Here we used morphometrics and population genomics to determine whether morphological change is ongoing, investigate the genetic basis of morphological variation, and analyze population structure in these populations. We generated a genome assembly of B. balteatus. We then analyzed whole-genome sequencing data and morphometric measurements of 580 samples of both species from seven high-altitude localities. Out of 281 samples originally identified as B. sylvicola, 67 formed a separate genetic cluster comprising a newly-discovered cryptic species ("incognitus"). However, an absence of genetic structure within species suggests that gene flow is common between mountains. We found a significant decrease in tongue length between bees collected between 2012-2014 and in 2017, indicating that morphological shifts are ongoing. We did not discover any genetic associations with tongue length, but a SNP related to production of a proteolytic digestive enzyme was implicated in body size variation. We identified evidence of covariance between kinship and both tongue length and body size, which is suggestive of a genetic component of these traits, although it is possible that shared environmental effects between colonies are responsible. Our results provide evidence for ongoing modification of a morphological trait important for pollination and indicate that this trait likely has a complex genetic and environmental basis.


title: A genomic and morphometric analysis of alpine bumblebees: ongoing reductions in tongue length but no clear genetic component


title: The unphosphorylated form of the PAQosome core subunit RPAP3 binds ribosomal preassembly complexes to modulate ribosome biogenesis.

abstract: The PAQosome (Particle for Arrangement of Quaternary structure) is a twelve-subunit HSP90 co-chaperone involved in the biogenesis of several human protein complexes. Two mechanisms of client selection have previously been identified, namely the selective recruitment of specific adaptors and the differential use of homologous core subunits. Here, we describe a third client selection mechanism by showing that RPAP3, one of the core PAQosome subunits, is phosphorylated at several Ser residues in HEK293 cells. Affinity purification coupled with mass spectrometry (AP-MS) using expression of tagged RPAP3 with single phospho-null mutations at Ser116, Ser119 or Ser121 reveals binding of the unphosphorylated form to several proteins involved in ribosome biogenesis. In vitro phosphorylation assays indicate that the kinase CK2 phosphorylates these RPAP3 residues. This finding is supported by data showing that pharmacological inhibition of CK2 enhances binding of RPAP3 to ribosome preassembly factors in AP-MS experiments. Moreover, silencing of PAQosome subunits interferes with ribosomal assembly factors interactome. Altogether, these results indicate that RPAP3 phosphate group addition/removal at specific residues modulates binding to subunits of preribosomal complexes and allows speculating that PAQosome posttranslational modifications is a mechanism of client selection.


title: Dynamic plasticity within the EMT spectrum, rather than static mesenchymal traits, drives tumor heterogeneity and metastatic progression of breast cancers

abstract: The Epithelial-to-Mesenchymal Transition (EMT) is a developmental cellular program frequently coopted by cancer cells1 and is a key contributor to both heterogeneity in solid tumors2-4 and later stage chemo-resistance and metastasis5,6. Rather than being a switch from an epithelial to a mesenchymal state, increasing evidence points to the existence of intermediate EMT states, wherein cells co-express both epithelial and mesenchymal traits7-13. Multiple stable intermediate EMT states possessing unique characteristics exist across the EMT spectrum7,8,14,15, contributing to the complex heterogeneity of tumors to promote metastasis16. While much work has been carried out identifying and characterizing EMT-inducing transcription factors17-20, the transcriptional and epigenetic networks responsible for the stability and maintenance of the midpoints along the EMT spectrum are poorly defined. In addition, there are currently no approaches to identifying and quantifying intermediate EMT subpopulations within patient tumors to evaluate their prognostic significance. Using clonally isolated derivatives of the SUM149PT breast cancer cell line, we systematically interrogate how each EMT state independently contributes to heterogeneity and influences metastatic progression, uncovering the role of RUNX2 in stabilizing certain intermediate states. Using SUM149PT-derived tumors as a training set, we develop an entropy-based model to quantify phenotypic heterogeneity and EMT status. Remarkably, the majority of cell states captured in the SUM149PT model are represented in a selection of patient tumors, laying the foundation for quantification of epithelial-mesenchymal heterogeneity and understanding the role of the intermediate EMT state in tumor progression.

category: cancer biology


title: Phenotypic heterogeneity driven by plasticity of the intermediate EMT state governs disease progression and metastasis in breast cancer


title: Wheels within wheels, Nested Function-value Traits as a Tool for Modeling Ontogeny

abstract: Plant morphologies exhibit a wide array of outcomes that have evolved as a consequence adapting to a wide array of ecological pressures. These disparate morphologies have provided a rich field for comparative morphologists, developmental biologists, and geneticists to explore. Ultimately the array of variation observed in nature across different plant species is built on the same functional unit, the phytomer, which is composed of a leaf, a node, and an internode. Sequentially produced phytomers exhibit heteroblasty, that is, a gradual or abrupt change in shape, either due to size changes or changes due to reproductive phase. The progression of shape change over time is often indirectly measured by sampling several stages of plant growth and comparing allometric relationships between shape variables. However, a more precise method is to use an absolute time scale and measure shape change of sequential organs directly. In this study we use such time-dependent measurements to build a general model of organ growth for several Setaria genotypes, for both leaves and internodes. We term this the second-order function-value trait (2FVT) model, because it generalizes individual function-value trait models generated for each organ. This model reduces phenotypic noise by averaging the general trend of ontogeny and provides a quantitative tool to describe where and when phenotypic shifts occur during the ontogenies of different genotypes. The ability to recognize how ontogenetic variation is distributed within equivalent positions of the body plan at the interspecific level can be used as a tool to explore various questions related to growth and form in plants both for comparative morphology and developmental genetics.


title: Understanding C4 photosynthesis in Setaria by a proteomic and kinetic approach

abstract:
Plants performing C4 photosynthesis have a higher productivity per crop area related to an optimized use of water and nutrients. This is achieved through a series of anatomical and biochemical features that allow the concentration of CO2 around RuBisCO. In C4 plants the photosynthetic reactions are distributed between two cell types, they initially fix the carbon to C4 acids within the mesophyll cells (M) and then transport these compounds to the bundle sheath cells (BS), where they are decarboxylated so that the resulting CO2 is incorporated into the Calvin cycle (CC).

This work is focused on the comparative analysis of the proteins present in M and BS of Setaria viridis, a C4 model close relative of several major feed, fuel, and bioenergy grasses. The integration of kinetic and proteomic approaches agrees that the C4 compound malate is mainly decarboxylated in the chloroplasts of BS cells by NADP-malic enzyme (NADP-ME). Besides, NAD-malic enzyme (NAD-ME) located in the mitochondria could also contribute to the C4 carbon shuttle. We presented evidence of metabolic strategies that involve chloroplastic, mitochondrial and peroxisomal proteins to avoid the leakage of C4 intermediates in order to sustain an efficient photosynthetic performance.

HighlightProteomic and kinetic analyses show metabolic strategies involving chloroplastic, mitochondrial and peroxisomal proteins to maintain the C4 cycle performance in parallel to other metabolic pathways.


title: Implementation and acceleration of optimal control for systems biology

abstract: Optimal control theory provides insight into complex resource allocation decisions. The forward-backward sweep method (FBSM) is an iterative technique commonly implemented to solve two-point boundary value problems (TPBVPs) arising from the application of Pontryagins Maximum Principle (PMP) in optimal control. In this review we discuss the PMP approach to optimal control and the implementation of the FBSM. By conceptualising the FBSM as a fixed point iteration process, we leverage and adapt existing acceleration techniques to improve its rate of convergence. We show that convergence improvement is attainable without prohibitively costly tuning of the acceleration techniques. Further, we demonstrate that these methods can induce convergence where the underlying FBSM fails to converge. All code used in this work to implement the FBSM and acceleration techniques is available on GitHub at https://github.com/Jesse-Sharp/Sharp2021.

category: systems biology


title: Real Time Face Recognition with limited training data: Feature Transfer Learning integrating CNN and Sparse Approximation

abstract: It is highly challenging to obtain high performance with limited and unconstrained data in real time face recognition applications. Sparse Approximation is a fast and computationally efficient method for the above application as it requires no training time as compared to deep learning methods. It eliminates the training time by assuming that the test image can be approximated by the sum of individual contributions of the training images from different classes and the class with maximum contribution is closest to the test image. The efficiency of the Sparse Approximation method can be further increased by providing high quality features as input for classification. Hence, we propose to integrate pre-trained CNN architecture to extract the highly discriminative features from the image dataset for Sparse classification. The proposed approach provides better performance even for one training image per class in complex environment as compared to the existing methods. Highlight of the present approach is the results obtained for LFW dataset with one and thirteen training images per class are 84.86% and 96.14% respectively, whereas the existing deep learning methods use a large amount of training data to achieve comparable results.


title: An inconvenient association between granzyme A and Nicotinamide Nucleotide Transhydrogenase

abstract: Granzyme A (GzmA) is a serine protease secreted by cytotoxic lymphocytes, with GzmA-/- mouse studies informing our understanding of GzmAs physiological function. We show herein that GzmA-/- mice have a mixed C57BL/6J and C57BL/6N background and retain the full length Nicotinamide Nucleotide Transhydrogenase (Nnt) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in GzmA-/- mice; however, the presence of Nnt, rather than loss of GzmA, was responsible for this phenotype by constraining lymphocyte infiltration. A new CRISPR active site mutant C57BL/6J GzmAS211A mouse provided the first insights into GzmAs bioactivity free of background issues, with circulating proteolytically active GzmA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that {approx}27% of Run Accessions and {approx}38% of Bioprojects listing C57BL/6J as the mouse strain, had Nnt sequencing reads inconsistent with a C57BL/6J background. The Nnt issue has clearly complicated our understanding of GzmA and may similarly have influenced studies across a broad range of fields.


title: Function of the Vibrio PomB plug region based on the stator rotation model for bacterial flagellar motor

abstract:
Bacterial flagella are the only real rotational motor organs in the biological world. The spiral-shaped flagellar filaments that extend from the cell surface rotate like a screw to create a propulsive force. The base of the flagellar filament has a protein motor consisting of a stator and a rotor embedded in the membrane. The motor part has stators composed of two types of membrane subunits, PomA(MotA) and PomB(MotB), which are energy converters coupled to the ion flow that assemble around the rotor. Recently, structures of the stator, in which two molecules of MotB stuck in the center of the MotA ring made of five molecules, were reported and a model in which the MotA ring rotates with respect to MotB, which is coupled to the influx of ions, was proposed. We focused on the Vibrio PomB plug region, which has been reported to control the activation of flagellar motors. We searched for the plug region, which is the interacting region, through site-directed photo-cross-linking and disulfide cross-linking experiments. Our results demonstrated that it interacts with the extracellular short loop region of PomA, which is between transmembrane 3 and 4. Although the motor halted following cross-linking, its function was recovered with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion inflow by stopping the rotation of the rotor.

ImportanceThe flagellar biological motor resembles a mechanical motor, which is composed of stator and rotor and where the rotational force is transmitted by gear-like movements. We hypothesized that the flagellar the rotation of stator that the pentamer of A subunits revolves around the axis of the B subunit dimer with ion flow. The plug region of the B subunit has been shown to regulate the ion flow. Herein, we demonstrated that the ion flow was terminated by the crosslinking between the plug region and the A subunit. These finding support the rotation hypothesis and explain the role of the plug region in terminating the rotation.


title: Estimating occupancy dynamics and encounter rates with species misclassification: a semi-supervised individual-level approach

abstract:
O_LILarge-scale, long-term biodiversity monitoring is essential to meeting conservation and land management goals and identifying threats to biodiversity. However, multispecies surveys are prone to various types of observation error, including false positive/negative detection, and misclassification, where a species is encountered but its species identity is not correctly identified. Previous methods assume an imperfect classifier produces species-level classifications, but in practice, particularly with human observers, we may end up with extraspecific classifications including "unknown", morphospecies designations, and taxonomic identifications coarser than species. Disregarding these types of species misclassification in biodiversity monitoring datasets can bias estimates of ecologically important quantities such as demographic rates, occurrence, and species richness.
C_LIO_LIHere we develop an occupancy model that accounts for species non-detection and misclassification. Our framework accommodates extinction and colonization dynamics, allows for additional uncertain  morphospecies designations in the imperfect species classifications, and makes use of individual specimen with known species identities in a semi-supervised setting. We compare the performance of our joint classification-occupancy model to a reduced classification model that discards information about occupancy and encounter rate on a withheld test set. We illustrate our model with an empirical case study of the carabid beetle (Carabidae) community at the National Ecological Observatory Network Niwot Ridge Mountain Research Station, west of Boulder, CO, USA, and quantify taxonomist identification error by accounting for classification probabilities.
C_LIO_LISpecies occupancy varied through time and across sites and species. The model yielded high probabilities (30 to 92% medians) of classification where the imperfect classifier matched the true species. The classification model informed by occupancy and encounter rates outperformed the classification that was not, and these differences were most pronounced for abundant species.
C_LIO_LIOur probabilistic framework can be applied to datasets with imperfect species detection and classification. This model can identify commonly misclassified species, helping biodiversity monitoring organizations systematically prioritize which samples need validation by an expert. Our Bayesian approach propagates classification uncertainty to offer an alternative to making conservation decisions based on point estimates
C_LI


title: Phylotranscriptomics points to multiple independent origins of multicellularity and cellular differentiation in the volvocine algae

abstract: The volvocine algae, which include the single-celled species Chlamydomonas reinhardtii and the colonial species Volvox carteri, serve as a model in which to study the evolution of multicellularity and cellular differentiation. Studies reconstructing the evolutionary history of this group have often relied on datasets of one to a few genes for phylogenetic inference and ancestral character state reconstruction. These studies suggest that multicellularity evolved only once in the volvocine algae, that each of its three colonial families is monophyletic, and that there have been at least three independent origins of cellular differentiation in the group. We performed RNA-Seq on 55 strains representing 47 volvocine algal species and obtained similar data from curated databases on 13 additional strains. We compiled a dataset consisting of transcripts for 40 single-copy, protein-coding, nuclear genes, then subjected the predicted amino acid sequences of these genes to maximum likelihood, Bayesian inference, and coalescent-based analyses. These analyses show that multicellularity independently evolved at least twice in the volvocine algae and that the colonial family Goniaceae is not monophyletic. Our data further indicate that cellular differentiation independently arose at least four and possibly as many as six times within the group. Altogether, these results show how multicellularity and cellular differentiation are evolutionarily labile in the volvocine algae, affirming their importance for the study of major transitions in the history of life.


title: Lichen fungi do not depend on the alga for ATP production

abstract: Lichen fungi live in a symbiotic association with unicellular phototrophs and have no known aposymbiotic stage. A recent study postulated that some of them have lost mitochondrial oxidative phosphorylation and rely on their algal partners for ATP. This claim originated from an apparent lack of ATP9, a gene encoding one subunit of ATP synthase, from a few mitochondrial genomes. Here we show that while these fungi indeed have lost the mitochondrial ATP9, each retain a nuclear copy of this gene. Our analysis reaffirms that lichen fungi produce their own ATP.


title: Early-Emerging and Highly-Heritable Sensitivity to Human Communication in Dogs

abstract:
Dogs exhibit similarities to humans in their sensitivity to cooperative-communicative cues, but the extent to which they are biologically prepared for communication with humans is heavily debated. To investigate the developmental and genetic origins of these traits, we tested 375 eight-week-old dog puppies on a battery of social-cognitive measures. We hypothesized that if dogs social skills for cooperating with humans are biologically prepared, then these skills should emerge robustly in early development, not require extensive socialization or learning, and exhibit heritable variation. Puppies were highly skillful at using diverse human gestures and we found no evidence of learning across test trials, suggesting that they possess these skills prior to their first exposure to these cues. Critically, over 40% of the variation in dogs point-following abilities and attention to human faces was attributable to genetic factors. Our results suggest that these social skills in dogs emerge early in development and are under strong genetic control.

Highlights - Genetic factors account for nearly half of variation in dog social skills
- Puppies displayed social skills and interest in human faces from 8 weeks old
- Puppies successfully used human gestures from the very first trial


title: Anxiety-related frontocortical activity is associated with dampened stressor reactivity in the real world

abstract:
Negative affect is a fundamental dimension of human emotion. When extreme, it contributes to a variety of adverse outcomes--from physical and mental illness to divorce and premature death. Mechanistic work in animals and neuroimaging research in humans and monkeys has begun to reveal the broad contours of the neural circuits governing negative affect, but the relevance of these discoveries to everyday distress remains incompletely understood. Here we used a combination of approaches-- including neuroimaging assays of threat anticipation and emotional face perception and >10,000 momentary assessments of emotional experience--to demonstrate that individuals showing greater activation in a cingulo-opercular circuit during an anxiety-eliciting laboratory paradigm experience lower levels of stressor-dependent distress in their daily lives (n=202-208). Extended amygdala activation was not significantly related to momentary negative affect. These observations provide a framework for understanding the neurobiology of negative affect in the laboratory and in the real world.

STATEMENT OF RELEVANCEAnxiety, sadness, and other negative emotions are hallmarks of the human condition. When extreme, they contribute to a variety of adverse outcomes--from physical and mental illness to divorce and premature death--pointing to the need to develop a better understanding of the underlying brain circuitry. Recent work has begun to reveal the neural systems governing negative affect, but the relevance of these tantalizing laboratory discoveries to the real world has remained unclear. Here we used a combination of brain imaging and smartphone-based survey techniques to show that individuals marked by greater activation in a cingulo-opercular circuit during an anxiety-promoting laboratory paradigm tend to experience diminished distress in response to everyday stressors. These observations provide new insights into the brain systems most relevant to moment-by-moment fluctuations in negative mood, underscoring the importance of more recently evolved cortical association areas.


title: Characterisation of a novel ACE2-based therapeutic with enhanced rather than reduced activity against SARS-CoV2 variants

abstract: The human angiotensin-converting enzyme 2 acts as the host cell receptor for SARS-CoV-2 and the other members of the Coronaviridae family SARS-CoV-1 and HCoV-NL63. Here we report the biophysical properties of the SARS-CoV-2 spike variants D614G, B.1.1.7 and B.1.351 with affinities to the ACE2 receptor and infectivity capacity, revealing weaknesses in the developed neutralising antibody approaches. Furthermore, we report a pre-clinical characterisation package for a soluble receptor decoy engineered to be catalytically inactive and immunologically inert, with broad neutralisation capacity, that represents an attractive therapeutic alternative in light of the mutational landscape of COVID-19. This construct efficiently neutralised four SARS-CoV-2 variants of concern. The decoy also displays antibody-like biophysical properties and manufacturability, strengthening its suitability as a first-line treatment option in prophylaxis or therapeutic regimens for COVID-19 and related viral infections.


title: Identification of ACE2 mutations that modulate SARS-CoV-2 spike binding across multiple mammalian species

abstract: Understanding how SARS-CoV-2 interacts with different mammalian angiotensin-converting enzyme II (ACE2) cell entry receptors elucidates determinants of virus transmission and facilitates development of vaccines for humans and animals. Yeast display-based directed evolution identified conserved ACE2 mutations that increase spike binding across multiple species. Gln42Leu increased ACE2-spike binding for human and four of four other mammalian ACE2s; Leu79Ile had a effect for human and three of three mammalian ACE2s. These residues are highly represented, 83% for Gln42 and 56% for Leu79, among mammalian ACE2s. The above findings can be important in protecting humans and animals from existing and future SARS-CoV-2 variants.


abstract: Since SARS-CoV-2 emerged in 2019, genomic sequencing has identified mutations in the viral RNA including in the receptor-binding domain of the Spike protein. Structural characterisation of the Spike carrying point mutations aids in our understanding of how these mutations impact binding of the protein to its human receptor, ACE2, and to therapeutic antibodies. The Spike G485R mutation has been observed in multiple isolates of the virus and mutation of the adjacent residue E484 to lysine is known to contribute to antigenic escape. Here, we have crystallised the SARS-CoV-2 Spike receptor-binding domain with a G485R mutation in complex with human ACE2. The crystal structure shows that while the G485 residue does not have a direct interaction with ACE2, its mutation to arginine affects the structure of the loop made by residues 480-488 in the receptor-binding motif, disrupting the interactions of neighbouring residues with ACE2 and with potential implications for antigenic escape from vaccines, antibodies and other biologics directed against SARS-CoV-2 Spike.


title: SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway

abstract: Pathogenesis of COVID-19 is associated with a hyperinflammatory response; however, the precise mechanism of SARS-CoV-2-induced inflammation is poorly understood. Here we investigated direct inflammatory functions of major structural proteins of SARS-CoV-2. We observed that spike (S) protein potently induces inflammatory cytokines and chemokines including IL-6, IL-1{beta}, TNFa, CXCL1, CXCL2, and CCL2, but not IFNs in human and mouse macrophages. No such inflammatory response was observed in response to membrane (M), envelope (E), and neucleocapsid (N) proteins. When stimulated with extracellular S protein, human lung epithelial cells A549 also produce inflammatory cytokines and chemokines. Interestingly, epithelial cells expressing S protein intracellularly are non-inflammatory, but elicit an inflammatory response in macrophages when co-cultured. Biochemical studies revealed that S protein triggers inflammation via activation of the NF-{kappa}B pathway in a MyD88-dependent manner. Further, such an activation of the NF-{kappa}B pathway is abrogated in Tlr2-deficient macrophages. Consistently, administration of S protein induces IL-6, TNF-a, and IL-1 {beta} in wild-type, but not Tlr2-deficient mice. Together these data reveal a mechanism for the cytokine storm during SARS-CoV-2 infection and suggest that TLR2 could be a potential therapeutic target for COVID-19.


title: Longitudinal characterization of humoral and cellular immunity in hospitalized COVID-19 patients reveal immune persistence up to 9 months after infection

abstract:
BackgroundInsights into early, specific humoral and cellular responses to infection with SARS-CoV-2, as well as the persistence and magnitude of resulting immune memory is important amidst the ongoing pandemic. The combination of humoral and cellular immunity will most likely contribute to protection from reinfection or severe disease.

MethodsHere, we conducted a longitudinal study on hospitalized moderate and severe COVID-19 patients from the acute phase of disease into convalescence at five- and nine-months post symptom onset. Utilizing flow cytometry, serological assays as well as B cell and T cell FluoroSpot assays, we assessed the magnitude and specificity of humoral and cellular immune memory during and after human SARS-CoV-2 infection.

FindingsDuring acute COVID-19, we observed an increase in germinal center activity, a substantial expansion of antibodysecreting cells, and the generation of SARS-CoV-2-neutralizing antibodies. Despite gradually decreasing antibody levels, we show persistent, neutralizing antibody titers as well as robust specific memory B cell responses and polyfunctional T cell responses at five- and nine-months after symptom onset in both moderate and severe COVID-19 patients. Long-term SARS-CoV-2 specific responses were marked by preferential targeting of spike over nucleocapsid protein.

ConclusionsOur findings describe the initiation and, importantly, persistence of cellular and humoral SARS-CoV-2 specific immunological memory in hospitalized COVID-19 patients long after recovery, likely contributing towards protection against reinfection.


title: Subnuclear localisation is associated with gene activation not repression or parental origin at the imprinted Dlk1-Dio3 locus

abstract:
At interphase, de-condensed chromosomes have a non-random three-dimensional architecture within the nucleus, however, little is known about the extent to which nuclear organisation might influence expression or vice versa. Here, using imprinting as a model, we use 3D RNA- and DNA-fluorescence-in-situ-hybridisation in normal and mutant mouse embryonic stem cells to assess the relationship between imprinting control, gene expression and allelic distance from the nuclear periphery. We compared the two parentally inherited imprinted domains at the Dlk1-Dio3 domain and find a small but reproducible trend for the maternally inherited domain to be further away from the periphery if the maternally expressed gene Gtl2/Meg3 is active compared to when it is silenced. Using Zfp57KO ES cells, which harbour a paternal to maternal epigenotype switch, we observe active alleles significantly further away from the nuclear periphery with the distance from the periphery being proportional to the number of alleles active within the cell. This distribution of alleles suggests an activating effect of the nuclear interior rather than a repressive association with the nuclear periphery. Although we see a trend for the paternally inherited copy of the locus to be closer to the nuclear periphery, this appears to be linked to stochastic gene expression differences rather than parental origin. Our results suggest that transcriptional activity, rather than transcriptional repression or parental origin, defines sub-nuclear localisation at an endogenous imprinted domain.

Author summaryGenomic imprinting is an epigenetically regulated process that results in the preferential expression of a subset of developmentally regulated genes from maternally or paternally inherited chromosomes. We have used imprinted genes as a model system to investigate the relationship between the localisation of genes within the cell nucleus and their active expression while at the same time distinguishing gene repression by genomic imprinting, and gene repression by other mechanisms that act on the active allele. We find that there is a significant correlation between transcription and distance to the edge of the nucleus for the Gtl2/Meg3 gene in the imprinted Dlk1-Dio3 region. However, this correlation has a very small effect size and the nuclear envelope, which is commonly thought to act as a repressive environment for gene expression, does not appear to play a major role. We show that position effects, which have been shown for artificially lamina-targeted genes, also exist for endogenous loci and consider the possible biological relevance of the observed small effect.


title: Disruption of origin chromatin structure by helicase activation in the absence of DNA replication

abstract: Prior to initiation of DNA replication, the eukaryotic helicase, Mcm2-7, must be activated to unwind DNA at replication start sites in early S-phase. To study helicase activation within origin chromatin, we constructed a conditional mutant of the polymerase  subunit Cdc17 (or Pol1) to prevent priming and block replication. Recovery of these cells at permissive conditions resulted in the generation of unreplicated gaps at origins, likely due to helicase activation prior to replication initiation. We used micrococcal nuclease (MNase)-based chromatin occupancy profiling under restrictive conditions to study chromatin dynamics associated with helicase activation. Helicase activation in the absence of DNA replication resulted in the disruption and disorganization of chromatin which extends up to one kilobase from early, efficient replication origins. The CMG holo-helicase complex also moves the same distance out from the origin, producing single-stranded DNA that activates the intra-S-phase checkpoint. Loss of the checkpoint did not regulate the progression and stalling of the CMG complex, but rather resulted in the disruption of chromatin at both early and late origins. Finally, we found that the local sequence context regulates helicase progression in the absence of DNA replication, suggesting that the helicase is intrinsically less processive when uncoupled from replication.


title: Transcriptome analysis of the NR1H3 mouse model of multiple sclerosis reveals a pro-inflammatory phenotype with dysregulation of lipid metabolism and immune response genes

abstract:
BackgroundThe development of effective treatments for multiple sclerosis (MS), and in particular its progressive forms, is hampered by the lack of etiologically relevant cellular and animal models of human disease. Models that recapitulate the biological and pathological processes leading to the onset and progression of MS in patients are likely to afford better translational efficacy. Following the discovery of the NR1H3 p.Arg415Gln pathogenic mutation for progressive MS in two Canadian families, we developed a knock-in mouse model harboring a homologous mutation in the endogenous gene to provide a more physiologically relevant model of human MS.

MethodsGene expression was evaluated in constitutive heterozygote (which recapitulates the human disease genotype) and homozygote Nr1h3 p.Arg413Gln knock-in mice on a C57BL/6 background, and compared to wild-type littermates. AmpliSeq Transcriptome Mouse Gene Expression kits analyzed on an Ion Proton sequencer were used to generate the gene expression profiles of spleen, liver, brain and spinal cord tissue from three-month-old male and female mice. Differential expression between genotypes was assessed with DESeq2, and Gene Ontologies pathways enrichment analysis performed with DAVID v6.8. Benjamini-Hochberg false discovery rate (FDR) correction for multiple testing was applied.

ResultsTranscriptome analysis of spleen tissue from Nr1h3 p.Arg413Gln mice revealed 23 significantly dysregulated genes (FDR<0.05) with greater than a two-fold change in expression. These include CD5 antigen-like (Cd5l), complement component 6 (C6), procollagen C-endopeptidase enhancer 2 (Pcolce2), interleukin 22 receptor, alpha 2 (Il22ra2), and T cell immunoglobulin and mucin domain containing 4 (Timd4). Gene Ontology enrichment analysis support upregulation of cell cycle pathways and downregulation of immune system response in splenic cells. The liver transcriptome identified 27 significantly dysregulated genes with greater than a two-fold change in expression compared to wild-type littermates. Cd5l, Timd4, C-C motif chemokine receptor 3 (Ccr3), ADAM metallopeptidase domain 11 (Adam11) and macrophage expressed 1 (Mpeg1) were amongst those most significantly dysregulated. Enrichment analysis supported altered immune function with upregulation of sterol and steroid metabolic processes and downregulation of fatty acid biosynthesis and inflammatory and immune system responses. Although brain and spinal cord transcriptome profiles identified several genes significantly dysregulated in Nr1h3 mice compared to wild-type littermates (FDR<0.05), none presented greater than two-fold changes in gene expression.

DiscussionThe analysis of the Nr1h3 p.Arg413Gln mouse model of MS suggests that the predominance of a pro-inflammatory over a healing or reparative phenotype, combined with deficiencies in myelination and remyelination, are the biological mechanisms implicated in the onset of MS and the development of a more severe progressive disease course observed in patients with NR1H3 mutations. Association of NR1H3 common variants with MS risk indicates that the disruption of these biological and immunological processes is not only informative for familial forms of disease but MS patients at large. Differences in transcriptome profiles underline the value of this model for the development and validation of novel therapeutic strategies and ultimately treatments with the potential to delay or even halt the onset of progressive MS and to ameliorate the severity of clinical symptoms.


title: Mitochondrial fission is required for proper nucleoid distribution within mitochondrial networks

abstract:
Mitochondrial DNA (mtDNA) maintenance is essential to sustain a functionally healthy population of mitochondria within cells. Proper mtDNA replication and distribution within mitochondrial networks are essential to maintain mitochondrial homeostasis. However, the fundamental basis of mtDNA segregation and distribution within mitochondrial networks is still unclear. To address these questions, we developed an algorithm, Mitomate tracker to unravel the global distribution of nucleoids within mitochondria. Using this tool, we decipher the semi-regular spacing of nucleoids across mitochondrial networks. Furthermore, we show that mitochondrial fission actively regulates mtDNA distribution by controlling the distribution of nucleoids within mitochondrial networks. Specifically, we found that primary cells bearing disease-associated mutations in the fission proteins DRP1 and MYH14 show altered nucleoid distribution, and acute enrichment of enlarged nucleoids near the nucleus. Further analysis suggests that the altered nucleoid distribution observed in the fission mutants is the result of both changes in network structure and nucleoid density. Thus, our study provides novel insights into the role of mitochondria fission in nucleoid distribution and the understanding of diseases caused by fission defects.

Significance statementMutation or deletion of mitochondrial DNA (mtDNA) is associated with a large number of human diseases. However, the mechanisms controlling mtDNA replication and segregation are still poorly understood. Here, we have developed a new computational method to quantify the distribution of nucleoids (mtDNA with associated proteins) and define how nucleoid distribution is affected by changes in mitochondrial network structure. We demonstrate that mitochondrial fission is required for the proper distribution of nucleoids across mitochondrial networks, cells from patients with fission defects showing irregular nucleoid distribution and perinuclear accumulation. Nonetheless, each fission mutant behaved in a distinct manner, indicating a complex relationship between mitochondrial dynamics and nucleoid distribution.


title: iPSC-derived pancreatic progenitors are an optimal model system to study T2D regulatory variants active during fetal development of the pancreas

abstract: Pancreatic progenitor cells (PPCs), which are multipotent cells that have the potential to give rise to endocrine, exocrine and epithelial cells, provide a powerful model system to examine the molecular characteristics of differentiating, fetal-like pancreas cells and to study the genetics of pancreatic disease. We differentiated ten induced pluripotent stem cell-derived pancreatic progenitor cell (iPSC-PPC) lines and, using single cell RNA-seq and single nuclear ATAC-seq, we found that they show varying levels of cellular maturity, including the presence of early PPC, endocrine and exocrine cells. We evaluated iPSC-PPC as a model system to study type 2 diabetes (T2D) and found that, of 380 T2D risk loci, 208 overlapped regulatory elements active in iPSC-PPC, including 55 fetal-specific. These loci are associated with transcription factor binding site alterations and allele-specific expression, suggesting that iPSC-PPC are an optimal model system to annotate GWAS variants that are not functional in the adult pancreas.


title: 4polar-STORM polarized super-resolution imaging of actin filament organization in cells

abstract: Advances in single-molecule localization microscopy are providing unprecedented insights into the nanometer-scale organization of protein assemblies in cells and thus a powerful means for interrogating biological function. However, localization imaging alone does not contain information on protein conformation and orientation, which constitute additional key signatures of protein function. Here, we present a new microscopy method which combines for the first time Stochastic Optical Reconstruction Microscopy (STORM) super-resolution imaging with single molecule orientation and wobbling measurements using a four polarization-resolved image splitting scheme. This new method, called 4polar-STORM, allows us to determine both single molecule localization and orientation in 2D and to infer their 3D orientation, and is compatible with high labelling densities and thus ideally placed for the determination of the organization of dense protein assemblies in cells. We demonstrate the potential of this new method by studying the nanometer-scale organization of dense actin filament assemblies driving cell adhesion and motility, and reveal bimodal distributions of actin filament orientations in the lamellipodium, which were previously only observed in electron microscopy studies. 4polar-STORM is fully compatible with 3D localization schemes and amenable to live-cell observations, and thus promises to provide new functional readouts by enabling nanometer-scale studies of orientational dynamics in a cellular context.


title: Co-condensation of proteins with single- and double-stranded DNA

abstract: Biomolecular condensates provide distinct compartments that can localize and organize biochemistry inside cells. Recent evidence suggests that condensate formation is prevalent in the cell nucleus. To understand how different components of the nucleus interact during condensate formation is an important challenge. In particular, the physics of co-condensation of proteins together with nucleic acids remains elusive. Here, we use optical tweezers to study how the prototypical prion-like protein Fused-in-Sarcoma (FUS) forms liquid-like assemblies in vitro, by co-condensing together with individual DNA molecules. Through progressive DNA unpeeling, buffer exchange and force measurements, we show that FUS adsorbing in a single layer on DNA effectively generates a sticky FUS-DNA polymer that can collapse to form a liquid-like FUS-DNA co-condensate. Condensation occurs at constant DNA tension for double-stranded DNA, which is a signature of phase separation. We suggest that co-condensation mediated by protein adsorption on nucleic acids is an important mechanism for intracellular compartmentalization.


title: Membrane Hydrophobicity Determines the Activation Free Energy of Passive Lipid Transport

abstract:
The collective behavior of lipids with diverse chemical and physical features determines a membranes thermodynamic properties. Yet, the influence of lipid physicochemical properties on lipid dynamics, in particular interbilayer transport, remains underexplored. Here, we systematically investigate how the activation free energy of passive lipid transport depends on lipid chemistry and membrane phase. Through all-atom molecular dynamics simulations of 11 chemically distinct glycerophos-pholipids, we determine how lipid acyl chain length, unsaturation, and headgroup influence the free energy barriers for two elementary steps of lipid transport, lipid desorption, which is rate-limiting, and lipid insertion into a membrane. Consistent with previous experimental measurements, we find that lipids with longer, saturated acyl chains have increased activation free energies compared to lipids with shorter, unsaturated chains. Lipids with different headgroups exhibit a range of activation free energies; however, no clear trend based solely on chemical structure can be identified, mirroring difficulties in the interpretation of previous experimental results. Compared to liquid-crystalline phase membranes, gel phase membranes exhibit substantially increased free energy barriers. Overall, we find that the activation free energy depends on a lipids local hydrophobic environment in a membrane and that the free energy barrier for lipid insertion depends on a membranes interfacial hydrophobicity. Both of these properties can be altered through changes in lipid acyl chain length, lipid headgroup, and membrane phase. Thus, the rate of lipid transport can be tuned through subtle changes in local membrane composition and order, suggesting an unappreciated role for nanoscale membrane domains in regulating cellular lipid dynamics.

SIGNIFICANCECell homeostasis requires spatiotemporal regulation of heterogeneous membrane compositions, in part, through non-vesicular transport of individual lipids between membranes. By systematically investigating how the chemical diversity present in glycerophospholipidomes and variations in membrane order influence the free energy barriers for passive lipid transport, we discover a correlation between the activation free energy and membrane hydrophobicity. By demonstrating how membrane hydrophobicity is modulated by local changes in membrane composition and order, we solidify the link between membrane physicochemical properties and lipid transport rates. Our results suggest that variations in cell membrane hydrophobicity may be exploited to direct non-vesicular lipid traffic.


title: Programmable pattern formation in cellular systems with local signaling

abstract: Diverse complex systems, ranging from developing embryos to systems of locally communicating agents, display an apparent capability of "programmable" pattern formation: They reproducibly form a target pattern, but this target can be readily changed. A distinguishing feature of such systems, as compared to simpler physical pattern forming systems, is that their subunits are capable of information processing. Here, we explore schemes for programmable pattern formation within a theoretical framework, in which subunits process discrete local signals to update their internal state according to logical rules. We study systems with different update rules, different topologies, and different control schemes, to assess their ability to perform programmable pattern formation and their susceptibility to errors. Only a small subset of systems permits local organizer cells to dictate any target pattern. These systems follow a common principle, whereby a temporal pattern is transcribed into a spatial pattern, reminiscent of the clock-and-wavefront mechanism underlying vertebrate somitogenesis. An alternative scheme employing several different rules can only form a fraction of patterns but is robust with respect to the timing of organizer cell inputs. Our results establish a basis for the design of synthetic systems, and for more detailed models of programmable pattern formation closer to real systems.

category: synthetic biology


title: CRISPR-Targeted CAR Gene Insertion Using Cas9/RNP and AAV6 Enhances Anti-AML Activity of Primary NK Cells

abstract: Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein (RNP) complexes. Here we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of homology arms for homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. For proof-of-concept, we generated mCherry-expressing primary NK cells and determined that sc vectors with 300bp homology arms had optimal transduction efficiency. Then, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3{zeta} and NKG2D/2B4+CD3{zeta}) and expanded them on CSTX002 feeder cells. Expansion kinetics were unaltered and the expanded NK cells maintained high CAR expression (mean 68% CAR+). The CD33-CAR-NK cells showed increased activation markers and enhanced antileukemic activity with improved killing kinetics against CD33-positive acute myeloid leukemia (AML) cell lines and primary samples. Using targeted sequencing we demonstrated the accuracy of CAR gene insertion in human primary NK cells genome. Site-directed insertion using RNP and scAAV6 is an efficient method for stable genetic transfer into primary NK cells that has broad potential for fundamental discovery and therapeutic applications.


title: A squalene-hopene cyclase in Schizosaccharomyces japonicus represents a eukaryotic adaptation to sterol-independent anaerobic growth

abstract:
Biosynthesis of sterols, which are key constituents of canonical eukaryotic membranes, requires molecular oxygen. Anaerobic protists and deep-branching anaerobic fungi are the only eukaryotes in which a mechanism for sterol-independent growth has been elucidated. In these organisms, tetrahymanol, formed through oxygen-independent cyclization of squalene by a squalene-tetrahymanol cyclase, acts as a sterol surrogate. This study confirms an early report (Bulder (1971), Antonie Van Leeuwenhoek, 37, 353-358) that Schizosaccharomyces japonicus is exceptional among yeasts in growing anaerobically on synthetic media lacking sterols and unsaturated fatty acids. Mass spectrometry of lipid fractions of anaerobically grown Sch. japonicus showed the presence of hopanoids, a class of cyclic triterpenoids not previously detected in yeasts, including hop-22(29)-ene, hop-17(21)-ene, hop-21(22)-ene and hopan-22-ol. A putative gene in Sch. japonicus showed high similarity to bacterial squalene-hopene cyclase (SHC) genes and in particular to those of Acetobacter species. No orthologs of the putative Sch. japonicus SHC were found in other yeast species. Expression of the Sch. japonicus SHC gene (Sjshc1) in Saccharomyces cerevisiae enabled hopanoid synthesis and supported ergosterol-independent anaerobic growth, thus confirming that one or more of the hopanoids produced by SjShc1 can act as ergosterol surrogate in anaerobic yeast cultures. Use of hopanoids as sterol surrogates represents a previously unknown adaptation of eukaryotic cells to anaerobic growth. The fast sterol-independent anaerobic growth of Sch. japonicus is an interesting trait for developing robust fungal cell factories for application in anaerobic industrial processes.

Significance statementBiosynthesis of sterols requires oxygen. This study identifies a previously unknown evolutionary adaptation in a eukaryote, which enables anaerobic growth in absence of exogenous sterols. A squalene-hopene cyclase, proposed to have been acquired by horizontal gene transfer from an acetic acid bacterium, is implicated in a unique ability of the yeast Schizosaccharomyces japonicus to synthesize hopanoids and grow in anaerobic, sterol-free media. Expression of this cyclase in S. cerevisiae confirmed that at least one of its hopanoid products acts as sterol-surrogate. The involvement of hopanoids in sterol-independent growth of this yeast provides new leads for research into the structure and function of eukaryotic membranes, and into the development of sterol-independent yeast cell factories for application in anaerobic processes.


title: Synthesis of the NarP response regulator of nitrate respiration in Escherichia coli is regulated at multiple levels by Hfq and small RNAs

abstract:
Two-component systems (TCS) and small RNAs (sRNA) are widespread regulators that participate in the response and the adaptation of bacteria to their environments. They mostly act at the transcriptional and post-transcriptional levels, respectively, and can be found integrated in regulatory circuits, where TCSs control sRNAs transcription and/or sRNAs post-transcriptionally regulate TCSs synthesis.

In response to nitrate and nitrite, the paralogous NarQ-NarP and NarX-NarL TCSs regulate the expression of genes involved in anaerobic respiration of these alternative electron acceptors. In addition to the previously reported repression of NarP synthesis by the SdsN137 sRNA, we show here that RprA, another Hfq-dependent sRNA, also negatively controls narP. Interestingly, the repression of narP by RprA actually relies on two independent controls. The first is via the direct pairing of the central region of RprA to the narP translation initiation region and presumably occurs at the translation initiation level. In contrast, the second control requires only the very 5 end of the narP mRNA, which is targeted, most likely indirectly, by the full-length or the shorter, processed, form of RprA. In addition, our results raise the possibility of a direct role of Hfq in narP control, further illustrating the diversity of post-transcriptional regulation mechanisms in the synthesis of TCSs.


title: Epigallocatechin Gallate from Green Tea Effectively Blocks Infection of SARS-CoV-2 and New Variants by Inhibiting Spike Binding to ACE2 Receptor

abstract: As the COVID-19 pandemic rages on, the new SARS-CoV-2 variants have emerged in the different regions of the world. These newly emerged variants have mutations in their spike (S) protein that may confer resistance to vaccine-elicited immunity and existing neutralizing antibody therapeutics. Therefore, there is still an urgent need of safe, effective, and affordable agents for prevention/treatment of SARS-CoV-2 and its variant infection. Here, we demonstrated that green tea beverage (GTB) or its major ingredient, epigallocatechin gallate (EGCG), were highly effective in inhibiting infection of live SARS-CoV-2 and human coronavirus (HCoV OC43). In addition, infection of the pseudoviruses with spikes of the new variants (UK-B.1.1.7, SA-B.1.351, and CA-B.1.429) was efficiently blocked by GTB or EGCG. Among the 4 active green tea catechins at noncytotoxic doses, EGCG was the most potent in the action against the viruses. The highest inhibitory activity was observed when the viruses or the cells were pre-incubated with EGCG prior to the infection. Mechanistic studies revealed that EGCG blocked infection at the entry step through interfering with the engagement of the receptor binding domain (RBD) of the viral spikes to angiotensin-converting enzyme 2 (ACE2) receptor of the host cells. These data support further clinical evaluation and development of EGCG as a novel, safe, and cost-effective natural product for prevention/treatment of SARS-CoV-2 transmission and infection.