title: Gene Expression in Lung Epithelial Cells Following Interaction with Pneumocystis carinii and its Specific Life Forms Yields Insights into Host Gene Responses to Infection

abstract: Pneumocystis spp. interacts with epithelial cells in the alveolar spaces of the lung. It is thought that the binding of Pneumocystis to host cell epithelium is needed for life cycle completion and proliferation. The effect of this interaction on lung epithelial cells have previously shown that the trophic form of this organism greatly inhibits p34cdc2 activity, a serine/threonine kinase required for transition from G2 to M phase in the cell cycle. To gain further insight into the host response during Pneumocystis pneumonia (PCP), we used microarray technology to profile epithelial cell (A549) gene expression patterns following Pneumocystis carinii interaction. Furthermore, we isolated separate populations of cyst and trophic forms of P. carinii, which were then applied to the lung epithelial cells. Differential expression of genes involved in various cellular functions dependent on the specific P. carinii life form in contact with the A549 cell were identified. The reliability of our data was further confirmed by Northern blot analysis on a number of selected up or down regulated transcripts. The transcriptional response to P. carinii was dominated by cytokines, apoptotic, and anti-apoptotic related genes. These results reveal several previously unknown effects of P. carinii on the lung epithelial cell and provide insight into the complex interactions of host and pathogen.

category: microbiology


title: Dairy products as sources of methanogens for humans

abstract: Methanogens are detected in human gut from the first moments of life and there is a diversification of methanogens during infancy. However, the sources of acquisition of methanogens are not well elucidated. We therefore investigated 56 dairy products as potential sources of methanogens by applying molecular biology. In the presence of negative controls, we obtained an overall prevalence of methanogens in 85.7% (48/56) of samples by real-time PCR. Further PCR-sequencing identified 73.2% (41/56) of Methanobrevibacter smithii. We also found for the first time in dairy products 1.8% (1/56) of Methanobrevibacter oralis, 7.1% (4/56) of Methanobrevibacter millerae, 1.8% (1/56) of Methanobrevibacter ruminantium, 1.8% (1/56) of Methanocorpusculum sp. We observed a significant presence (p-value=0.001) of methanogens in fermented dairy products compared to unfermented dairy products. This study gives credit to the fact that dairy products could be considered as a source of methanogens for humans, especially for children.


title: Crosstalk between guanosine nucleotides regulates cellular heterogeneity in protein synthesis during nutrient limitation

abstract:
Phenotypic heterogeneity of microbial populations can facilitate survival in dynamic environments by generating sub-populations of cells that may have differential fitness in a future environment. Bacillus subtilis cultures experiencing nutrient limitation contain distinct sub-populations of cells exhibiting either comparatively high or low protein synthesis activity. This heterogeneity requires the production of phosphorylated guanosine nucleotides (pp)ppGpp by three synthases: SasA, SasB, and RelA. Here we show that these enzymes differentially affect this bimodality: RelA and SasB are necessary to generate the sub-population of cells exhibiting low protein synthesis whereas SasA is necessary to generate cells exhibiting comparatively higher protein synthesis. The RelA product (pppGpp) allosterically activates SasB and we find, in contrast, that the SasA product (pGpp) competitively inhibits this activation. Finally, we provide in vivo evidence that this antagonistic interaction mediates the observed heterogeneity in protein synthesis. This work therefore identifies the mechanism underlying phenotypic heterogeneity in the central physiological process of protein synthesis.

Author SummaryUpon encountering conditions that are unfavorable to growth, such as nutrient limitation, bacteria enter into a quiescent phenotype that is mediated by group of guanosine nucleotides collectively known as (pp)pGpp. These nucleotides direct the down-regulation of energy intensive processes and are essential for a striking heterogeneity in protein synthesis observed during exit from rapid growth. Here, we show that a network of (pp)pGpp synthases is responsible for this heterogeneity and describe a mechanism that allows for the integration of multiple signals into the decision to down regulate the most energy intensive process in a cell.


title: The Bacillus phage SPβ and its relatives: A temperate phage model system reveals new strains, species, prophage integration loci, conserved proteins and lysogeny management components

abstract: The Bacillus phage SP{beta} has been known for about 50 years, but only a few strains are avalible. We isolated four new wild type strains of the SPbeta species. Phage vB_BsuS-Goe14 introduces its prophage into the spoVK locus, previously not observed to be used by SP{beta}-like phages. We could also reveal the SP{beta}-like phage genome replication strategy, the genome packaging mode, and the phage genome opening point. We extracted 55 SP{beta}-like prophages from public Bacillus genomes, thereby discovering three more integration loci and one additional type of integrase. The identified prophages resembled four new species clusters and three species orphans in the genus Spbetavirus. The determined core proteome of all SP{beta}-like prophages consists of 38 proteins. The integration cassette proved to be not conserved even though present in all strains. It consists of distinct integrases. Analysis of SP{beta} transcriptomes revealed three conserved genes, yopQ, yopR, and yokI, to be transcribed from a dormant prophage. While yopQ and yokI could be deleted from the prophage without activating the prophage, damaging of yopR led to a clear-plaque phenotype. Under the applied laboratory conditions, the yokI mutant showed an elevated virion release implying the YokI protein being a component of the arbitrium system.


title: A portable and high-integrated 3D microfluidic chip for bacterial quantification and antibiotic susceptibility testing

abstract: On-site single-cell antibiotic susceptibility testing (sc-AST) provides unprecedented technical potential to improve the treatment of bacterial infections and study heterogeneous resistance to antibiotics. Herein, we developed a portable and high-integrated 3D polydimethylsiloxane (PDMS) chip to perform fast and on-site bacteria quantification and sc-AST. The 3D arrangement of the chambers significantly improved the integration of reaction units ([~]10000/cm2) and widened the dynamic range to 5 orders of magnitude. A capillary valve-based flow distributor was adopted for flow equidistribution in 64 parallel channels and uniform sample loading in as short as 2 s. The degassed PDMS enabled this device to independently dispense the sample into 3D chamber array with almost 100% efficiency. The quantification of Escherichia coli (E. coli) strains with various activity was accomplished in 0.5-2 h, shortened by 20 h in comparison to the traditional plate counting. The functionality of our platform was demonstrated with several effective antibiotics by determining minimum inhibitory concentrations at single-cell level. Furthermore, we utilized the lyophilization of test reagents and needle-mediated reagents rehydration to realize one-step on-site sc-AST. The results indicate that the proposed sc-AST platform is portable, highly sensitive, fast, accurate and user-friendly, thus it has the potential to facilitate precise therapy in time and monitor the treatment. Meanwhile, it could serve as an approach for investigating the mechanisms of heteroresistance at single-cell resolution.


title: Simple and efficient differentiation of human iPSCs into contractible skeletal muscles for muscular disease modeling

abstract:
Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patients pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) can potentially recapitulate disease pathology more accurately than existing disease models when differentiated into affected cell types. Thus, successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection procedures, and clonal variations must be overcome. Moreover, their functionality to exhibit muscular contraction has never been reported. Here, we demonstrated that bulk MYOD1- hiPSCs established with puromycin selection, but not with G418 selection, showed high differentiation efficiency, generating more than 80% Myogenin (MyoG)+ and Myosin heavy chain (MHC)+ muscle cells within seven days. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties compared with those of clonally established MYOD1- hiPSCs, suggesting that the bulk method may minimize the effects of clonal variations. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical pulse stimulation, indicating their functionality. Together, the findings indicate that our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and facilitates the generation of muscular disease models.

Graphical Abstract

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category: cell biology


title: Re-entry into mitosis and regeneration of intestinal stem cells through enteroblast dedifferentiation in Drosophila midguts

abstract: Many adult tissues and organs including the intestine rely on resident stem cells to maintain homeostasis. In mammalian intestines, upon ablation of resident stem cells, the progenies of intestinal stem cells (ISCs) such as secretory cells and tuft cells can dedifferentiate to generate ISCs to drive epithelial regeneration, but whether and how the ISC progenies dedifferentiate to generate ISCs under physiological conditions remains unknown. Here we show that infection of pathogenic bacteria induces enteroblasts (EBs) as one type of ISC progenies to re-enter the mitotic cycle in the Drosophila intestine. The re-entry into mitosis is dependent on epithermal growth factor receptor (EGFR)-Ras signaling and ectopic activation of EGFR-Ras signaling in EBs is sufficient to drive EBs cell-autonomously to re-enter into mitosis. In addition, we examined whether EBs gain ISC identity as a prerequisite to divide, but the immunostaining with stem cell marker Delta shows that these dividing EBs do not gain ISC identity. After employing lineage tracing experiments, we further demonstrate that EBs dedifferentiate to generate functional ISCs after symmetric divisions of EBs. Together, our study in Drosophila intestines uncovers a new role of EGFR-Ras signaling in regulating re-entry into mitosis and dedifferentiation during regeneration and reveals a novel mechanism by which ISC progenies undergo dedifferentiation through a mitotic division, which has important implication to mammalian tissue homeostasis and tumorigenesis.


title: ACE2 expression and localization are regulated by CFTR: implications beyond cystic fibrosis

abstract:
As an inherited disorder characterized by severe pulmonary disease, cystic fibrosis (CF) could be considered a comorbidity for coronavirus disease 2019 (COVID-19)1. Instead, CF seems to constitute an advantage in COVID-19 infection2-5.

To clarify whether host factors expressed by the CF epithelia may influence COVID-19 progression, we investigated the expression of SARS-CoV-2 receptor and coreceptors in primary airway epithelial cells. We found that angiotensin converting enzyme 2 (ACE2) expression and localization are regulated by cystic fibrosis transmembrane conductance regulator (CFTR) channels. Consistently, our results indicate that dysfunctional CFTR channels alter susceptibility to SARS-CoV-2 infection, resulting in reduced viral infection in CF cells. Depending on the pattern of ACE2 expression, the SARS-CoV-2 spike (S) protein induced high levels of Interleukin (IL)-6 in healthy donor-derived primary airway epithelial cells but a very weak response in primary CF cells. Collectively, these data support the hypothesis that CF condition is unfavorable for SARS-CoV-2 infection.


title: Investigation of Human Endogenous Retrovirus-K (ERVK) Expression and Function in Normal Placentation and Preterm Pregnancy

abstract:
BackgroundThere is a growing body of evidence indicating the importance of endogenous retrovirus (ERV) derived proteins during early development and reproduction in mammals. Recently, a protein derived from the youngest ERV in humans, ERVK (HML2), was shown to be expressed during human placentation. Since a number of highly similar ERVK proviral loci exist across the human genome, locus-specific analysis of ERVK transcription and identification of the coding sequence expressed in the human placenta is difficult. Thus, despite its activity in early human development, the native expression and function of ERVK in the human placenta remains largely uncharacterized.

ResultsIn this study, we comprehensively examined locus-specific ERVK transcription across several human placental tissues and cell types. Through a combination of RNA-seq and siRNA knock-down analyses, we identified the expression of a single ERVK locus, ERVK11q23.3, as (1) being significantly upregulated in preterm compared to term placenta, (2) predominantly expressed by mononuclear trophoblasts, (3) capable of encoding a truncated viral-like envelope protein, and (4) contributing to the expression cytokines involved in both antiviral and anti-inflammatory innate immune responses in human placental trophoblasts and BeWo choriocarcinoma cells, respectively.

ConclusionsCollectively, the results of this study highlight the utility of studying locus-specific ERVK expression, provide a thorough characterization of locus-specific ERVK transcription from human placental tissues, and indicate that altered expression of placental ERVK11q23.3 influences interferon antiviral response, which may contribute to preterm birth and other pregnancy complications.

category: developmental biology


title: Two decades of suspect evidence for adaptive molecular evolution - Negative selection confounding positive selection signals

abstract: There is a large literature in the last two decades affirming adaptive DNA sequences evolution between species. The main lines of evidence are from i) the McDonald-Kreitman (MK) test, which compares divergence and polymorphism data, and ii) the PAML test, which analyzes multi-species divergence data. Here, we apply these two tests concurrently on the genomic data of Drosophila and Arabidopsis. To our surprise, the >100 genes identified by the two tests do not overlap beyond random expectation. Because the non-concordance could be due to low powers leading to high false-negatives, we merge every 20 - 30 genes into a "supergene". At the supergene level, the power of detection is large but the calls still do not overlap. We rule out methodological reasons for the non-concordance. In particular, extensive simulations fail to find scenarios whereby positive selection can only be detected by either MK or PAML, but not both. Since molecular evolution is governed by positive and negative selection concurrently, a fundamental assumption for estimating one (say, positive selection) is that the other is constant. However, in a broad survey of primates, birds, Drosophila and Arabidopsis, we found that negative selection rarely stays constant for long in evolution. As a consequence, the variation in negative selection is often mis-construed as signals of positive selection. In conclusion, MK, PAML or any method that examines genomic sequence evolution has to explicitly address the variation in negative selection before estimating positive selection. In a companion study, we propose a possible path forward in two stages - first, by mapping out the changes in negative selection and then using this map to estimate positive selection. For now, the large literature on positive selection between species has to await the re-assessment.

category: evolutionary biology


title: SARS-CoV-2 papain-like protease PLpro in complex with natural compounds reveal allosteric sites for antiviral drug design

abstract: SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in evading the hosts innate immune responses. We established a high-throughput X-ray screening to identify inhibitors by elucidating the native PLpro structure refined to 1.42 [A] and performing co-crystallization utilizing a diverse library of selected natural compounds. We identified three phenolic compounds as potential inhibitors. Crystal structures of PLpro inhibitor complexes, obtained to resolutions between 1.7-1.9 [A], show that all three compounds bind at the ISG15/Ub-S2 allosteric binding site, preventing the essential ISG15-PLpro molecular interactions. All compounds demonstrate clear inhibition in a deISGylation assay, two exhibit distinct antiviral activity and one inhibited a cytopathic effect in a non-cytotoxic concentration range. These results highlight the druggability of the rarely explored ISG15/Ub-S2 PLpro allosteric binding site to identify new and effective antiviral compounds. Importantly, in the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

category: biochemistry


title: Theoretical Guarantees for Phylogeny Inference from Single-Cell Lineage Tracing

abstract: CRISPR-Cas9 lineage tracing technologies have emerged as a powerful tool for investigating development in single-cell contexts, but exact reconstruction of the underlying clonal relationships in experiment is plagued by data-related complications. These complications are functions of the experimental parameters in these systems, such as the Cas9 cutting rate, the diversity of indel outcomes, and the rate of missing data. In this paper, we develop two theoretically grounded algorithms for reconstruction of the underlying phylogenetic tree, as well as asymptotic bounds for the number of recording sites necessary for exact recapitulation of the ground truth phylogeny at high probability. In doing so, we explore the relationship between the problem difficulty and the experimental parameters, with implications for experimental design. Lastly, we provide simulations validating these bounds and showing the empirical performance of these algorithms. Overall, this work provides a first theoretical analysis of phylogenetic reconstruction in the CRISPR-Cas9 lineage tracing technology.

category: bioinformatics


title: Fine-grained, nonlinear image registration of live cell movies reveals spatiotemporal organization of diffuse molecular processes

abstract:
We present an application of non-linear Image registration that allows spatiotemporal analysis of extremely noisy and diffuse molecular processes across the entire cell. To produce meaningful local tracking of the spatially coherent portion of diffuse protein dynamics, we improved upon existing non-linear image registration to compensate for cell movement and deformation. The registration relies on a subcellular fiducial marker, a cell motion mask, and a topological regularization that enforces diffeomorphism on the registration without significant loss of granularity. We demonstrate the potential of this approach in conjunction with stochastic time-series analysis through the discovery of distinct zones of coherent Profillin dynamics in symmetry-breaking U2OS cells. Further analysis of the resulting Profilin dynamics revealed strong relationships with the underlying actin organization. This study thus provides a framework for extracting functional interactions between cell morphodynamics, protein distributions, and signaling in cells undergoing continuous shape changes.

Author SummaryBy adapting optical flow based nonlinear image registration we created a method specific for live cell movies that preserves the dynamics of a signal of interest by remapping using a separate measurable subcellular location fiducial. This is an extension as well on our labs previously published method of cell edge-based referencing of subcellular locations that was incapable of extracting interpretable subcellular time series more than a few microns away from the cell edge. We showed that our method overcomes this key limitation and allows sampling of subcellular time series from every subcellular location through our discovery of organized profilin dynamics in moving cell and that these profilin dynamics are related to actin dynamics due to their ability to bind growing actin structures likely through actin polymerizing factors. Most importantly, our method is applicable to discovering subcellular organization and coordination in a widely used form of live cell microscopy data that hitherto has been largely limited to anecdotal analysis.


title: Ions and lipids drive aggregation of surface-functionalized gold nanoparticles on lipid membranes

abstract: The control of the aggregation of biomedical nanoparticles (NP) in physiological conditions is crucial as clustering may change completely the way they interact with the biological environment. Here we show that Au nanoparticles, functionalized by an anionic, amphiphilic shell, spontaneously aggregate in fluid zwitterionic lipid bilayers. We use Molecular Dynamics and enhanced sampling techniques to disentangle the short-range and long-range driving forces of aggregation. At short inter-particle distances, ion-mediated, charge-charge interactions (ion bridging) stabilize the formation of large NP aggregates, as confirmed by cryo-electron microscopy. Lipid depletion and membrane curvature are the main membrane deformations driving long-range NP-NP attraction. Ion bridging, lipid depletion, and membrane curvature stem from the configurational flexibility of the nanoparticle shell. Our simulations show, more in general, that the aggregation of same-charge membrane inclusions can be expected as a result of intrinsically nanoscale effects taking place at the NP-NP and NP-bilayer soft interfaces.

category: biophysics


title: New fossil wasp species from the earliest Eocene Fur Formation has its closest relatives in late Eocene ambers (Hymenoptera, Ichneumonidae, Pherhombinae)

abstract: Darwin wasps (Ichneumonidae) are one of the most species-rich insect families, but also one of the most understudied ones, both in terms of their extant and extinct diversity. We here use morphometrics of wing veins and an integrative Bayesian analysis to place a new rock fossil species from the Danish Fur Formation ([~]54 Ma) in the tree of Darwin wasps. The new species, Pherhombus parvulus n. sp., is placed firmly in Pherhombinae, an extinct subfamily so far only known from Baltic and Rovno-Ukranian ambers, which are estimated to be 34-48 Ma and 34-38 Ma, respectively. Our phylogenetic analysis recovers a subfamily clade within the higher Ophioniformes formed by Pherhombinae, Townesitinae and Hybrizontinae, in accordance with previous suggestions. Due to the placement of the new species as sister to the remaining members of Pherhombinae, we argue that our finding is not at odds with a much younger, late Eocene age ([~]34-41 Ma) of Baltic amber and instead demonstrates that Pherhombus existed over a much longer period than previously thought. Our results also exemplify the power of wing vein morphometrics and integrative phylogenetic analyses in resolving the placement even of poorly preserved fossil specimens.

category: paleontology


title: Characterization of Reactive Oxygen Species Signaling changes in a cell culture model of skeletal muscle ageing, and its application to screening pharmacokinetically-relevant exposures of dietary polyphenols for bioactivity.

abstract: Age-related frailty is a significant health and social care burden, however treatment options are limited. There is currently a lack of suitable cell culture model for screening large numbers of test compounds to identifying those which can potentially promote healthy skeletal muscle function. This paper describes the characterization of reactive oxygen and nitrogen species (RONS) signalling changes in young and aged myoblasts and myotubes using the C2C12 cell line, and the application of aged myoblast and myotube cultures to assess the effect of dietary polyphenols on RONS signalling. Aged myoblasts and myotubes were observed to have significantly increased reactive oxygen species levels (p<0.01 and p<0.001 respectively), increases in nitric oxide levels (p<0.05 for myoblasts and myotubes), and lipid peroxidation markers (p<0.05 for myoblasts and myotubes). A panel of nine polyphenols were assessed in aged myoblasts and myotubes using concentrations and incubation times consistent with known pharmacokinetic parameters for these compounds. Of these, although several polyphenols were seen to reduce single markers of RONS signalling, only kaempferol and resveratrol consistently reduced multiple markers of RONS signalling with statistical significance in both cell models. Modulation of cellular enzymatic antioxidant activities (superoxide and catalase) was assessed as a possible mechanism of action for these polyphenols, and although both superoxide and catalase activities were significantly reduced in aged (versus young) myotubes (p<0.01 and p<0.05 respectively), no effect of polyphenol treatment on these enzyme activities were observed. Overall, this research has shown the utility of the C2C12 model, as both myoblasts and myotubes, as a suitable cell model for screening compounds for modulating RONS signalling in aged muscle, and that resveratrol and kaempferol (using pharmacokinetically-informed exposures) can modulate RONS signalling in skeletal muscle cells after an acute exposure.

category: pharmacology and toxicology


title: Heat stress prevented the biomass and yield stimulation caused by elevated CO2 in two well-watered wheat cultivars

abstract:
To investigate the interactive effects of elevated CO2 and heat stress (HS), we grew two contrasting wheat cultivars, early-maturing Scout and high-tillering Yitpi, under non-limiting water and nutrients at ambient (aCO2, 450 ppm) or elevated (eCO2, 650 ppm) CO2 and 22{degrees}C in the glasshouse. Plants were exposed to two 3-day HS cycles at the vegetative (38.1{degrees}C) and/or flowering (33.5{degrees}C) stage.

At aCO2, both wheat cultivars showed similar responses of photosynthesis and mesophyll conductance to temperature and produced similar grain yield. Relative to aCO2, eCO2 enhanced photosynthesis rate and reduced stomatal conductance and maximal carboxylation rate (Vcmax). During HS, high temperature stimulated photosynthesis at eCO2 in both cultivars, while eCO2 stimulated photosynthesis in Scout. Electron transport rate (Jmax) was unaffected by any treatment. eCO2 equally enhanced biomass and grain yield of both cultivars in control, but not HS, plants. HS reduced biomass and yield of Scout at eCO2. Yitpi, the cultivar with higher grain nitrogen, underwent a trade-off between grain yield and nitrogen. In conclusion, eCO2 improved photosynthesis of control and HS wheat, and improved biomass and grain yield of control plants only. Under well-watered conditions, HS was not detrimental to photosynthesis or growth but precluded a yield response to eCO2.

Key messageHigh temperatures increased photosynthetic rates only at eCO2 and photosynthesis was upregulated after recovery from heat stress at eCO2 in Scout suggesting that eCO2 increased optimum temperature of photosynthesis.

category: physiology


title: Materials on Plant Leaf Surfaces are Deliquescent in a Variety of Environments

abstract: Many materials on plant leaf surfaces are hygroscopic, and they impact penetration of foliar-applied agrochemicals, foliar water uptake, gas exchange and stomatal density. Few studies are available on the nature of these substances, and we quantify how hygroscopic these materials are. Water vapor sorption experiments on twelve leaf washes were conducted and analyzed with inductively coupled plasma-optical emission spectroscopy and X-ray diffraction. Oils were found in all Eucalyptus samples studied. For mangroves that excrete salt to the leaf surfaces, significant sorption occurred at high humidity of a total of 316 mg ([~] 0.3 mL) over 6-10 leaves, and fitted a Guggenheim, Anderson, and de Boer sorption isotherm. All plant materials studied were hygroscopic. Materials on the plant leaf surface can deliquesce and form an aqueous solution in a variety of environments where plants grow, including glasshouses and by the ocean, which is an important factor when considering plant-atmosphere relations.

category: plant biology


title: Comparative transcriptional analysis of the satellite glial cell injury response

abstract: Satellite glial cells (SGCs) tightly surround and support primary sensory neurons in the peripheral nervous system and are increasingly recognized for their involvement in the development of neuropathic pain following nerve injury. The SGCs are difficult to investigate due to their flattened shape and tight physical connection to neurons in vivo and their rapid changes in phenotype and protein expression when cultured in vitro. Consequently, several aspects of SGC function under normal conditions as well as after a nerve injury remain to be explored. The recent advance in single cell RNAseq technologies has enabled a new approach to investigate SGCs. Here we publish a dataset from mice subjected to sciatic nerve injury as well as a dataset from dorsal root ganglia cells after 3 days in culture. We use a meta-analysis approach to compare the injury response with that in other published datasets and conclude that SGCs share a common signature following sciatic nerve crush and sciatic ligation, involving transcriptional regulation of cholesterol biosynthesis. We also observed a considerable transcriptional change when culturing SGCs, suggesting that some differentiate into a specialised in vitro state, while others start resembling Schwann cell-like precursors. The datasets are available via the Broad Institute Single Cell Portal.

category: neuroscience


title: Self-assembling short immunostimulatory duplex RNAs with broad spectrum antiviral activity

abstract: The current COVID-19 pandemic highlights the need for broad-spectrum antiviral therapeutics. Here we describe a new class of self-assembling immunostimulatory short duplex RNAs that potently induce production of type I and type III interferon (IFN-I and IFN-III), in a wide range of human cell types. These RNAs require a minimum of 20 base pairs, lack any sequence or structural characteristics of known immunostimulatory RNAs, and instead require a unique conserved sequence motif (sense strand: 5-C, antisense strand: 3-GGG) that mediates end-to-end dimer self-assembly of these RNAs by Hoogsteen G-G base-pairing. The presence of terminal hydroxyl or monophosphate groups, blunt or overhanging ends, or terminal RNA or DNA bases did not affect their ability to induce IFN. Unlike previously described immunostimulatory siRNAs, their activity is independent of TLR7/8, but requires the RIG-I/IRF3 pathway that induces a more restricted antiviral response with a lower proinflammatory signature compared with poly(I:C). Immune stimulation mediated by these duplex RNAs results in broad spectrum inhibition of infections by many respiratory viruses with pandemic potential, including SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza A, as well as the common cold virus HCoV-NL63 in both cell lines and human Lung Chips that mimic organ-level lung pathophysiology. These short dsRNAs can be manufactured easily, and thus potentially could be harnessed to produce broad-spectrum antiviral therapeutics at low cost.

category: immunology


title: Characterization of SARS-CoV-2 public CD4+ αβ T cell clonotypes through reverse epitope discovery

abstract:
The amount of scientific data and level of public sharing produced as a consequence of the COVID-19 pandemic, as well as the speed at which these data were produced, far exceeds any previous effort against a specific disease condition. This unprecedented situation allows for development and application of new research approaches. One of the major technical hurdles in immunology is the characterization of HLA-antigen-T cell receptor (TCR) specificities. Most approaches aim to identify reactive T cells starting from known antigens using functional assays. However, the need for a reverse approach identifying the antigen specificity of orphan TCRs is increasing.

Utilizing large public single-cell gene expression and TCR datasets, we identified highly public CD4+ T cell responses to SARS-CoV-2, covering >75% of the analysed population. We performed an integrative meta-analysis to deeply characterize these clonotypes by TCR sequence, gene expression, HLA-restriction, and antigen-specificity, identifying strong and public CD4+ immunodominant responses with confirmed specificity. CD4+ COVID-enriched clonotypes show T follicular helper functional features, while clonotypes depleted in SARS-CoV-2 individuals preferentially had a central memory phenotype. In total we identify more than 1200 highly public CD4+ T cell clonotypes reactive to SARS-CoV-2. TCR similarity analysis showed six prominent TCR clusters, for which we predicted both HLA-restriction and cognate SARS-CoV-2 immunodominant epitopes. To validate our predictions we used an independent cohort of TCR repertoires before and after vaccination with ChAdOx1, a replication-deficient simian adenovirus-vectored vaccine, encoding the SARS-CoV-2 spike protein. We find statistically significant enrichment of the predicted spike-reactive TCRs after vaccination with ChAdOx1, while the frequency of TCRs specific to other SARS-CoV-2 proteins remains stable. Thus, the CD4-associated TCR repertoire differentiates vaccination from natural infection.

In conclusion, our study presents a novel reverse epitope discovery approach that can be used to infer HLA- and antigen-specificity of orphan TCRs in any context, such as viral infections, antitumor immune responses, or autoimmune disease.

HighlightsO_LIIdentification of highly public CD4+ T cell responses to SARS-CoV-2
C_LIO_LISystematic prediction of exact immunogenic HLA class II epitopes for CD4+ T cell response
C_LIO_LIMethodological framework for reverse epitope discovery, which can be applied to other disease contexts and may provide essential insights for future studies and clinical applications
C_LI

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: Cooperative binding of TCR and CD4 to pMHC enhances TCR sensitivity

abstract: Antigen recognition of CD4+ T cells by the T cell receptor (TCR) can be greatly enhanced by the coreceptor CD41-7. Yet, understanding of the molecular mechanism is hindered by the ultra-low affinity of CD4 binding to class-II peptide-major histocompatibility complexes (pMHC)1,7-10. Using two-dimensional (2D) mechanical-based assays, we determined a CD4-pMHC interaction to have 3-4 logs lower affinity than cognate TCR-pMHC interactions8, and to be susceptible to increased dissociation by forces (slip bond)5,8,11. In contrast, CD4 binds TCR-prebound pMHC at 5-6 logs higher affinity, forming TCR-pMHC-CD4 trimolecular bonds that are prolonged by force (catch bond)5,8,11 and modulated by protein mobility on the cell membrane, indicating profound TCR-CD4 cooperativity. Consistent with a tri-crystal structure12, using DNA origami as a molecular ruler to titrate spacing between TCR and CD4 indicates 7-nm proximity enables optimal trimolecular bond formation with pMHC. Our results reveal how CD4 augments TCR antigen recognition.


title: Inhibition of the TLR4 signalling pathway with TAK-242 reduces RSV infection and cytokine release in primary airway epithelial cells

abstract:
Respiratory syncytial virus (RSV) infection is the leading cause of hospitalisation in children worldwide, but there is still no vaccine or anti-viral treatment available. RSV has been implicated in the development of respiratory diseases such as asthma. Toll like receptor 4 (TLR4) has been well characterised in the immune responses to RSV. However, the role of TLR4 in RSV infection remains unclear. To study RSV in the lung epithelium, where RSV preferentially infects ciliated cells, we used a well-differentiated primary airway epithelial cell (WD-PAEC) model: a pseudostratified epithelium that produces mucus and beating cilia. We demonstrate in this physiologically relevant model that TLR4 is a pro-viral factor. Inhibition of TLR4 using TAK-242 significantly reduces RSV titres in WD-PAECs in a dose-dependent manner but has no effect on RSV growth kinetics in a range of immortalised respiratory-derived cell lines. Specific inhibition of a range of downstream effectors of TLR4 signalling in the WD-PAEC model identified p38 MAPK as a pro-viral factor, whereas inhibition of MEK1/2 significantly increased RSV titres. Our data demonstrate a role for TLR4 in RSV infection and highlight the importance of biologically relevant models to study virus-host interactions.

Author summaryRespiratory Syncytial Virus (RSV) can cause severe respiratory infection in young children and is responsible for approximately 200,000 deaths worldwide every year. Despite decades of research since the identification of this virus in the 1950s there is still no vaccine or treatment available. Advances in research have led to the development of cell cultures that are very similar to the cells that line human airways. These cultures provide an opportunity to study how viruses interacts with airway cells in a representative model and may provide insights that traditional research models have not yet been able to answer. Using this experimental model we show that a drug, TAK-242 which targets a pathogen recognition receptor on the surface of cells, reduces growth of RSV and dampens the immune response to infection in these airway cells. Our data demonstrate potential targets for RSV treatments and also highlight the importance of using relevant experimental models.


title: Klebsiella pneumoniae mutants resistant to ceftazidime/avibactam plus aztreonam, imipenem/relebactam, meropenem/vaborbactam and cefepime/taniborbactam.

abstract: Using modified Klebsiella pneumoniae clinical isolates, we show that ramR plus ompK36 mutation together with production of the V239G variant KPC-3 confirs resistance to ceftazidime/avibactam plus aztreonam, imipenem/relebactam and meropenem/vaborbactam, but not cefepime/taniborbactam. This is because the V239G variant does not generate collateral {beta}-lactam susceptibility as do many other KPC-3 variants associated with ceftazidime/avibactam resistance. Additional mutation of ompK35 and carriage of a plasmid expressing the OXA-48-like carbapenemase OXA-232 was required to confer cefepime/taniborbactam resistance.


title: Target recognition in tandem WW domains: complex structures for parallel and antiparallel ligand orientation in h-FBP21 tandem WW

abstract: Protein-protein interactions often rely on specialized recognition domains, such as WW domains, which bind to specific proline-rich sequences. The specificity of these protein-protein interactions can be increased by tandem repeats, i.e. two WW domains connected by a linker. With a flexible linker, the WW domains can move freely with respect to each other. Additionally, the tandem WW domains can bind in two different orientations to their target sequences. This makes the elucidation of complex structures of tandem WW domains extremely challenging. Here, we identify and characterize two complex structures of the tandem WW domain of human formin-binding protein 21 and a peptide sequence from its natural binding partner, the core-splicing protein SmB/B. The two structures differ in the ligand orientation, and consequently also in the relative orientation of the two WW domains. We analyze and probe the interactions in the complexes by molecular simulations and NMR experiments. The workflow to identify the complex structures uses molecular simulations, density-based clustering and peptide docking. It is designed to systematically generate possible complex structures for repeats of recognition domains. These structures will help us to understand the synergistic and multivalency effects that generate the astonishing versatility and specificity of protein-protein interactions.

category: molecular biology


title: Target Recognition in Tandem WW Domains: Complex Structures for Parallel and Antiparallel Ligand Orientation in h-FBP21 Tandem WW


title: Induction and inhibition of Drosophila X chromosome gene expression are both impeded by the dosage compensation complex

abstract: Sex chromosomes frequently differ from the autosomes in the frequencies of genes with sexually dimorphic or tissue-specific expression. Multiple hypotheses have been put forth to explain the unique gene content of the X chromosome, including selection against male-beneficial X-linked alleles, expression limits imposed by the haploid dosage of the X in males, and interference by the dosage compensation complex (DCC) on expression in males. Here, we investigate these hypotheses by examining differential gene expression in Drosophila melanogaster following several treatments that have widespread transcriptomic effects: bacterial infection, viral infection, and abiotic stress. We found that genes that are induced (up-regulated) by these biotic and abiotic treatments are frequently under-represented on the X chromosome, but so are those that are repressed (down-regulated) following treatment. We further show that whether a gene is bound by the DCC in males can largely explain the paucity of both up- and down-regulated genes on the X chromosome. Specifically, genes that are bound by the DCC, or close to a DCC high-affinity site, are unlikely to be up- or down-regulated after treatment. This relationship, however, could partially be explained by a correlation between differential expression and breadth of expression across tissues. Nonetheless, our results suggest that DCC binding, or the associated chromatin modifications, inhibit both up- and down-regulation of X chromosome gene expression within specific contexts. We propose multiple possible mechanisms of action for the effect, including a role of Males absent on the first (Mof), a component of the DCC, as a dampener of gene expression variance in both males and females. This effect could explain why the Drosophila X chromosome is depauperate in genes with tissue-specific or induced expression, while the mammalian X has an excess of genes with tissue-specific expression.

category: genomics


title: Inferring ongoing cancer evolution from single tumour biopsies using synthetic supervised learning

abstract: Variant allele frequencies (VAF) encode ongoing evolution and subclonal selection in growing tumours. However, existing methods that utilize VAF information for cancer evolutionary inference are compressive, slow, or incorrectly specify the underlying cancer evolutionary dynamics. Here, we provide a proof-of-principle synthetic supervised learning method, TumE, that integrates simulated models of cancer evolution with Bayesian neural networks, to infer ongoing selection in bulk-sequenced single tumour biopsies. Analyses in synthetic and patient tumours show that TumE significantly improves both accuracy and inference time per sample when detecting positive selection, deconvoluting selected subclonal populations, and estimating subclone frequency. Importantly, we show how transfer learning can leverage stored knowledge within TumE models for related evolutionary inference tasks -- substantially reducing data and computational time for further model development and providing a library of recyclable deep learning models for the cancer evolution community. This extensible framework provides a foundation and future directions for harnessing progressive computational methods for the benefit of cancer genomics and, in turn, the cancer patient. TumE is publicly available for use at https://github.com/tomouellette/TumE.


title: Immune escape facilitation by mutations of epitope residues in RdRp of SARS-CoV-2

abstract: SARS-CoV-2 has considerably higher mutation rate. SARS-CoV-2 possesses a RNA dependent RNA polymerase (RdRp) which helps to replicate its genome. The mutation P323L in RdRp is associated with the loss of a particular epitope (321-327) from this protein which may influence the pathogenesis of the concern SARS-CoV-2 through the development of antibody escape variants. We consider the effect of mutations in some of the epitope regions including the naturally occurring mutation P323L on the structure of the epitope and their interface with paratope using all-atom molecular dynamics (MD) simulation studies. P323L mutations cause conformational changes in the epitope region by opening up the region associated with increase in the radius of gyration and intramolecular hydrogen bonds, making the region less accessible. Moreover, the fluctuations in the dihedral angles in the epitope:paratope (IgG) interface increase which destabilize the interface. Such mutations may help in escaping antibody mediated immunity of the host.


title: Sampling the conformational landscapes of transporters and receptors with AlphaFold2

abstract: Equilibrium fluctuations and triggered conformational changes often underlie the functional cycles of membrane proteins. For example, transporters mediate the passage of molecules across cell membranes by alternating between inward-facing (IF) and outward-facing (OF) states, while receptors undergo intracellular structural rearrangements that initiate signaling cascades. Although the conformational plasticity of these proteins has historically posed a challenge for traditional de novo protein structure prediction pipelines, the recent success of AlphaFold2 (AF2) in CASP14 culminated in the modeling of a transporter in multiple conformations to high accuracy. Given that AF2 was designed to predict static structures of proteins, it remains unclear if this result represents an underexplored capability to accurately predict multiple conformations and/or structural heterogeneity. Here, we present an approach to drive AF2 to sample alternative conformations of topologically diverse transporters and G-protein coupled receptors (GPCRs) that are absent from the AF2 training set. Whereas models generated using the default AF2 pipeline are conformationally homogeneous and nearly identical to one another, reducing the depth of the input multiple sequence alignments (MSAs) by stochastic subsampling led to the generation of accurate models in multiple conformations. In our benchmark, these conformations spanned the range between two experimental structures of interest, with models at the extremes of these conformational distributions observed to be among the most accurate (average template modeling (TM)-score of 0.94). These results suggest a straightforward approach to identifying native-like alternative states, while also highlighting the need for the next generation of deep learning algorithms to be designed to predict ensembles of biophysically relevant states.


title: In vitro assessment of elevated soil iron on germinability and germination characteristics of Sorghum bicolor (L.) Moench after chemo-priming

abstract: The commercial importance of Sorghum (Sorghum bicolor (L.) Moench) has attracted breeders to increase its seed yield using various breeding approach. Adverse soil factors however hampered progress made in crop development, especially micronutrient toxicity. Plant growth stimulators (PGS) have a significant role in enhancing growth parameters in Sorghum. In the present study, seeds were primed in 50, 150, and 250 ppm of each of gibberellic acid, indole acetic acid, and ascorbic acid respectively for 1 hr before sowing in Petri dishesmoistened with 10 ml of the iron-rich solution obtained as filtrate from a mix of distilled water and ferruginous soil (1:1 v/w). Results showed that although germination percentage in ferruginous medium was significantly reduced, there was enhancement in germination percentagewhen the seeds were primed in gibberellic acid (GA). Germinability in the iron-rich medium was 31.2 hrs; this was significantly reduced to 19.6 to 21.1hrs when these seeds were primed with growth stimulators.Although, shoot length was significantly reduced in plants exposed to ferruginous solutions, the root parameters were however enhanced. They were no significant changes in the total number of root branches regardless of ferrugenic status or use of growth stimulating agents. The utilization of growth stimulators as priming agents is called for to reduce stress impacts imposed by ferruginous soils during germination.


title: Influence of different anionic charges in lead compounds in the remediation of lead by Eleusine indica (Gaertn)

abstract: The study was carried out to investigate the remediative capacity of Eleusine indica in lead-polluted soil. Soil samples were collected near student hostel (hall 5) in the University of Benin. The soil samples were sun dried to constant weight and was pulverized with wooden roller and sieved with a hand sieve of 2 mm mesh size. The sieved soil was spiked with 0.625 g lead nitrate (PbNO3), lead sulphate (PbSO4), lead carbonate (PbCO3), lead acetetrahydrate (PbC2H6) and lead chloride (PbCl2) separately in three replicate using aqueous standard solutions. Tillers of Eleusine Indica were placed in the metal - polluted soil immediately and the experiment was allowed to stay for 15 weeks. The result showed that the uptake efficiency for Eleusineindica in both shoots and roots for lead nitrate was 0.016% and 0.8%, lead sulphate 0.016 % and 0.352 %, lead carbonate 0.064% and 0.496 %, lead acetetrahydrate 0.032 % and 0.688 %, and lead chloride 0.08 % and 0.72 % respectively, indicating that the plant might have sequestered the metal in the soil rather than accumulating it in the leaves. This was evident in the presentation of the metal sequestration factor of over 70 % irrespective of the nature of the metal. Microbial count of soil before and after contamination with lead nitrate was 1.9x104 and 0.4x104 cfu/g indicating a reduction. The study therefore revealed that Eleusine indica is a high efficient plant in sequestering lead in polluted soil.


title: Long-term decreased cannabinoid type-1 receptor activity restores specific neurological phenotypes in the Ts65Dn mouse model of Down syndrome

abstract: Intellectual disability is the most prevalent and limiting hallmark of Down syndrome (DS), without any pharmacological treatment available. Neurodegeneration and neuroinflammation are relevant neurological features of DS reaching to early development of Alzheimers disease. Preclinical evidence suggests that the endocannabinoid system, an important neuromodulator on cognition and neuroinflammation, could act as beneficial target in DS. Indeed, cannabinoid type-1 receptor (CB1R) activity was enhanced in the hippocampus of young-adult trisomic Ts65Dn mice, a well-characterized surrogate model of DS. In previous studies, inhibition of CB1R, was able to restore key neurological deficits in this mouse model. To determine the possible clinical relevance of this target, it is mandatory to evaluate the long-term consequences of attenuated CB1R activity and to minimize the possible side-effects associated to this mechanism. We found that CB1R expression was significantly enhanced in the hippocampus brains of aged DS subjects. Similarly, middle-aged trisomic mice showed enhanced CB1R expression. Long-term oral administration of a low dose of the CB1R specific antagonist rimonabant was administered to male and female Ts65Dn trisomic and wild-type mice from the time of weaning to 10 months, an age when signs of neurodegeneration have been described in the model. CB1R inhibition resulted in significant cognitive improvement in novel object-recognition memory in trisomic male and female mice, reaching a similar performance to that of wild-type littermates. Interestingly, this long-term rimonabant treatment modify locomotor activity, anxiety-like behavior, body weight or survival rates. Brain analysis at 10 months of age revealed noradrenergic and cholinergic neurodegeneration signs in trisomic mice that were not modified by the treatment, although the alterations in hippocampal microglia morphology shown by vehicle-treated trisomic mice was normalized in trisomic mice exposed to rimonabant. Altogether, our results demonstrate a sustained pro-cognitive effect of CB1R inhibition at doses that do not produce major side effects that could be associated to an anti-inflammatory action, suggesting a potential interest in this target of to preserve cognitive functionality in DS.


title: All-optical electrophysiology with improved genetically encoded voltage indicators reveals interneuron network dynamics in vivo

abstract: All-optical electrophysiology can be a powerful tool for studying neural dynamics in vivo, as it offers the ability to image and perturb membrane voltage in multiple cells simultaneously. The "Optopatch" constructs combine a red-shifted archaerhodopsin (Arch)-derived genetically encoded voltage indicator (GEVI) with a blue-shifted channelrhodopsin actuator (ChR). We used a video-based pooled screen to evolve Arch-derived GEVIs with improved signal-to-noise ratio (QuasAr6a) and kinetics (QuasAr6b). By combining optogenetic stimulation of individual cells with high-precision voltage imaging in neighboring cells, we mapped inhibitory and gap junction-mediated connections, in vivo. Optogenetic activation of a single NDNF-expressing neuron in visual cortex Layer 1 significantly suppressed the spike rate in some neighboring NDNF interneurons. Hippocampal PV cells showed near-synchronous spikes across multiple cells at a frequency significantly above what one would expect from independent spiking, suggesting that collective inhibitory spikes may play an important signaling role in vivo. By stimulating individual cells and recording from neighbors, we quantified gap junction coupling strengths. Together, these results demonstrate powerful new tools for all-optical microcircuit dissection in live mice.


title: Simultaneous assessment of eight phosphorylated STAT residues in T-cells by flow cytometry.

abstract: Signal transducer and activator of transcription (STAT) proteins are a family of transcription factors controlling functions in immune responses and other cell types. Given their importance, we developed a flow cytometry panel to assess eight phosphorylated STAT residues in human T-cells, including six tyrosine residues across six STAT proteins (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT6) and additional serine residues on STAT1 and STAT3. We applied this protocol to test the in vitro induction of pSTATs in response to CD3/CD28 activation and a panel of recombinant cytokines. We also assessed the pSTAT expression profiles of naive CD4+ T-cells polarized to Th1, Th2, Th17 or iTregs. pSTAT1(S727), pSTAT2(Y689) and pSTAT3(S727) were constitutively expressed in most T-cells, even in the absence of stimulation. For pSTAT1(S727) and pSTAT3(S727), we observed two positive states, high and low. Conversely, expression of pSTAT1(Y701), pSTAT3(Y705), pSTAT4(Y693) and pSTAT6(Y641) were absent in resting T-cells and only expressed with CD3/CD28 activation or with specific cytokines. Variable frequencies of pSTAT5a(Y694) expression were observed in resting T-cells, which increased with activation or specific cytokine stimulation (e.g. IL-2). IFN{beta} stimulation enhanced frequencies of expressing cells for all pSTATs. Correlations among several pSTATs, particularly pSTAT1(S727)high and pSTAT3(S727)high were observed. While polarization resulted in increases in canonically associated pSTATs, other non-canonical pSTAT changes were also observed. Collectively, we developed, optimized, and tested a sensitive and rapid approach for simultaneously assessing phosphorylation of six STAT proteins. Using this approach, we made several novel observations of T-cell pSTAT induction in response to stimuli.


title: A Field-Deployable Diagnostic Assay for the Visual Detection of Misfolded Prions

abstract: Chronic Wasting Disease (CWD), a prion disease of cervids, has been identified across North America, Northern Europe and Korea. Diagnostic tools for the rapid and reliable detection of prion diseases are limited. Here, we combine gold nanoparticles (AuNPs) and quaking induced conversion (QuIC) technologies for the visual detection of amplified misfolded prion proteins from tissues of wild white-tailed deer infected with Chronic Wasting Disease (CWD). Our newly developed diagnostic test, MN-QuIC, enables both naked-eye and light-absorbance measurements for the detection of misfolded prions. The MN-QuIC assay leverages basic laboratory equipment that is cost-effective and portable, thus facilitating real-time prion diagnostics across a variety of settings. To test the portability of our assay, we deployed to a rural field station in southeastern Minnesota and tested for CWD on site. We successfully demonstrated that MN-QuIC is functional in a non-traditional laboratory setting by performing a blinded analysis in the field and correctly identifying all CWD positive and CWD not detected (independently confirmed with ELISA and/or IHC tests) animals at the field site, thus documenting the portability of the assay. Additionally, we show that electrostatic forces and concentration effects help govern the AuNP/prion interactions and contribute to the differentiation of CWD-prion positive and negative samples. We examined 17 CWD-positive and 24 CWD-not-detected white-tailed deer tissues that were independently tested using ELISA, IHC, and RT-QuIC technologies, and results secured with MN-QuIC were 100% consistent with these tests. We conclude that hybrid AuNP and QuIC assays, such as MN-QuIC, have great potential for sensitive, field-deployable diagnostics for a variety of protein misfolding diseases.


title: Long distance migration is a major factor driving local adaptation at continental scale in Coho Salmon

abstract: Inferring the genomic basis of local adaptation is a long-standing goal of evolutionary biology. Beyond its fundamental evolutionary implications, such knowledge can guide conservation decisions for populations of conservation and management concern. Here, we investigated the genomic basis of local adaptation in the Coho salmon (Oncorhynchus kisutch) across its entire North American range. We hypothesized that extensive spatial variation in environmental conditions and the species homing behavior may promote the establishment of local adaptation. We genotyped 7,829 individuals representing 217 sampling locations at more than 100,000 high-quality RADseq loci to investigate how recombination might affect the detection of loci putatively under selection and took advantage of the precise description of the demographic history of the species from our previous work to draw accurate population genomic inferences about local adaptation. Results indicated that genetic differentiation scans and genetic-environment association analyses were both significantly affected by variation in recombination rate as low recombination regions displayed an increased number of outliers. By taking these confounding factors into consideration, we revealed that migration distance was the primary selective factor driving local adaptation and partial parallel divergence among distant populations. Moreover, we identified several candidates SNP associated with long distance migration and altitude including a gene known to be involved in adaptation to altitude in other species. The evolutionary implications of our findings are discussed along with conservation applications.


title: Quantification of vascular networks in photoacoustic mesoscopy

abstract: Mesoscopic photoacoustic imaging (PAI) enables non-invasive visualisation of tumour vasculature and has the potential to assess prognosis and therapeutic response. Currently, evaluating vasculature using mesoscopic PAI involves visual or semi-quantitative 2D measurements, which fail to capture 3D vessel network complexity, and lack robust ground truths for assessment of segmentation accuracy. Here, we developed an in silico, phantom, in vivo, and ex vivo-validated end-to-end framework to quantify 3D vascular networks captured using mesoscopic PAI. We applied our framework to evaluate the capacity of rule-based and machine learning-based segmentation methods, with or without vesselness image filtering, to preserve blood volume and network structure by employing topological data analysis. We first assessed segmentation performance against ground truth data of in silico synthetic vasculatures and a photoacoustic string phantom. Our results indicate that learning-based segmentation best preserves vessel diameter and blood volume at depth, while rule-based segmentation with vesselness image filtering accurately preserved network structure in superficial vessels. Next, we applied our framework to breast cancer patient-derived xenografts (PDXs), with corresponding ex vivo immunohistochemistry. We demonstrated that the above segmentation methods can reliably delineate the vasculature of 2 breast PDX models from mesoscopic PA images. Our results underscore the importance of evaluating the choice of segmentation method when applying mesoscopic PAI as a tool to evaluate vascular networks in vivo.

category: bioengineering


title: Identification of biological processes and signaling pathways for the knockout of REV-ERB in mouse brain

abstract: REV-ERB is an orphan nuclear receptor that is widely expressed in the brain and inhibits transcriptional activities. A variety of genes affect the activity and expression of REV-ERB. In this study, our objective is to identify significant signaling pathways and biological processes in the knockout of the REV-ERB mouse brain. The GSE152919 dataset was originally created by using the Illumina HiSeq 4000 (Mus musculus). The KEGG and GO analyses suggested that biological processes "PPAR signaling", "Hippo signaling", and "Hypertrophic cardiomyopathy (HCM)" are mostly affected in the knockout of REV-ERB. Furthermore, we identified a number of genes according to the PPI network including NPAS2, CRY2, BMAL1, and CRY1 which were involved in the lack of REV-ERB in the brain. Therefore, our study provides further insights into the study of circadian clocks.


title: Influenza A virus hemagglutinin prevents extensive membrane damage upon dehydration

abstract: While the molecular mechanisms of virus infectivity are rather well known, the detailed consequences of environmental factors on virus biophysical properties are poorly understood. Seasonal influenza outbreaks are usually connected to the low winter temperature, but also to the low relative air humidity. Indeed, transmission rates increase in cold regions during winter. While low temperature must slow degradation processes, the role of low humidity is not clear. We studied the effect of relative humidity on a model of influenza A H1N1 virus envelope, a supported lipid bilayer containing the surface glycoprotein hemagglutinin (HA), which is present in the viral envelope in very high density. For complete cycles of hydration, dehydration and rehydration, we evaluate the membrane properties in terms of structure and dynamics, which we assess by combining confocal fluorescence microscopy, raster image correlation spectroscopy, line-scan fluorescence correlation spectroscopy and atomic force microscopy. Our findings indicate that the presence of HA prevents macroscopic membrane damage after dehydration. Without HA, fast membrane disruption is followed by irreversible loss of lipid and protein mobility. Although our model is principally limited by the membrane composition, the macroscopic effects of HA under dehydration stress reveal new insights on the stability of the virus at low relative humidity.


title: EZH2 inhibition in glioblastoma stem cells increases the expression of neuronal genes and the neuronal developmental regulators ZIC2, ZNF423 and MAFB

abstract: Glioblastoma multiforme (GBM) is an aggressive brain cancer with a very poor prognosis. It has been shown that GBM stem cells within a GBM tumour have increased resistance to standard therapies, so new approaches are needed to increase the range of treatment options available. Here we use two GBM stem cell lines, representing the classical/pro-neural and mesenchymal GBM subtypes, to investigate the effects of three different EZH2 inhibitors on GBM stem cell survival and gene expression: EPZ6438, GSK343 and UNC1999. EZH2 is the catalytic component of the PRC2 chromatin repressor complex, which represses transcription through methylation of histone H3 at lysine 27. Both cell lines showed significantly reduced colony formation after 48-hour exposure to the inhibitors, indicating they were sensitive to all three EZH2 inhibitors. RNA-seq analysis revealed that all three EZH2 inhibitors led to increased expression of genes related to neurogenesis and/or neuronal structure in both GBM stem cell lines. Chromatin immunoprecipitation (ChIP-Seq) was used to identify potential direct targets of the histone methylation activity of EZH2 that might be driving the increase in neuronal gene expression. Three genes were identified as candidate regulatory targets common to both cell lines: MAFB, ZIC2 and ZNF423. These transcription factors all have known roles in regulating neurogenesis, brain development and/or neuronal function. Through analysis of three different EZH2 inhibitors and two GBM stem cell lines, this study demonstrates a common underlying mechanism for how inhibition of EZH2 activity reduces GBM stem cell proliferation and survival.

category: cancer biology


title: Acute SARS-CoV-2 infection in pregnancy is associated with placental ACE-2 shedding

abstract: Human placental tissues have variable rates of SARS-CoV-2 invasion resulting in consistently low rates of fetal transmission suggesting a unique physiologic blockade against SARS-CoV-2. Angiotensin-converting enzyme (ACE)-2, the main receptor for SARS-CoV-2, is expressed as cell surface and soluble forms regulated by a metalloprotease cleavage enzyme, ADAM17. ACE-2 is expressed in the human placenta, but the regulation of placental ACE-2 expression in relation to timing of maternal SARS-CoV-2 infection in pregnancy is not well understood. In this study, we evaluated ACE-2 expression, ADAM17 activity and serum ACE-2 abundance in a cohort of matched villous placental and maternal serum samples from Control pregnancies (SARS-CoV-2 negative, n=8) and pregnancies affected by symptomatic maternal SARS-CoV-2 infections in the 2nd trimester ("2ndTri COVID", n=8) and 3rd trimester ("3rdTri COVID", n=8). In 3rdTri COVID as compared to control and 2ndTri-COVID villous placental tissues ACE-2 mRNA expression was remarkably elevated, however, ACE-2 protein expression was significantly decreased with a parallel increase in ADAM17 activity. Soluble ACE-2 was also significantly increased in the maternal serum from 3rdTri COVID infections as compared to control and 2ndTri-COVID pregnancies. These data suggest that in acute maternal SARS-CoV-2 infections, decreased placental ACE-2 protein may be the result of ACE-2 shedding. Overall, this work highlights the importance of ACE-2 for ongoing studies on SARS-CoV-2 responses at the maternal-fetal interface.

category: pathology


title: Thermodynamic Constraints on Electromicrobial Protein Production

abstract: Global consumption of protein is projected to double by the middle of the 21st century1. However, protein production is one of the most energy intensive and environmentally damaging parts of the food supply system today. Electromicrobial production technologies that combine renewable electricity and CO2-fixing microbial metabolism could dramatically increase the energy efficiency of commodity chemical production2-5. Here we present a molecular-scale model that sets an upper limit on the performance of any organism performing electromicrobial protein production. We show that engineered microbes that fix CO2 and N2 using reducing equivalents produced by H2-oxidation or extracellular electron uptake could produce amino acids with energy inputs as low as 64 MJ kg-1. This work provides a roadmap for development of engineered microbes that could significantly expand access to proteins produced with a low environmental footprint.

category: synthetic biology


title: Stochastic Simulation Algorithm for effective spreading dynamics on Time-evolving Adaptive NetworX (SSATAN-X)

abstract:
Modelling and simulating the dynamics of pathogen spreading has been proven crucial to inform public heath decisions, containment strategies, as well as cost-effectiveness calculations. Pathogen spreading is often modelled as a stochastic process that is driven by pathogen exposure on time-evolving contact networks. In adaptive networks, the spreading process depends not only on the dynamics of a contact network, but vice versa, infection dynamics may alter risk behaviour and thus feed back onto contact dynamics, leading to emergent complex dynamics. However, stochastic simulation of pathogen spreading processes on adaptive networks is currently computationally prohibitive.

In this manuscript, we propose SSATAN-X, a new algorithm for the accurate stochastic simulation of pathogen spreading on adaptive networks. The key idea of SSATAN-X is to only capture the contact dynamics that are relevant to the spreading process. We show that SSATAN-X captures the contact dynamics and consequently the spreading dynamics accurately. The algorithm achieves up to 100 fold speed-up over the state-of-art stochastic simulation algorithm (SSA). The speed-up with SSATAN-X further increases when the contact dynamics are fast in relation to the spreading process, i.e. if contacts are short-lived and per-exposure infection risks are small, as applicable to most infectious diseases.

We envision that SSATAN-X may extend the scope of analysis of pathogen spreading on adaptive networks. Moreover, it may serve to create benchmark data sets to validate novel numerical approaches for simulation, or for the data-driven analysis of the spreading dynamics on adaptive networks. A C++ implementation of the algorithm is available at https://github.com/nmalysheva/SSATAN-X.

Author summaryModelling and simulating of infectious disease spreading supports public heath decisions, such as prevention and containment strategies and allows to perform cost-effectiveness calculations. Detailed modelling approaches consider stochastic pathogen spreading on time-evolving contact networks. In adaptive networks, the spreading process depends not only on the dynamics of a contact network, but vice versa, infection dynamics may alter risk behaviour and thus feed back onto contact dynamics.

Stochastic simulation of these complex dynamics is currently computationally prohibitive.

We propose a new algorithm (SSATAN-X) that can significantly speed up stochastic simulations on adaptive networks, while being accurate at the same time. Our algorithm achieves this speed-up by only considering the contact dynamics that are relevant to the spreading process. The benefit of algorithm is particularly pronounced when contacts are short-lived and per-exposure infection risks are small, which is applicable to most infectious diseases.

We envision that SSATAN-X may allow simulation and analysis of pathogen spreading on more complex adaptive networks than previously possible. Moreover, data sets may be created with SSATAN-X that are useful for benchmarking novel numerical schemes and analytic approaches.

category: systems biology


title: Chronic Physical and Vicarious Psychosocial Stress Alter Fentanyl Consumption and Nucleus Accumbens Rho GTPases in Male and Female C57BL/6 Mice

abstract: Chronic stress can increase the risk of developing a substance use disorder in vulnerable individuals. Numerous models have been developed to probe the underlying neurobiological mechanisms, however most prior work has been restricted to male rodents, conducted only in rats, or introduces physical injury that can complicate opioid studies. Here we sought to establish how chronic psychosocial stress influences fentanyl consumption in male and female C57BL/6 mice. We used chronic social defeat stress (CSDS), or the modified vicarious chronic witness defeat stress (CWDS), and used social interaction to stratify mice as stress-susceptible or resilient. We then subjected mice to a 15 day fentanyl drinking paradigm in the home cage that consisted of alternating forced and choice periods with increasing fentanyl concentrations. Male mice susceptible to either CWDS or CSDS consumed more fentanyl relative to unstressed mice, and exhibited increased preference for fentanyl. CWDS-susceptible female mice did not differ from unstressed mice during the forced periods, but showed increased preference for fentanyl. We also found decreased expression of nucleus accumbens Rho GTPases in male, but not female mice following stress and fentanyl drinking. We also compare fentanyl drinking behavior in mice that had free access to plain water throughout. Our results indicate that stress-sensitized fentanyl consumption is dependent on both sex and behavioral outcomes to stress.


title: Identification of a genomic DNA sequence that quantitatively modulates KLF1 expression in differentiating human hematopoietic cells

abstract: Expression of the {beta}-like globin genes is under strict developmental control, with both direct and indirect inputs responsible for this effect. One of the major players regulating their transition is KLF1/EKLF, where even a two-fold difference in its level alters the regulation of globin switching. We have reproduced this change in KLF1 expression in both cell lines and primary human cells, thus demonstrating that directed, quantitative control of KLF1 expression can be attained by genomic manipulation, and suggest a new way in which modulation of transcription factor levels may be used for clinical benefit.


title: Single organoid RNA-sequencing reveals high organoid-to-organoid variability

abstract: Over the last decades, organoids have been established from the majority of tissue resident stem and iPS cells. They hold great promise for our understanding of mammalian organ development, but also for the study of disease or even personalized medicine. In recent years, several reports hinted at intraculture organoid variability, but a systematic analysis of such a heterogeneity has not been performed before. Here, we used RNA-seq of individual organoids to address this question. Importantly, we find that batch-to-batch variation is very low, even when prepared by different researchers. On the other hand, there is organoid-to-organoid variability within a culture. Using differential gene expression, we did not identify specific pathways that drive this variability, pointing towards possible effects of the microenvironment within the culture condition. Taken together, our study provides a framework for organoid researchers to properly consider experimental design.

category: cell biology


title: The role of indirect effects in coevolution as mutualism transitions into antagonism

abstract: Species interactions have evolved from antagonistic to mutualistic and back several times throughout lifes history. Yet, it is unclear how changes in the type of interaction between species alter the coevolutionary dynamics of entire communities. This is a pressing matter, as transitions from mutualisms to antagonisms may be becoming more common with human-induced global change. Here, we combine network and evolutionary theory to simulate how shifts in interaction types alter the coevolution of empirical communities. We show that as mutualistic networks shift to antagonistic, selection imposed by direct partners begins to outweigh that imposed by indirect partners. This weakening of indirect effects is associated with communities losing their tight integration of traits and increasing their rate of adaptation. The above changes are more pronounced when specialist consumers are the first species to switch to antagonism. A shift in the outcome of species interactions may therefore reverberate across communities and alter the direction and speed of coevolution.

category: evolutionary biology


title: Genome Assembly and Population Resequencing Reveal the Geographical Divergence of 'Shanmei'(Rubus corchorifolius)

abstract: Rubus corchorifolius ( Shanmei or mountain berry, 2n =14) is widely distributed in China, and its fruit has high nutritional and medicinal values. Here, we report a high-quality chromosome-scale genome assembly of Shanmei, with a size of 215.69 Mb and encompassing 26,696 genes. Genome comparisons among Rosaceae species show that Shanmei and Fupenzi (Rubus chingii Hu) are most closely related, and then is blackberry (Rubus occidentalis). Further resequencing of 101 samples of Shanmei collected from four regions in provinces of Yunnan, Hunan, Jiangxi, and Sichuan in South China reveals that the Hunan population of Shanmei possesses the highest diversity and may represent the relatively more ancestral population. Moreover, the Yunnan population undergoes strong selection based on nucleotide diversity, linkage disequilibrium, and the historical effective population size analyses. Furthermore, genes from candidate genomic regions that show strong divergence are significantly enriched in flavonoid biosynthesis and plant hormone signal transduction, indicating the genetic basis of adaptation of Shanmei to the local environments. The high-quality genome sequences and the variome dataset of Shanmei provide valuable resources for breeding applications and for elucidating the genome evolution and ecological adaptation of Rubus species.


title: A Neurostimulator System for Real, Sham, and Multi-Target Transcranial Magnetic Stimulation

abstract:
AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackgroundC_ST_ABSTranscranial magnetic stimulation (TMS) is a clinically effective therapeutic instrument used to modulate neural activity. Despite three decades of research, two challenging issues remain, the possibility of changing the 1) stimulated spot and 2) stimulation type (real or sham) without physically moving the coil.

ObjectiveIn this study, a second-generation programmable TMS (pTMS2) device with advanced stimulus shaping is introduced that uses a 5-level cascaded H-bridge inverter and phase-shifted pulse-width modulation (PWM). The principal idea of this research is to obtain real, sham, and multi-locus stimulation with the same TMS system.

MethodsWe propose a two-channel modulation-based magnetic pulse generator and a novel coil arrangement, consisting of two circular coils with a physical distance of 20 mm between the coils and a control method for modifying the effective stimulus intensity, which leads to the live steerability of the location and type of stimulation.

ResultsBased on the measured system performance, the stimulation profile can be steered {+/-} 20 mm along a line from the centroid of the coil locations by modifying the modulation index.

ConclusionThe proposed system supports electronic control of the stimulation spot without physical coil movement, resulting in tunable modulation of targets, which is a crucial step towards automated TMS machines.


title: Polyelectrolyte Nanocomplex Formation Combined with Electrostatic Self-Assembly Enables the Co-Delivery of Synergistic Antimicrobials to Treat Bacterial Biofilms

abstract: New approaches are needed to treat bacterial biofilm infections, particularly those of Pseudomonas aeruginosa (PA). PA biofilms have high rates of antimicrobial resistance and are commonly found in chronic wound and cystic fibrosis lung infections. The use of combination therapeutics that act synergistically is a promising treatment strategy; however, the delivery of multiple therapeutics at relevant dosages is challenging in the clinic. We therefore developed a new nanoscale drug carrier by combining approaches from both polyelectrolyte nanocomplex (NC) formation and layer-by-layer electrostatic self-assembly. This strategy led to NC drug carriers functionalized with both tobramycin antibiotics and antimicrobial silver nanoparticles with high loading efficiencies. AgTob-NCs displayed synergistic enhancements in antimicrobial activity against planktonic and biofilm PA cultures, with positively charged NCs demonstrating complete biofilm eradication. Overall, this approach shows promise in the co-delivery of diverse classes of antimicrobials to overcome antimicrobial resistance and treat bacterial biofilm infections.


title: Simplified, Shear Induced Generation of Double Emulsions for Robust Compartmentalization during Single Genome Analysis

abstract: Drop microfluidics has driven innovations for high throughput, low input analysis techniques such as single-cell RNA-seq. However, the instability of single emulsion (SE) drops occasionally causes significant merging during drop processing, limiting most applications to single-step reactions in drops. Here, we show that double emulsion (DE) drops address this critical limitation and completely prevent content mixing, which is essential for single entity analysis. DEs show excellent stability during thermal cycling. More importantly, DEs undergo rupture into the continuous phase instead of merging, preventing content mixing and eliminating unstable drops from the downstream analysis. Due to the lack of drop merging, the monodispersity of drops is maintained throughout a workflow, enabling the deterministic manipulation of drops downstream. We also developed a simple, one-layer fabrication method for DE drop makers. This design is powerful as it allows robust production of single-core DEs at a wide range of flow rates and better control over the shell thickness, both of which have been significant limitations of conventional two-layer devices. This approach makes the fabrication of DE devices much more accessible, facilitating its broader adoption. Finally, we show that DE droplets effectively maintain the compartmentalization of single virus genomes during PCR-based amplification and barcoding, while SEs mixed contents due to merging. With their resistance to content mixing, DE drops have key advantages for multistep reactions in drops, which is limited in SEs due to merging and content mixing.


title: In Diverse Conditions Intrinsic Chromatin Condensates Have Liquid-like Material Properties

abstract: Eukaryotic nuclear DNA is wrapped around histone proteins to form nucleosomes, which further assemble to package and regulate the genome. Understanding of the physical mechanisms that contribute to higher order chromatin organization is limited. Previously, we reported the intrinsic capacity of chromatin to undergo phase separation and form dynamic liquid-like condensates, which can be regulated by cellular factors. Recent work from Hansen, Hendzel, and colleagues suggested these intrinsic chromatin condensates are solid in all but a specific set of conditions. Here we show that intrinsic chromatin condensates are fluid in diverse solutions, without need for specific buffering components. Exploring experimental differences in sample preparation and imaging between these two studies, we suggest what may have led Hansen, Hendzel, and colleagues to mischaracterize the innate properties of chromatin condensates. We also describe how liquid-like in vitro behaviors can translate to the locally dynamic but globally constrained movement of chromatin in cells.


title: Hypertonic saline and aprotinin based blockage of SARS-CoV-2 specific furin site cleavage by inhibition of nasal protease activity

abstract: SARS-CoV-2 enters into the human body mainly through the nasal epithelial cells. Cell entry of SARS-CoV-2 needs to be pre-activated by S1/S2 boundary furin motif cleavage by furin and/or relevant proteases. It is important to locally block SARS-CoV-2 S1/S2 site cleavage caused by furin and other relevant protease activity in the nasal cavity. We tested hypertonic saline and aprotinin-based blockage of SARS-CoV-2 specific furin site cleavage by furin, trypsin and nasal swab samples containing nasal proteases. Our results show that saline and aprotinin block SARS-Cov-2 specific furin site cleavage and that a saline and aprotinin combination could significantly reduce SARS-Cov-2 wild-type and P681R mutant furin site cleavage by inhibition of nasal protease activity.


title: Loss of hepatic Lgr4 and Lgr5 promotes nonalcoholic fatty liver disease

abstract:
Background & AimsThe Rspo-Lgr4/5-Znrf3/Rnf43 module is a master regulator of hepatic Wnt/{beta}-catenin signaling and metabolic zonation, but its impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. We studied whether liver-specific loss of the Wnt/{beta}-catenin modulators Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 4/5 (Lgr4/5) promotes nonalcoholic fatty liver disease (NAFLD).

MethodsMice with liver-specific deletion of both receptors Lgr4/5 (Lgr4/5dLKO) were fed with normal diet (ND) or high fat diet (HFD). Livers of these mice were analyzed for lipid and fibrotic content by tissue staining and immunohistochemistry (IHC), and lipoproteins, inflammation and liver enzyme markers were measured in blood. Mechanistic insights into hepatic lipid accumulation were obtained by using ex vivo primary hepatocyte cultures derived from the Lgr4/5dLKO mice. Lipid analysis of mouse livers was performed by mass spectrometry (MS)-based untargeted lipidomic analysis.

ResultsWe demonstrated that liver-specific ablation of Lgr4/5-mediated Wnt signaling resulted in hepatic steatosis, impaired bile acid (BA) secretion and predisposition to liver fibrosis. Under HFD conditions, we observed progressive intrahepatic fat accumulation, developing into macro-vesicular steatosis. Serum lipoprotein levels in HFD-fed Lgr4/5dLKO mice were decreased, rather than increased, suggesting that accumulation of fat in the liver was due to impaired lipid secretion by hepatocytes. Our lipidome analysis revealed a severe alteration of several lipid species in livers of Lgr4/5dLKO mice, including triacylglycerol estolides (TG-EST), a storage form of bioactive free fatty acid (FA) esters of hydroxy FAs (FAHFAs).

ConclusionsLoss of hepatic Wnt/{beta}-catenin activity by Lgr4/5 deletion led to deregulation of lipoprotein pathways, loss of BA secretion, intrinsic alterations of lipid homeostasis and the onset of NAFLD.

Lay summaryThe Wnt/{beta}-catenin pathway plays an important role during development and tissue homeostasis. Loss of Wnt/{beta}-catenin activity in mouse liver leads to loss of liver zonation, but the impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. We show that livers of mice developed steatosis upon deletion of the positive pathway regulators Lgr4/5. Livers of knock-out (KO) mice exhibited altered lipid composition due to impaired lipid secretion. Furthermore, livers of these mice developed a nonalcoholic steatohepatitis (NASH)-like phenotype and fibrotic features derived from activated hepatic stellate cells. Our data demonstrate a protective role of Wnt/{beta}-catenin pathway activity towards the development of NAFLD.

HighlightsO_LIAbrogation of hepatic Wnt/{beta}-catenin activity and liver zonation upon Lgr4/5 deletion in mice led to hepatic steatosis.
C_LIO_LILiver fat accumulation was caused by impaired lipid secretion from hepatocytes.
C_LIO_LISteatotic livers contained increased levels of diverse lipid species, including polyunsaturated fatty acids and triglycerol-estolides.
C_LIO_LIThese data confirmed that a decrease in Wnt/{beta}-catenin signaling led to the development of nonalcoholic fatty liver disease (NAFLD) in mice.
C_LI


title: Implications of soil nitrogen enhancement on the yield performance of soybean (Glycine max) in cadmium-polluted soil

abstract: The growth, development and yield of important crop plants like soybean (Glycine max) are constantly under threat by continuous inputs of cadmium in the biosphere as a result of various industrial activities. This study investigated the level to which, addition of nitrogen fertilization can enhance plant survival, growth and yield development in a cadmium-polluted. Three accessions of Glycine max (TGm - 1, TGm - 2 and TGm - 3) were sown in a 12 mg/kg-cadmium polluted, which was thereafter amended with urea (FU), ammonia (FA), and ammonium nitrate (FN) singly and in combinations of equal proportions. A non-fertilized Cd-polluted soil and a general control constituted the negative and positive controls. Results showed that although N application did not enhance yield dispositions of soybean in Cd-polluted soil, significant impact on vegetative development was noteworthy. Compared to yield of control plants, cadmium pollution imposed a 26.1 % reduction in per plant yield in TGm-1, compared 1.71 % in TGm-3. Generally, addition of nitrogenous fertilizer further suppressed crop yield by as much as 80 % in plants sown in cadmium-polluted soil. However, application of ammonia fertilizer to TGm-2 improved its yield performances in the cadmium-polluted soil.


title: Electrophysiological correlates of confidence differ across correct and erroneous perceptual decisions

abstract:
Every decision we make is accompanied by an estimate of the probability that our decision is accurate or appropriate. This probability estimate is termed our degree of decision confidence. Recent work has uncovered event-related potential (ERP) correlates of confidence both during decision formation and after a decision has been made. However, the interpretation of these findings is complicated by methodological issues related to ERP amplitude measurement that are prevalent across existing studies. To more accurately characterise the neural correlates of confidence, we presented participants with a difficult perceptual decision task that elicited a broad range of confidence ratings. We identified a frontal ERP component within an onset prior to the behavioural response, which exhibited more positive-going amplitudes in trials with higher confidence ratings. This frontal effect also biased measures of the centro-parietal positivity (CPP) component at parietal electrodes via volume conduction. Amplitudes of the error positivity (Pe) component that followed each decision were negatively associated with confidence for trials with decision errors, but not for trials with correct decisions, with Bayes factors providing moderate evidence for the null in the latter case. We provide evidence for both pre- and post-decisional neural correlates of decision confidence that are observed in trials with correct and erroneous decisions, respectively. Our findings suggest that certainty in having made a correct response is associated with frontal activity during decision formation, whereas certainty in having committed an error is instead associated with the post-decisional Pe component. These findings also highlight the possibility that some previously reported associations between decision confidence and CPP/Pe component amplitudes may have been a consequence of ERP amplitude measurement-related confounds. Re-analysis of existing datasets may be useful to test this hypothesis more directly.

Highlights- We mapped the event-related potential correlates of decision confidence
- A frontal component was associated with confidence during decision formation
- The error positivity component was associated with confidence in error trials
- The error positivity was not associated with confidence in correct response trials


title: The gendered self: Evidence for differences in whole-brain dynamics

abstract:
How the brain constructs gender identity is largely unknown, but some neural differences have recently been discovered. Here, we used an intrinsic-ignition framework to investigate if gender identity changes the propagation of the neural activity across the whole-brain network and within resting-state networks. Studying 29 transmen and 17 transwomen with gender incongruence, 22 ciswomen, and 19 cismen, we computed the capability of a given brain area in space to propagate activity to other areas (mean-ignition) and its variability across time (node-metastability). We found that both measures differentiated all four groups across the whole-brain network. Furthermore, at the network level, we found that compared to the other groups, cismen showed higher mean-ignition of the dorsal attention network and node-metastability of the dorsal and ventral attention, executive control, and temporal parietal networks. We also found mean-ignition differences between cismen and ciswomen within the executive control network, but higher in ciswomen than cismen and transmen for the default-mode network. For the node-metastability, this was higher in cismen compared to ciswomen in the somatomotor network, while both mean-ignition and node-metastability were higher for cismen than transmen in the limbic network. Finally, we computed correlations between both measures and their body image scores. Transmen dissatisfaction, cismen, and ciswomen satisfaction towards their own body image were distinctively associated with specific networks per group. Overall, the study of the whole-brain network dynamical complexity discriminates binary gender identity groups, and functional connectivity dynamics approaches are needed to disentangle the complex understanding of the gendered self.

Significance statementThe study of sex/gender differences may be enriched by the heterogeneity of other gender minority groups, such as transgender. Functional connectivity measures capturing the spatio-temporal oscillations of the brain can provide insights on how the brain cooperates. This is the first study investigating how the whole-brain network propagates information across the brain, spatially and temporally, in binary gender groups (cisgender and transgender) by means of the intrinsic-ignition framework. We found four whole-brain unique phenotypes pertaining to each gender group, namely cismen, ciswomen, transmen and transwomen. Novel functional connectivity dynamics frameworks can contribute to disentangle the complex experience of a gendered-self.


title: Human visual gamma for color stimuli: When LGN drive is equalized, red is not special

abstract: Strong gamma-band oscillations in primate early visual cortex can be induced by spatially homogeneous, high-contrast stimuli, such as color surfaces. Compared to other hues, particularly strong gamma oscillations have been reported for red stimuli. However, precortical color processing and the resultant strength of input to V1 has often not been fully controlled for. This leaves the possibility that stronger responses to some hues were due to differences in V1 input strength. We presented stimuli that had equal luminance and color contrast levels in a color coordinate system based on color responses of the lateral geniculate nucleus, the main input source for area V1. With these stimuli, we recorded magnetoencephalography in 30 human subjects. We found narrowband color-induced gamma oscillations in early visual cortex, which, contrary to previous reports, did not differ between red and green stimuli of equal L-M cone contrast. Notably, blue stimuli with contrast exclusively on the S-cone axis induced very weak gamma responses, as well as smaller event-related fields and poorer change detection performance. The strength of human color gamma responses could be well explained by the strength of thalamic input induced by each hue and does not show a clear red bias when this input strength is properly equalized.


title: Adaptive coding of stimulus information in human fronto-parietal cortex during visual classification

abstract: The neural mechanisms of how frontal and parietal brain regions support flexible adaptation of behavior remain poorly understood. Here, we used functional magnetic resonance imaging (fMRI) and model-based representational similarity analysis (RSA) to investigate frontoparietal representations of stimulus information during visual classification under varying task demands. Based on prior research, we predicted that increasing perceptual task difficulty should lead to more categorical coding of stimulus information, and that exemplar-level stimulus coding would be restricted to posterior, sensory brain regions. Counter to our expectations, however, we found frontoparietal regions encoded exemplar-level stimulus information. Interestingly, the anterior intraparietal sulcus (aIPS) encoded stimuli equally well regardless of perceptual difficulty, and these representations were directly related to choice behavior (proportion of guessing). Overall, these findings reveal unexpected exemplar-level stimulus coding in frontoparietal cortex, and highlight the role of aIPS in supporting adaptive behavior.