title: Dynamic cortico-cortical information transfer regimes during vocalization

abstract: The mammalian frontal and auditory cortices are important for vocal behaviour. Here, using local field potential recordings, we demonstrate for the first time that the timing and spatial pattern of oscillations in the fronto-auditory cortical network of vocalizing bats (Carollia perspicillata) predict the purpose of vocalization: echolocation or communication. Transfer entropy analyses revealed predominantly top-down (frontal-to-auditory cortex) information flow during spontaneous activity and pre-vocal periods. The dynamics of information flow depended on the behavioural role of the vocalization and on the timing relative to vocal onset. Remarkably, we observed the emergence of predominantly bottom-up (auditory-to-frontal cortex) information transfer patterns specific echolocation production, leading to self-directed acoustic feedback. Electrical stimulation of frontal areas selectively enhanced responses to echolocation sounds in auditory cortex. These results reveal unique changes in information flow across sensory and frontal cortices, potentially driven by the purpose of the vocalization in a highly vocal mammalian model.


title: Echolocation reverses information flow in a cortical vocalization network

category: neuroscience


title: Host F-box protein 22 enhances the uptake of Brucella by macrophages and drives a sustained release of pro-inflammatory cytokines through degradation of the anti-inflammatory effector proteins of Brucella.

abstract:
Brucella species are intracellular bacterial pathogens, causing the world-wide zoonotic disease, brucellosis. Brucella invade professional and non-professional phagocytic cells, followed by resisting intracellular killing and establishing a replication permissive niche. Brucella also modulate the innate and adaptive immune responses of the host for their chronic persistence. The complex intracellular cycle of Brucella majorly depends on multiple host factors but limited information is available on host and bacterial proteins that play essential role in the invasion, intracellular replication and modulation of host immune responses. By employing an siRNA screening, we identified a role for the host protein, FBXO22 in Brucella-macrophage interaction. FBXO22 is the key element in the SCF E3 ubiquitination complex where it determines the substrate specificity for ubiquitination and degradation of various host proteins. Downregulation of FBXO22 by siRNA or CRISPR-Cas9 system, resulted diminished uptake of Brucella into macrophages, which was dependent on NF-{kappa}B-mediated regulation of phagocytic receptors. FBXO22 expression was upregulated in Brucella-infected macrophages that resulted induction of phagocytic receptors and enhanced production of pro-inflammatory cytokines through NF-{kappa}B. Furthermore, we found that FBXO22 recruits the effector proteins of Brucella, including the anti-inflammatory proteins, TcpB and OMP25 for degradation through the SCF complex. We did not observe any role for another F-box containing protein of SCF complex, {beta}-TrCP in Brucella-macrophage interaction. Our findings unravel novel functions of FBXO22 in host-pathogen interaction and its contribution to pathogenesis of infectious diseases.

Author SummaryBrucellosis is a major zoonotic disease world-wide that poses a serious veterinary and public health problem in various countries, impacting their economic development. Brucellosis is caused by the species of intracellular bacterial pathogen, Brucella that replicates in professional and non-professional phagocytic cells. Brucella is considered as a stealthy pathogen as it invades/suppresses host defense responses using various virulence strategies. Brucella hijacks many cellular processes for gaining entry into the target cells, followed by establishing a replication permissive niche. However, host proteins that are involved in Brucella-macrophage interaction remains obscure. Here, we identified the host protein, FBXO22 that recruits target proteins to SCF E3 ubiquitination complex for their ubiquitination and degradation. We found that down-regulation and upregulation of FBXO22 decreased and enhanced the uptake of Brucella by macrophages, respectively. Our subsequent studies revealed that Brucella induces the expression of FBXO22 that resulted activation of NF-{kappa}B and the concomitant upregulation of phagocytic receptors that might have contributed to the enhanced uptake of Brucella. The Brucella-induced expression of FBXO22 resulted enhanced production of pro-inflammatory cytokines. We have also found that FBXO22 targets Brucella effectors, including the anti-inflammatory effector proteins for degradation through the SCF complex. Our experimental data reveals that FBXO22 plays an important role in the uptake of microbial pathogens by macrophages and pathogenesis of infectious diseases that is resulting from overt inflammatory responses.

category: microbiology


title: Trans -cinnamaldehyde attenuates Enterococcus faecalis virulence and inhibits biofilm formation

abstract: Enterococcus faecalis as an important nosocomial pathogen is critically implicated in the pathogenesis of endocarditis, urinary tract and surgical wound infections. Its major virulence attributes (biofilm formation, production of proteases and hemolytic toxins) enable it to cause extensive host tissue damage. With the alarming increase in enterococcal resistance to antibiotics, novel therapeutics are required to inhibit E. faecalis biofilm formation and virulence. Trans-cinnamaldehyde (TC), the main phytochemical in cinnamon essential oils has demonstrated promising activity against a wide range of pathogens. Here, we comprehensively investigated the effect of TC on planktonic growth, biofilm formation, proteolytic and hemolytic activities, as well as gene regulation in E. faecalis. Our findings revealed that sub-inhibitory concentrations of TC reduced biofilm formation, biofilm exopolysaccharides as well as its proteolytic and hemolytic activities. Mechanistic studies revealed significant down regulation of the quorum sensing fsr locus and downstream gelE, which are major virulence regulators in E. faecalis. Taken together, our study highlights the potential of TC to inhibit E. faecalis biofilm formation and its virulence.


title: Arginine induced Streptococcus gordonii biofilm detachment using a novel rotating-disc rheometry method

abstract: Oral diseases are one of the most common pathologies affecting human health. These diseases are typically associated with dental plaque-biofilms, through either build-up of the biofilm or dysbiosis of the microbial community. Arginine can disrupt dental plaque-biofilms, and maintain plaque homeostasis, making it an ideal therapeutic to combat the development of oral disease. Despite our understanding of the actions of arginine towards dental plaque-biofilms, it is still unclear how or if arginine effects the mechanical integrity of the dental plaque-biofilm. Here we adapted a rotating-disc rheometry assay, which is routinely used in marine microbial ecology, to study how arginine treatment of Streptococcus gordonii biofilms influences biofilm detachment from surfaces. We demonstrate that the assay is highly sensitive at quantifying the presence of biofilm and the detachment or rearrangement of the biofilm structure as a function of shear stress. We demonstrate that arginine treatment leads to earlier detachment of the biofilm, indicating that arginine treatment weakens the biofilm, making it more suspectable to removal by shear stresses. Our results add to the understanding that arginine targets biofilms by multifaceted mechanisms, both metabolic and physical, further promoting the potential of arginine as an active compound in dentifrices to maintain oral health.


title: Antibody evasion by the Brazilian P.1 strain of SARS-CoV-2

abstract: Terminating the SARS-CoV-2 pandemic relies upon pan-global vaccination. Current vaccines elicit neutralizing antibody responses to the virus spike derived from early isolates. However, new strains have emerged with multiple mutations: P.1 from Brazil, B.1.351 from South Africa and B.1.1.7 from the UK (12, 10 and 9 changes in the spike respectively). All have mutations in the ACE2 binding site with P.1 and B.1.351 having a virtually identical triplet: E484K, K417N/T and N501Y, which we show confer similar increased affinity for ACE2. We show that, surprisingly, P.1 is significantly less resistant to naturally acquired or vaccine induced antibody responses than B.1.351 suggesting that changes outside the RBD impact neutralisation. Monoclonal antibody 222 neutralises all three variants despite interacting with two of the ACE2 binding site mutations, we explain this through structural analysis and use the 222 light chain to largely restore neutralization potency to a major class of public antibodies.


title: High Content and High Throughout Phenotypic Assay for the Hourly Resolution of the Malaria Parasite Erythrocytic Cycle

abstract: Over the last 20 years increased funding for malaria research has resulted in very significant technical advances to study the biology of Plasmodium species. High throughput phenotypic assays have been developed to screen millions of compounds and identify small molecules with antiparasitic activity. At the same time, advances in malaria genetic have greatly facilitated the generation of genetically modified parasites, and whole genome genetic screens are now feasible in Plasmodium species. Finally, there has been an increased interest to study malaria parasites at the population level, in particular in the area of drug resistance. Drug resistant field isolates have been collected around the world, and drug resistant strains are routinely generated in the lab to study the mechanisms of drug resistance. As a result, one of the current bottlenecks in malaria research is our ability to quickly characterize the phenotype associated with compound treatment or genetic modification, or to quickly compare differences in intracellular development between strains. Here, we present a high content/high throughput phenotypic assay that combines highly selective RNA, DNA, and RBC membrane dyes to provide hourly resolution of the full erythrocytic cycle for both P. falciparum and P. knowlesi. A flow cytometry assay allows the analysis of samples in a 384-well format and a quick way to determine the parasite developmental stage. On the other hand, the fluorescence microscopy format allows for a detailed visualization of parasite morphology. Finally, using open source software we have developed protocols for the automated cluster analysis of microscopy images. This assay can be applied to any Plasmodium species, requires very little amount of sample, is performed with fixed cells, and is easily scalable. Overall, we believe this assay will be a great tool for the malaria community to study Plasmodium species.


title: An ecological basis for dual genetic code expansion in marine deltaproteobacteria

abstract: Marine benthic environments may be shaped by anthropogenic and other localized events, leading to changes in microbial community composition evident decades after a disturbance. Marine sediments in particular harbor exceptional taxonomic diversity and can shed light on distinctive evolutionary strategies. Genetic code expansion may increase the structural and functional diversity of proteins in cells, by repurposing stop codons to encode noncanonical amino acids: pyrrolysine (Pyl) and selenocysteine (Sec). Here, we show that the genomes of abundant Deltaproteobacteria from the sediments of a deep-ocean chemical waste dump site, have undergone genetic code expansion. Pyl and Sec in these organisms appear to augment trimethylamine (TMA) and one-carbon metabolism, representing key drivers of their ecology. The inferred metabolism of these sulfate-reducing bacteria places them in competition with methylotrophic methanogens for TMA, a contention further supported by earlier isotope tracer studies and reanalysis of metatranscriptomic studies. A survey of genomic data further reveals a broad geographic distribution of a niche group of similarly specialized Deltaproteobacteria including at sulfidic sites in the Atlantic Ocean, Gulf of Mexico, Guayamas Basin, and North Sea, as well as in terrestrial and estuarine environments. These findings reveal an important biogeochemical role for specialized Deltaproteobacteria at the interface of the carbon, nitrogen and sulfur cycles, with their niche adaptation and ecological success seemingly enabled by genetic code expansion.


title: Primary brain cell infection by Toxoplasma gondii reveals the extent and dynamics of parasite differentiation and impact on neuron biology.

abstract: Toxoplasma gondii is a eukaryotic parasite that forms latent cysts in the brain of immunocompetent individuals. The latent parasite infection of the immune-privileged central nervous system is linked to most complications. With no drug currently available to eliminate the latent cysts in the brain of infected hosts, the consequences of neurons long-term infection are unknown. It has long been known that T. gondii specifically differentiates into a latent form (bradyzoite) in neurons, but how the infected neuron responds to the infection remains to be elucidated. We have established a new in vitro model resulting in the production of mature bradyzoite cysts in brain cells. Using dual, host and parasite RNA-seq, we characterized the dynamics of differentiation of the parasite, revealing the involvement of key pathways in this process. Moreover, we identified how the infected brain cells responded to the parasite infection revealing the drastic changes that take place. We showed that neuronal-specific pathways are strongly affected, with synapse signaling being particularly affected, especially glutamatergic synapse signaling. The establishment of this new in vitro model allows investigating both the dynamics of parasite differentiation and the specific response of neurons to the long-term infection by this parasite.


title: Primary brain cell infection by Toxoplasma gondii reveals the extent and dynamics of parasite differentiation and its impact on neuron biology.


title: Shed the light on virus: virucidal effects of 405 nm visible light on SARS-CoV-2 and influenza A virus.

abstract: Germicidal potential of specific wavelengths within the electromagnetic spectrum is an area of growing interest. While ultra-violet (UV) based technologies have shown satisfactory virucidal potential, the photo-toxicity in humans coupled with UV associated polymer degradation limit its use in occupied spaces. Alternatively, longer wavelengths with less irradiation energy such as visible light (405 nm) have largely been explored in the context of bactericidal and fungicidal applications. Such studies indicated that 405 nm mediated inactivation is caused by the absorbance of porphyrins within the organism creating reactive oxygen species which result in free radical damage to its DNA and disruption of cellular functions. The virucidal potential of visible-light based technologies has been largely unexplored and speculated to be ineffective given the lack of porphyrins in viruses. The current study demonstrated increased susceptibility of lipid-enveloped respiratory pathogens of importance such as SARS-CoV-2 (causative agent of COVID-19) as well as the influenza A virus to 405nm, visible light in the absence of exogenous photosensitizers indicating a potential porphyrin-independent alternative mechanism of visible light mediated viral inactivation. These results were obtained using less than expected irradiance levels which are generally safe for humans and commercially achievable. Our results support further exploration of the use of visible light technology for the application of continuous decontamination in occupied areas within hospitals and/or infectious disease laboratories, specifically for the inactivation of respiratory pathogens such as SARS-CoV-2 and Influenza A.


title: Lighting a better future: the virucidal effects of 405 nm visible light on SARS-CoV-2 and influenza A virus


title: The virucidal effects of 405 nm visible light on SARS-CoV-2 and influenza A virus


title: The PfAP2-HS transcription factor protects malaria parasites from febrile temperatures

abstract: Periodic fever is the most characteristic clinical feature of human malaria1-3, but how parasites survive febrile episodes is not known. While Plasmodium spp. genomes encode a full complement of chaperones4, they lack an ortholog of the conserved transcription factor HSF1, which in most eukaryotes activates the expression of key chaperones upon heat shock (HS)5-8. Here we identified PfAP2-HS, a transcription factor of the ApiAP2 family9-11, as the key regulator of the P. falciparum protective HS response. The PfAP2-HS-dependent HS response is largely restricted to rapid activation of hsp70-1, the predominant direct target of PfAP2-HS, and hsp90. Deletion of PfAP2-HS dramatically reduced HS survival and also resulted in severe growth defects at 37{degrees}C, but not at 35{degrees}C, and increased sensitivity to imbalances in protein homeostasis (proteostasis) produced by artemisinin, the current frontline antimalarial drug12,13. These results demonstrate that PfAP2-HS contributes to general maintenance of proteostasis and drives a rapid chaperone-based protective response against febrile temperatures. While several ApiAP2 transcription factors regulate life cycle transitions in malaria parasites11,14,15, PfAP2-HS is the first identified Plasmodium transcription factor that controls a protective response to a within-host environmental challenge.


title: Cross-species evolutionary rewiring in the enteric bacterium Campylobacter

abstract: The lateral transfer of genes among bacterial strains and species has opposing effects, conferring potentially beneficial adaptations whilst introducing disharmony in coadapted genomes. The prevailing outcome will depend upon the fitness cost of disrupting established epistatic interactions between genes. It is challenging to understand this in nature because it requires population-scale analysis of recombination and genomic coadaptation, and laboratory confirmation of the functional significance of genotype variation. By assigning the ancestry of DNA in the genomes of two species of the worlds most common enteric bacterial pathogen, we show that up to 28% of the Campylobacter coli genome has been recently introgressed from Campylobacter jejuni. Then, by quantifying covariation across the genome we show that >83% of putative epistatic links are between introgressed C. jejuni genes in divergent genomic positions (>20kb apart), consistent with independent acquisition. Much of this covariation is between 16 genes, with just 5 genes accounting for 99% of epistatic SNP pairs. Laboratory mutagenesis and complementation cloning assays demonstrated functional links between these genes, specifically related to formate dehydrogenase (FDH) activity. These findings suggest that the genetic confederations that define genomic species may be transient. Even for complex traits such as central metabolism in the bacterial cell, conditions can arise where epistatic genes can be decoupled, transferred and reinstated in a new genetic background.


title: Vtc5 is localized to the vacuole membrane by the conserved AP-3 complex to regulate polyphosphate synthesis in budding yeast

abstract:
Polyphosphates (polyP) are energy-rich polymers of inorganic phosphates assembled into chains ranging from 3-1000s of residues in length. They are thought to exist in all cells on earth and play roles in an eclectic mix of functions ranging from phosphate homeostasis to cell signaling, infection control, and blood clotting. In the budding yeast Saccharomyces cerevisiae, polyP chains are synthesized by the vacuole-bound VTC complex, which synthesizes polyP while simultaneously translocating it into the vacuole lumen where it is stored at high concentrations. VTCs activity is promoted by an accessory subunit called Vtc5. In this work, we find that the conserved AP-3 complex is required for proper Vtc5 localization to the vacuole membrane. In human cells, previous work has demonstrated that mutation of AP-3 subunits gives rise to Hermansky-Pudlak Syndrome, a rare disease with molecular phenotypes that include decreased polyP accumulation in platelet dense granules. In yeast AP-3 mutants, we find that Vtc5 is rerouted to the vacuole lumen by the ESCRT complex, where it is degraded by the vacuolar protease Pep4. Cells lacking functional AP-3 have decreased levels of polyP, demonstrating that membrane localization of Vtc5 is required for its VTC stimulatory activity in vivo. Our work provides insight into the molecular trafficking of a critical regulator of polyP metabolism in yeast. We speculate that AP-3 may also be responsible for the delivery of polyP regulatory proteins to platelet dense granules in higher eukaryotes.

HIGHLIGHTSVtc5 localization to the vacuole membrane depends on the AP-3 complex

The ESCRT pathway brings mislocalized Vtc5 to the vacuole lumen where it is degraded

Decreased polyP levels in AP-3 mutants are explained by Vtc5 mislocalization

Deletion of DOA4 restores wild-type localization of Vtc5 without restoring polyP levels

category: molecular biology


title: A Bioinformatic and Empiric Exploration of Prokaryotic Argonautes as Novel Programmable Endonuclease Systems

abstract: Argonautes are nucleases that can be programmed by short oligonucleotides to cleave complementary sequences. Here, we performed an unbiased bioinformatic search to mine bacterial genomes for prokaryotic Argonautes (pAgos) harboring a PIWI domain. Our search identified 3,033 pAgos in total, of which 1,464 portend to the subgroup of long pAgos with more than 600 amino acids. We purified a subset of 49 pAgos which were found in proximity to helicases and tested their nuclease activity in vitro. Ten of these were active towards single-stranded DNA substrates and this activity could be programmed by exogenous guide DNAs or RNAs. Cleavage of double-stranded plasmid DNA was much less readily observed and was fostered by elevated temperatures or exogenous addition of a DNA single-strand binding protein (ET-SSB). The efficiency of pAgo-mediated plasmid cleavage was dependent on the DNA target sequence as well as the surrounding sequence, suggesting that unwinding of the DNA double helix was a limiting factor. Intriguingly, we identified a cluster of pAgos from the Clostridial clade which was active at 37{degrees}C and activity was enhanced by exogenous ET-SSB. This suggests that Clostridial pAgos may be particularly suited to catalyze DNA double-strand cleavage and implies that such pAgos may be repurposed as gene editing tools in future.


title: Implications of cholesterol and sphingomyelin in STING phosphorylation by TBK1

abstract: Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA from virus or self-DNA from mitochondria/nuclei. In response to emergence of such DNAs in the cytosol, STING translocates from the endoplasmic reticulum (ER) to the Golgi, and activates TANK-binding kinase 1 (TBK1) at the trans-Golgi network (TGN). Activated TBK1 then phosphorylates STING at Ser365, generating an interferon regulatory factor 3 (IRF3)-docking site on STING. How this reaction proceeds specifically at the TGN remains poorly understood. Here we report a cell-free reaction in which endogenous STING is phosphorylated by TBK1. The reaction utilizes microsomal membrane fraction prepared from TBK1-knockout (KO) cells and recombinant TBK1. We observed agonist-, TBK1-, "ER-to-Golgi" traffic-, and palmitoylation-dependent phosphorylation of STING at Ser365, mirroring the nature of STING phosphorylation in vivo. Treating the microsomal membrane fraction with sphingomyelinase or methyl-{beta}-cyclodextrin, an agent to extract cholesterol from membranes, suppressed the phosphorylation of STING by TBK1. Given the enrichment of sphingomyelin and cholesterol in the TGN, these results may provide the molecular basis underlying the specific phosphorylation reaction of STING at the TGN.

category: cell biology


title: Cytoplasmic forces functionally reorganize nuclear condensates in oocytes

abstract:
Cells remodel their cytoplasm with force-generating cytoskeletal motors1. Their activity generates random forces that stir the cytoplasm, agitating and displacing membrane-bound organelles like the nucleus in somatic2-4 and germ5-7 cells. These forces are transmitted inside the nucleus4,7, yet their consequences on liquid-like biomolecular condensates8-10 residing in the nucleus remain unexplored. Here, we probe experimentally and computationally diverse nuclear condensates, that include splicing speckles, Cajal bodies, and nucleoli, during cytoplasmic remodeling of female germ cells named oocytes. We discover that growing mammalian oocytes deploy cytoplasmic forces to timely impose multiscale reorganization of condensates inside the nucleus. We determine that cytoplasmic forces accelerate nuclear condensate collision-coalescence and molecular kinetics within condensates. Inversely, disrupting the forces decelerates nuclear condensate reorganization on both scales. We link the molecular deceleration found in mRNA-processing splicing speckles to reduced and altered splicing of mRNA, which in oocytes impedes fertility11. We establish that different sources of cytoplasmic forces can reorganize nuclear condensates and that this cytoplasmic aptitude for subnuclear reorganization is evolutionary conserved in insects. Our work implies that cells evolved a mechanism, based on cytoplasmic force tuning, to functionally regulate a broad range of nuclear condensates across scales. This finding opens new perspectives when studying condensate-associated pathologies like cancer, neurodegeneration and viral infections12.

One sentence summaryCytoplasmic random forces in growing oocytes drive multiscale reorganization of nuclear liquid-like biomolecular condensates.


title: DNA methylation clocks show slower progression of aging in naked mole-rat queens

abstract: Naked mole-rats (NMRs) live an exceptionally long life, appear not to exhibit age-related decline in physiological capacity, and are seemingly resistant to age-related diseases. However, it has been unknown whether NMRs also evade aging according to a primary hallmark of aging: epigenetic changes. To address this question, we generated DNA methylation profiles from 329 tissues from animals of known age, at loci that are highly conserved between mammalian species, using a custom Infinium array (HorvathMammalMethylChip40). We observed strong aging effects on NMR DNA methylation, from which we developed seven highly accurate age estimators (epigenetic clocks) for several tissues (pan-tissue, blood, kidney clock, liver clock, skin clock) and two dual species (human-NMR) clocks. By identifying age-related cytosine methylation that are shared between NMR and humans, but not with the mouse, we identified genes and cellular pathways that impinge on developmental and metabolic processes that are potentially involved in NMR and human longevity. The NMR epigenetic clocks revealed that breeding NMR queens age more slowly than non-breeders, a feature that is also observed in some eusocial insects. CpGs associated with queen status were located near developmental genes and those that are regulated by the LHX3 transcription factor that controls pituitary development. In summary, our study demonstrates that despite a phenotype of reduced senescence, the NMR ages epigenetically through developmental and metabolic processes, and that NMR queens age more slowly than non-breeders.

category: developmental biology


title: Epiblast morphogenesis is controlled by selective mRNA decay triggered by LIN28A relocation

abstract: The embryonic progression from naive to primed pluripotency is accompanied by the rapid decay of pluripotency-associated mRNAs and a concomitant radical morphogenetic sequence of epiblast polarization, rosette formation and lumenogenesis. The mechanisms triggering and linking these events remain poorly understood. Guided by machine learning and metabolic RNA sequencing, we identified RNA binding proteins (RBPs), especially LIN28A, as primary mRNA decay factors. Using mRNA-RBP interactome capture, we revealed a dramatic increase in LIN28A mRNA binding during the naive-rosette-primed pluripotency transition, driven by its nucleolar-to-cytoplasmic translocation. Cytoplasmic LIN28A binds to 3UTRs of pluripotency-associated mRNAs to directly stimulate their decay and drive lumenogenesis. Accordingly, forced nuclear retention of LIN28A impeded lumenogenesis, impaired gastrulation, and caused an unforeseen embryonic multiplication. Selective mRNA decay, driven by nucleo-cytoplasmic RBP translocation, therefore acts as an intrinsic mechanism linking cell identity switches to the control of embryonic growth and morphogenesis.


title: Modeling the bistable transition between cell phenotypes during limb precartilage condensation

abstract: The tetrapod limb skeleton is initiated in unpatterned limb bud mesenchyme by the formation of precartilage condensations. Here, based on time-lapse videographic analysis of a forming condensation in a high-density culture of chicken limb bud mesenchyme, we observe a phase transition to a more fluidized state for cells within spatial compacted foci (protocondensations that will progress to condensations), as reflected in their spatial confinement, cell-substratum interaction and speed of motion. Previous work showed that galectin-8 and galectin-1A, two proteins of the galactoside-binding galectin family, are the earliest determinants of this process in the chicken limb bud, and that their interactions in forming skeletogenic patterns of condensations can be interpreted mathematically through a reaction-diffusion-adhesion framework. Based on this framework, we use an ordinary differential equation-based approach to analyze the core switching modality of the galectin reaction network and characterize the states of the network independent of the diffusive and adhesive arms of the patterning mechanism. We identify two steady states where the concentrations of both galectins are respectively, negligible, and very high. An explicit Lyapunov function shows that there are no periodic solutions. For sigmoidal galectin production terms, the model exhibits a bistable switch that arises from a monostable state via saddle-node bifurcation. Our model therefore predicts that the galectin network exists in low and high expression states separated in space or time without any intermediate states. This provides a causal basis for the observed outside vs. inside transition observed in the in vitro video data. We performed a quantitative analysis of the distribution of galectin-1A in cultures of condensing chick limb mesenchymal cells and found that the interior of the protocondensations had concentrations of this protein (compared to the immediate exterior) over and above that expected from its higher cell density, consistent with the models predictions. The galectin-based patterning network is thus suggested, on theoretical grounds, to incorporate a core switch independent of any spatial or temporal dynamics, that drives the chondrogenic cell state transition in limb skeletogenesis.


title: Precartilage condensation during limb skeletogenesis occurs by tissue phase separation controlled by a bistable cell-state switch with suppressed oscillatory dynamics


title: Traction forces exerted by the developing optic cup mediate cell movements and axon extension in the olfactory placode

abstract: While the chemical signals guiding neuronal migration and axon elongation have been extensively studied, the influence of mechanical cues on these processes remains poorly studied in vivo. Here, we investigate how mechanical forces exerted by surrounding tissues steer neuronal movements and axon extension during the morphogenesis of the olfactory placode in zebrafish. We mainly focus on the mechanical contribution of the adjacent eye tissue, which develops underneath the placode through extensive evagination and invagination movements. Using quantitative analysis of cell movements and biomechanical manipulations, we show that the developing eye exerts lateral traction forces on the olfactory placode through extracellular matrix, mediating proper morphogenetic movements and axon extension within the placode. Our data shed new light on the key participation of intertissue mechanical interactions in the sculpting of neuronal circuits.


title: Intertissue mechanical interactions shape the olfactory circuit in zebrafish


title: Evaluation of substrate composition and exogenous hormone application on vegetative propagule rooting success of essential oil hemp (Cannabis sativa L.)

abstract: To support the rapidly expanding industrial hemp industry, a commercial supply of high-quality starter plants with low genetic variability from nurseries will be key to consistent and efficient cultivation efforts. Rooting success was evaluated across four propagation medias, five rooting hormones, and eight commercially available high-cannabidiol (CBD) essential oil hemp cultivars. Cuttings were placed in a climate-controlled room and assessed for rooting success 12 days after cloning. Rooting success was determined by quantifying total root number, cumulative total root length, and total root mass. Propagation media had the greatest effect on rooting success (13-80 %). Rockwool had the highest rooting success resulting in 10-fold increases in rooting traits over the next highest scoring medium (Berger BM6). Hormone applications significantly improved (15-to 18-fold) rooting success compared to no hormone application, while non-statistical differences were observed across auxin hormone concentrations and application methods. Genetic variation in rooting response was observed between cultivars with  Cherry Wine outperforming all other cultivars with an approximate 20% increase in rooting success over the next highest rooting cultivar,  Wife. Although the ideal combination was not specifically identified in this study, findings provide insight into how rooting hormone application and medium selection impact vegetative propagule rooting success of essential oil hemp.

category: plant biology


title: Computational Design and Analysis of Modular Cells for Large Libraries of Exchangeable Product Synthesis Modules

abstract: Microbial metabolism can be harnessed to produce a large library of useful chemicals from renewable resources such as plant biomass. However, it is laborious and expensive to create microbial biocatalysts to produce each new product. To tackle this challenge, we have recently developed modular cell (ModCell) design principles that enable rapid generation of production strains by assembling a modular (chassis) cell with exchangeable production modules to achieve overproduction of target molecules. Previous computational ModCell design methods are limited to analyze small libraries of around 20 products. In this study, we developed a new computational method, named ModCell-HPC, capable of designing modular cells for large libraries with hundredths of products with a highly-parallel and multi-objective evolutionary algorithm. We demonstrated ModCell-HPC to design Escherichia coli modular cells towards a library of 161 endogenous production modules. From these simulations, we identified E. coli modular cells with few genetic manipulations that can produce dozens of molecules in a growth-coupled manner under different carbons sources. These designs revealed key genetic manipulations at the chassis and module levels to accomplish versatile modular cells. Furthermore, we used ModCell-HPC to identify design features that allow an existing modular cell to be re-purposed towards production of new molecules. Overall, ModCell-HPC is a useful tool towards more efficient and generalizable design of modular cells to help reduce research and development cost in biocatalysis.

category: synthetic biology


title: A chemogenetic platform for controlling plasma membrane signaling and synthetic signal oscillation

abstract: Chemogenetic methods that enable the rapid translocation of specific signaling proteins in living cells using small molecules are powerful tools for manipulating and interrogating intracellular signaling networks. However, existing techniques rely on chemically induced dimerization of two protein components and have certain limitations, such as a lack of reversibility, bioorthogonality, and usability. Here, by expanding our self-localizing ligand-induced protein translocation (SLIPT) approach, we have developed a versatile chemogenetic system for plasma membrane (PM)-targeted protein translocation. In this system, a novel engineered Escherichia coli dihydrofolate reductase in which a hexalysine (K6) sequence is inserted in a loop region (iK6DHFR) is used as a universal protein tag for PM-targeted SLIPT. Proteins of interest that are fused to the iK6DHFR tag can be specifically recruited from the cytoplasm to the PM within minutes by addition of a myristoyl-O_SCPLOWDC_SCPLOW-Cys-tethered trimethoprim ligand (mDcTMP). We demonstrated the broad applicability and robustness of this engineered protein-synthetic ligand pair as a tool for the conditional activation of various types of signaling molecules, including protein and lipid kinases, small GTPases, heterotrimeric G proteins, and second messengers. In combination with a competitor ligand and a culture-medium flow chamber, we further demonstrated the application of the system for chemically manipulating protein localization in a reversible and repeatable manner to generate synthetic signal oscillations in living cells. The present bioorthogonal iK6DHFR/mDcTMP-based SLIPT system affords rapid, reversible, and repeatable control of the PM recruitment of target proteins, offering a versatile and easy-to-use chemogenetic platform for chemical and synthetic biology applications.


title: Encoding innate ability through a genomic bottleneck

abstract: AO_SCPLOWBSTRACTC_SCPLOWAnimals are born with extensive innate behavioral capabilities, which arise from neural circuits encoded in the genome. However, the information capacity of the genome is orders of magnitude smaller than that needed to specify the connectivity of an arbitrary brain circuit, indicating that the rules encoding circuit formation must fit through a "genomic bottleneck" as they pass from one generation to the next. Here we formulate the problem of innate behavioral capacity in the context of artificial neural networks in terms of lossy compression of the weight matrix. We find that several standard network architectures can be compressed by several orders of magnitude, yielding pre-training performance that can approach that of the fully-trained network. Interestingly, for complex but not for simple test problems, the genomic bottleneck algorithm also captures essential features of the circuit, leading to enhanced transfer learning to novel data sets. Our results suggest that compressing a neural circuit through the genomic bottleneck serves as a regularizer, enabling evolution to select simple circuits that can be readily adapted to important real-world tasks. The genomic bottleneck also suggests how innate priors can complement conventional approaches to learning in designing algorithms for artificial intelligence.


title: A model of Ponto-Geniculo-Occipital waves supports bidirectional control of cortical plasticity across sleep-stages

abstract:
During sleep, cortical network connectivity likely undergoes both synaptic potentiation and depression through system consolidation and homeostatic processes. However, how these modifications are coordinated across sleep stages remains largely unknown. Candidate mechanisms are Ponto-Geniculo-Occipital (PGO) waves, propagating across several structures during Rapid Eye Movement (REM) sleep and the transitional stage from non-REM sleep to REM sleep (pre-REM), and exhibiting sleep stage-specific dynamic patterns. To understand their impact on cortical plasticity, we built an acetylcholine-modulated neural mass model of PGO wave propagation through pons, thalamus and cortex, reproducing a broad range of electrophysiological characteristics across sleep stages. Using a population model of Spike-Time-Dependent Plasticity, we show that cortical circuits undergo different transient regimes depending on the sleep stage, with different impacts on plasticity. Specifically, PGO-induced recurrent cortical activities lead to the potentiation of cortico-cortical synapses during pre-REM, and to their depression during REM sleep. Overall, these results shed light on how the dynamical properties of sleep events propagating to cortical circuits can favor different types of local plastic changes. The variety of events occurring across sleep stages may thus be instrumental in controlling the reorganization of cortical networks from one day to the next.

Significance statementConsiderable evidence supports rescaling of cortical synaptic connec-tions during sleep, requiring both long term potentiation to consolidate newly acquired memories, and long-term depression to maintain homeostatic levels of brain activity. However, which aspects of sleep activity contribute to this bidirectional control of plasticity remains unclear. This computational modeling study suggests that widespread transient phenomena called Ponto-geniculo-occipital (PGO) waves, have a sleep-stage dependent effect on plasticity. The alternation between sleep stages can thus be exploited in combination with spontaneously occurring transients, to trigger both up- and down-regulating effects on cortical connectivity, and may explain why the basic structure of sleep-cycles is a well-preserved property across mammalian species.


title: Morphological principles of neuronal mitochondria

abstract: In the highly dynamic metabolic landscape of a neuron, mitochondrial membrane architectures can provide critical insight into the unique energy balance of the cell. Current theoretical calculations of functional outputs like ATP and heat often represent mitochondria as idealized geometries and therefore can miscalculate the metabolic fluxes. To analyze mitochondrial morphology in neurons of mouse cerebellum neuropil, 3D tracings of complete synaptic and axonal mitochondria were constructed using a database of serial TEM tomography images and converted to watertight meshes with minimal distortion of the original microscopy volumes with a granularity of 1.6 nanometer isotropic voxels. The resulting in silico representations were subsequently quantified by differential geometry methods in terms of the mean and Gaussian curvatures, surface areas, volumes, and membrane motifs, all of which can alter the metabolic output of the organelle. Finally, we identify structural motifs that are present across this population of mitochondria; observations which may contribute to future modeling studies of mitochondrial physiology and metabolism in neurons.


title: Alignment and Integration of Spatial Transcriptomics Data

abstract:
Spatial transcriptomics (ST) is a new technology that measures mRNA expression across thousands of spots on a tissue slice, while preserving information about the spatial location of spots. ST is typically applied to several replicates from adjacent slices of a tissue. However, existing methods to analyze ST data do not take full advantage of the similarity in both gene expression and spatial organization across these replicates. We introduce a new method PASTE (Probabilistic Alignment of ST Experiments) to align and integrate ST data across adjacent tissue slices leveraging both transcriptional similarity and spatial distances between spots. First, we formalize and solve the problem of pairwise alignment of ST data from adjacent tissue slices, or layers, using Fused Gromov-Wasserstein Optimal Transport (FGW-OT), which accounts for variability in the composition and spatial location of the spots on each layer. From these pairwise alignments, we construct a 3D representation of the tissue. Next, we introduce the problem of simultaneous alignment and integration of multiple ST layers into a single layer with a low rank gene expression matrix. We derive an algorithm to solve the problem by alternating between solving FGW-OT instances and solving a Non-negative Matrix Factorization (NMF) of a weighted expression matrix. We show on both simulated and real ST datasets that PASTE accurately aligns spots across adjacent layers and accurately estimates a consensus expression matrix from multiple ST layers. PASTE outperforms integration methods that rely solely on either transcriptional similarity or spatial similarity, demonstrating the advantages of combining both types of information.

Code availabilitySoftware is available at https://github.com/raphael-group/paste

category: bioinformatics


title: Arpin regulates migration persistence by interacting with both tankyrases and the Arp2/3 complex

abstract: During cell migration, protrusion of the leading edge is driven by the polymerization of Arp2/3-dependent branched actin networks. Migration persistence is negatively regulated by the Arp2/3 inhibitory protein Arpin. To better understand Arpin regulation in the cell, we looked for interacting partners and identified both Tankyrase 1 and 2 (TNKS) using a yeast two hybrid screen and co-immunoprecipitation with full-length Arpin as a bait. Arpin interacts with ankyrin repeats of TNKS through a C-terminal binding site on its acidic tail overlapping with the Arp2/3 binding site. To uncouple the interactions of Arpin with TNKS and Arp2/3, we introduced point mutations in the Arpin tail and attempted to rescue the increased persistence of the Arpin knock-out using random plasmid integration or compensating knock-in at the ARPIN locus. Arpin mutations impairing either Arp2/3- or TNKS-interaction were insufficient to fully abolish Arpin activity. Only the mutation that affects both interactions rendered Arpin completely inactive, suggesting the existence of two independent pathways, by which Arpin controls migration persistence. Arpin was found to dissolve liquid-liquid phase separation of TNKS upon overexpression. Together these data suggest that TNKS might be mediating the function of Arpin rather than regulating Arpin.


title: Stiffness Regulates Intestinal Stem Cell Fate

abstract: Does fibrotic gut stiffening caused by inflammatory bowel diseases (IBD) direct the fate of intestinal stem cells (ISCs)? To address this question we first developed a novel long-term culture of quasi-3D gut organoids plated on hydrogel matrix of varying stiffness. Stiffening from 0.6kPa to 9.6kPa significantly reduces Lgr5high ISCs and Ki67+ progenitor cells while promoting their differentiation towards goblet cells. These stiffness-driven events are attributable to YAP nuclear translocation. Matrix stiffening also extends the expression of the stemness marker Olfactomedin 4 (Olfm4) into villus-like regions, mediated by cytoplasmic YAP. We next used single-cell RNA sequencing to generate for the first time the stiffness-regulated transcriptional signatures of ISCs and their differentiated counterparts. These signatures confirm the impact of stiffening on ISC fate and additionally suggest a stiffening-induced switch in metabolic phenotype, from oxidative phosphorylation to glycolysis. Finally, we used colon samples from IBD patients as well as chronic colitis murine models to confirm the in vivo stiffening-induced epithelial deterioration similar to that observed in vitro. Together, these results demonstrate stiffness-dependent ISC reprograming wherein YAP nuclear translocation diminishes ISCs and Ki67+ progenitors and drives their differentiation towards goblet cells, suggesting stiffening as potential target to mitigate gut epithelial deterioration during IBD.


title: VASP mediated actin dynamics activate and recruit a filopodia myosin

abstract: Filopodia are thin, actin-based structures that cells use to interact with their environments. Filopodia initiation requires a suite of conserved proteins but the mechanism remains poorly understood. The actin polymerase VASP and a MyTH-FERM (MF) myosin, DdMyo7 in amoeba, are essential for filopodia initiation. DdMyo7 is localized to dynamic regions of the actin-rich cortex. Analysis of VASP mutants and treatment of cells with anti-actin drugs shows that myosin recruitment and activation in Dictyostelium requires localized VASP-dependent actin polymerization. Targeting of DdMyo7 to the cortex alone is not sufficient for filopodia initiation; VASP activity is also required. The actin regulator locally produces a cortical actin network that activates myosin and together they shape the actin network to promote extension of parallel bundles of actin during filopodia formation. This work reveals how filopodia initiation requires close collaboration between an actin binding protein, the state of the actin cytoskeleton and MF myosin activity.


title: Inducible expression of Oct-3/4 reveals synergy with Klf4 in targeting Cyclin A2 to enhance proliferation during early reprogramming

abstract: During reprogramming of somatic cells, heightened proliferation is one of the earliest changes observed. While other early events such as mesenchymal-to-epithelial transition have been well studied, the mechanisms by which the cell cycle switches from a slow cycling state to a faster cycling state are still incompletely understood. To investigate the role of Oct-3/4 in this early feature of reprogramming, we created a 4-Hydroxytamoxifen dependent Oct-3/4 Estrogen Receptor fusion (OctER). We show that OctER can substitute for Oct-3/4 to reprogram mouse embryonic fibroblasts to induced pluripotent stem cells. While over-expression of OctER or Klf4 individually did not affect cell proliferation, in combination, these factors hasten the cell cycle, in a tamoxifen dose-dependent manner, supporting a key role for OctER. Oct-3/4 + Klf4 increased proliferation by enhancing expression of Cyclin A2. We verified occupancy of endogenous Oct-3/4 and Klf4 at bioinformatically identified binding sites in the Cyclin A2 promoter in mouse embryonic stem cells (mESC). Using inducible OctER along with Klf4, we show dose-dependent induction of Cyclin A2 promoter-reporter activity and mRNA levels. Taken together, our results provide further evidence of the interdependence of pluripotency and the rapid cell cycle seen in mESC, and identify CyclinA2 as a key early target.


title: Inhibition of the ubiquitin-proteasome system by a bioactivatable compound

abstract: Malignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a general accumulation of ubiquitylated proteins and caspase-dependent cell death. CBK77 caused accumulation of ubiquitin-dependent, but not ubiquitin-independent, reporter substrates of the UPS, suggesting a selective effect on ubiquitin-dependent proteolysis. In a genome-wide CRISPR interference screen, we identified the redox enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a critical mediator of CBK77 activity, and further demonstrated its role as the compound bioactivator. Through affinity-based proteomics, we found that CBK77 covalently interacts with ubiquitin. In vitro experiments showed that CBK77-treated ubiquitin conjugates were less susceptible to disassembly by deubiquitylating enzymes. In vivo efficacy of CBK77 was validated by reduced growth of NQO1-proficient human adenocarcinoma cells in nude mice treated with CBK77. This first-in-class NQO1-activatable UPS inhibitor suggests that it may be possible to exploit the intracellular environment in malignant cells for leveraging the impact of compounds that impair the UPS.


title: A high throughput SEC23 functional interaction screen reveals a role for focal adhesion and extracellular matrix signalling in the regulation of COPII subunit SEC23A

abstract: Proteins that enter the secretory pathway are transported from their place of synthesis in the Endoplasmic Reticulum to the Golgi complex by COPII coated carriers. The components of the COPII transport machinery have been well characterized, but the network of proteins that regulate these components in response to extracellular cues have remained largely elusive. The discovery of such regulatory proteins is crucial for understanding how cells integrate extracellular and intracellular information to fine-tune membrane traffic in the secretory pathway. A key group of proteins that plays a central role in the regulation of membrane traffic is associated with the cytoskeleton. Using high throughput microscopy of the well-established VSVG transport from the ER to the plasma membrane, we comprehensively screened 378 cytoskeleton-associated proteins for their functional interaction with the COPII components SEC23A and SEC23B using a double knockdown approach. Ranking of the transport effectors identified 20 cytoskeleton-associated proteins as the strongest functional interactors of SEC23, most of them not previously associated with the secretory pathway. Knockdown of a subgroup of these interactors (FERMT2, MACF1, MAPK8IP2, NGEF, PIK3CA, ROCK1), associated with cell adhesion, not only induced changes in focal adhesions and the expression of adhesion-related genes but led to the specific downregulation of SEC23A, a SEC23 paralogue that has been implicated in the secretion of extracellular matrix (ECM) components. Furthermore, SEC23A downregulation could also be recapitulated by plating cells on ECM and was dependent on focal adhesions function. Altogether, our results identify a network of cytoskeleton-associated proteins connecting focal adhesions and ECM-related signalling with the gene expression of the COPII secretory machinery.


title: Size and shape regional differentiation during the development of the spine in the nine-banded armadillo (Dasypus novemcinctus)

abstract: Xenarthrans (armadillos, anteaters, sloths and their extinct relatives) are unique among mammals in displaying a distinctive specialization of the posterior trunk vertebrae - supernumerary vertebral xenarthrous articulations. This study seeks to understand how xenarthry develops through ontogeny and if it may be constrained to appear within pre-existing vertebral regions. Using 3D geometric morphometrics on the neural arches of vertebrae, we explore phenotypic, allometric, and disparity patterns of the different axial morphotypes during ontogeny of nine-banded armadillos. Shape-based regionalisation analyses showed that adult thoracolumbar column is divided into three regions according to the presence or absence of ribs and the presence or absence of xenarthrous articulations. A three-region-division was retrieved in almost all specimens through development, although younger stages (e.g. foetuses, neonates) have more region boundary variability. In size-based regionalisation analyses, thoracolumbar vertebrae are separated into two regions: a pre-diaphragmatic, pre-xenarthrous region, and a post-diaphragmatic xenarthrous region. We show that posterior thoracic vertebrae grow at a slower rate, while anterior thoracics and lumbar grow at a faster rate relatively, with rates decreasing anteroposterioly in the former and increasing anteroposterioly in the latter. We propose that different proportions between vertebrae and vertebral regions might result from differences in growth pattern and timing of ossification.


title: Persistent ventricle partitioning in the adult zebrafish heart

abstract: The vertebrate heart integrates cells from the early-differentiating first heart field (FHF) and the later-differentiating second heart field (SHF) emerging from the lateral plate mesoderm. In mammals, this process forms the basis for the development of the left and right ventricle chambers and subsequent chamber septation. The single ventricle-forming zebrafish heart also integrates FHF and SHF lineages during embryogenesis, yet the contributions of these two myocardial lineages to the adult zebrafish heart remain incompletely understood. Here, we characterize the myocardial labeling of FHF descendants in both the developing and adult zebrafish ventricle. Expanding previous findings, late gastrulation-stage labeling using drl-driven CreERT2 recombinase with a myocardium-specific, myl7-controlled loxP reporter results in predominant labeling of FHF-derived outer curvature and the right side of the embryonic ventricle. Raised to adulthood, such lineage-labeled hearts retain broad areas of FHF cardiomyocytes in a region of the ventricle that is positioned at the opposite side to the atrium and encompasses the apex. Our data add to the increasing evidence for a persisting cell-based compartmentalization of the adult zebrafish ventricle even in the absence of any physical boundary.


title: Fmrp regulates oligodendrocyte lineage cell specification and differentiation

abstract: Neurodevelopment requires the precise integration of a wide variety of neuronal and glial cell types. During early embryonic development, motor neurons and then oligodendrocyte precursor cells (OPCs) are specified from neural progenitors residing in the periventricular pMN progenitor domain of the spinal cord. Following gliogenesis, OPCs can differentiate as oligodendrocytes (OLs) - the myelinating glial cells of the central nervous system - or remain as OPCs. To generate unique cell types capable of highly divergent functions, these specification and differentiation events require specialized gene expression programs. RNA binding proteins (RBPs) regulate mRNA localization and translation in the developing nervous system and are linked to many neurodevelopmental disorders. One example is Fragile X syndrome (FXS), caused by the loss of the RBP fragile X mental retardation protein (FMRP). Importantly, infants with FXS have reduced white matter and we previously showed that zebrafish Fmrp is autonomously required in OLs to promote myelin sheath growth. We now find that Fmrp regulates cell specification in pMN progenitor cells and subsequently promotes differentiation of OPCs, such that fmr1 mutant zebrafish embryos generate excess OPCs and fewer differentiating OLs in the developing spinal cord. Although the early patterning of spinal progenitor domains appears largely normal in fmr1 mutants during early embryogenesis, Shh signaling is greatly diminished. Taken together, these results suggest cell stage-specific requirements for Fmrp in the specification and differentiation of oligodendrocyte lineage cells.


title: Ecological correlates of gene family size: the draft genome of the redheaded pine sawfly Neodiprion lecontei

abstract: A central goal in evolutionary biology is to determine the predictability of adaptive genetic changes. Despite many documented cases of convergent evolution at individual loci, little is known about the repeatability of gene family expansions and contractions. To address this void, we examined gene family evolution in the redheaded pine sawfly Neodiprion lecontei, a non-eusocial hymenopteran and exemplar of a pine-specialized lineage evolved from angiosperm-feeding ancestors. After assembling and annotating a draft genome, we manually annotated multiple gene families with chemosensory, detoxification, or immunity functions and characterized their genomic distributions and evolutionary history. Our results suggest that expansions of bitter gustatory receptor (GR), clan 3 cytochrome P450 (CYP3), and antimicrobial peptide (AMP) subfamilies may have contributed to pine adaptation. By contrast, there was no evidence of recent gene family contraction via pseudogenization. Next, we compared the number of genes in these same families across insect taxa that vary in diet, dietary specialization, and social behavior. In Hymenoptera, herbivory was associated with small GR and olfactory receptor (OR) families, eusociality was associated with large OR and small AMP families, and--unlike investigations in more closely related taxa--ecological specialization was not related to gene family size. Overall, our results suggest that gene families that mediate ecological interactions may expand and contract predictably in response to particular selection pressures, however, the ecological drivers and temporal pace of gene gain and loss likely varies considerably across gene families.

category: evolutionary biology


title: Ecological correlates of gene family size in a pine-feeding sawfly genome and across Hymenoptera


title: OCD.py - Characterizing immunoglobulin inter-domain orientations

abstract:
SummaryInter-domain orientations between immunoglobulin domains are important for the modeling and engineering of novel antibody therapeutics. Previous tools to describe these orientations are applicable only to the variable domains of antibodies and T-cell receptors. We present the "Orientation of Cylindrical Domains (OCD)" tool, which employs a transferable approach to calculate inter-domain orientations for all immunoglobulin domains. Based on a reference structure, the OCD tool automatically builds a suitable reference coordinate system for each domain. Through alignment, the reference coordinate systems are transferred onto the sample to calculate six measures which fully characterize the inter-domain orientation.

Availability and implementationThe OCD approach is implemented as a stand-alone Python script, OCD.py, which can handle multiple types of data input for the analysis of single structures and molecular dynamics trajectories alike. OCD.py is available at https://github.com/liedllab/OCD under MIT license.

Supplementary InformationSupplementary information and data are available at Bioinformatics online.


title: Using machine learning and big data to explore the drug resistance landscape in HIV

abstract: The authors have withdrawn this manuscript due to a duplicate posting of manuscript number 435621. Therefore, the author does not wish this work to be cited as reference for the project. Please cite and refer to the preprint 435621 (DOI: https://doi.org/10.1101/2021.03.16.435621). If you have any questions, please contact the corresponding authors.


title: Indication-specific tumor evolution and its impact on neoantigen targeting and biomarkers for individualized cancer immunotherapies

abstract: Individualized neoantigen specific immunotherapy (iNeST) requires robustly expressed clonal neoantigens for efficacy, but tumor mutational heterogeneity, loss of neoantigen expression, and variable tissue sampling present challenges. To characterize these potential obstacles, we combined multi-region sequencing (MR-seq) analysis of five untreated, synchronously sampled metastatic solid tumors with re-analysis of published MR-seq data from 103 patients. Branching evolution in colorectal cancer and renal cell carcinoma led to fewer clonal neoantigens and to clade-specific neoantigens (those shared across a subset of tumor regions but not fully clonal), with the latter not being readily distinguishable in single tumor samples. Prioritizing mutations with higher purity- and ploidy-adjusted variant allele frequency enriched for globally clonal neoantigens (those found in all tumor regions), whereas estimated cancer cell fraction derived from clustering-based tools, surprisingly, did not. Neoantigen quality was associated with loss of neoantigen expression in the bladder cancer case, and HLA-allele loss was observed in the renal and non-small cell lung cancer cases. Our results show that indication type, multi-lesion sampling, neoantigen expression, and HLA allele retention are important factors for iNeST targeting and patient selection.

category: cancer biology


title: Late Anisian microbe-metazoan build-ups ('stromatolites') in the Germanic Basin -- aftermath of the Permian -- Triassic Crisis

abstract: The so-called Permian - Triassic mass extinction was followed by a prolonged period of ecological recovery that lasted until the Middle Triassic. Triassic stromatolites from the Germanic Basin seem to be an important part of the puzzle, but have barely been investigated so far. Here we analyzed late Anisian (upper Middle Muschelkalk) stromatolites from across the Germanic Basin by combining petrographic approaches (optical microscopy, micro X-ray fluorescence, Raman imaging) and geochemical analyses (sedimentary hydrocarbons, stable carbon and oxygen isotopes). Paleontological and sedimentological evidence, such as Placunopsis bivalves, intraclasts and disrupted laminated fabrics, indicate that the stromatolites formed in subtidal, shallow marine settings. This interpretation is consistent with {delta}13Ccarb of about -2.1{per thousand} to -0.4{per thousand}. Occurrences of calcite pseudomorphs after gypsum suggest slightly evaporitic environments, which is well in line with the relative rarity of fossils in the host strata. Remarkably, the stromatolites are composed of microbes (perhaps cyanobacteria and sulfate reducing bacteria) and metazoans such as non-spicular demosponges, Placunopsis bivalves, and/or Spirobis-like worm tubes. Therefore, these "stromatolites" should more correctly be referred to as microbe-metazoan build-ups. They are characterized by diverse lamination types, including planar, wavy, domal and conical ones. Microbial mats likely played an important role in forming the planar and wavy laminations. Domal and conical laminations commonly show clotted to peloidal features and mesh-like fabrics, attributed to fossilized non-spicular demosponges. Our observations not only point up that non-spicular demosponges are easily overlooked and might be mistakenly interpreted as stromatolites, but also demonstrate that microbe-metazoan build-ups were widespread in the Germanic Basin during Early to Middle Triassic times. In the light of our findings, it appears plausible that the involved organisms benefited from elevated salinities. Another (not necessarily contradictory) possibility is that the mutualistic relationship between microbes and non-spicular demosponges enabled these organisms to fill ecological niches cleared by the Permian - Triassic Crisis. If that is to be the case, it means that such microbe-metazoan associations maintained their advantage until the Middle Triassic.

category: paleontology


title: Broccoli sprout Sulforaphane affected hemodynamics and aorta myogenic spontaneous rhythmic contraction in sanitized water uptake mice

abstract: Drinking seawater erodes water source will lead to hemodynamic changes in cardiovascular system. The erosion affected vascular biomechanics further interrupt the blood supply in arterial network. In this study, we investigated the carotid arterial hemodynamics in salinity water fed mice, and the relative spontaneous contraction of aorta preparation. The biological effect of Broccoli sprout Sulforaphane was assessed in intake hemodynamic changes. Kunming mice were randomly divided into seawater feeding group, seawater + Sulforaphane group, freshwater feeding group, fresh water + Sulforaphane group. After 4 weeks of feeding, the pressure waveforms of common carotid artery were analyzed in vivo. The enhanced common carotid arterial pressures were calculated according to the breakpoint of systolic pressure rising phase. The ejection time was calculated according to the dicrotic notch. In vitro, the isolated aorta biomechanical features were tested on a micro stepping platform. The passive tension and relative myogenic spontaneous contraction were evaluated. The results indicated that in salinity water fed mice heart rate, ejection period were significantly accelerated. The systolic pressure breakpoint of the ascending phase was significantly increased; however, the central aortic pressure augment index was decreased. In vitro study, the isolated aorta preparations indicated remarkable myogenic spontaneous contraction in salinity water fed mice. The spontaneous contraction indicated a significant cycle pattern, the waveform cluster changes regularly in one cycle, maximal amplitude of myogenic autonomic contraction increased significantly. Spontaneous contraction became more active, however cycle duration shortened. In biological effect of Broccoli sprout supplement, Sulforaphane was effective in reducing the heart rate, prolonging ejection period, improving systolic pressure and pulse pressure amplitude in salinity water fed mice. We concluded that long-term salinity water uptake can form a new hypertension model in mice, which can affect the changes of carotid artery hemodynamics and local blood supply. The Broccoli sprout Sulforaphane can improve the high systolic blood pressure and ejection period of artery, and its mechanism needs further study.

category: physiology


title: Genome-wide association study of gastric cancer- and duodenal ulcer-derived Helicobacter pylori strains reveals discriminatory amino acid differences and novel oncoprotein candidates

abstract: Genome-wide association studies (GWASs) can reveal genetic variations associated with a phenotype in the absence of any hypothesis of candidate genes. The problem of false-positive sites linked with the responsible site might be bypassed in bacteria with a high homologous recombination rate, such as Helicobacter pylori, which causes gastric cancer (GC). We conducted a GWAS followed by regression-based prediction of GC and duodenal ulcer H. pylori strains. We identified 14 single nucleotide polymorphisms (11 amino acid changes) that, combined, allowed effective disease discrimination. They were often informative of the underlying molecular mechanisms, such as electric charge alteration at the ligand-binding pocket, alteration in subunit interaction, and mode-switching of DNA methylation. We also identified three novel virulence factors/oncoprotein candidates. These results provide both defined targets for further informatic and experimental analyses to gain insights into GC pathogenesis and a basis for identifying a set of biomarkers for application in clinical settings.


title: Deficiency of peroxisomal L-bifunctional protein (EHHADH) causes male-specific kidney hypertrophy and proximal tubular injury in mice

abstract:
BackgroundProximal tubular (PT) cells are enriched in mitochondria and peroxisomes. Whereas mitochondrial fatty acid oxidation (FAO) plays an important role in kidney function by supporting the high-energy requirements of PT cells, the role of peroxisomal metabolism remains largely unknown. EHHADH, also known as L-bifunctional protein, catalyzes the second and third step of peroxisomal FAO.

MethodsWe studied kidneys of WT and Ehhadh KO mice using histology, immunohistochemistry, immunofluorescence, immunoblot, RNA-sequencing, metabolomics and orchiectomy.

ResultsWe observed male-specific kidney hypertrophy and glomerular filtration rate reduction in adult Ehhadh KO mice. Transcriptome analysis unveiled a gene expression signature similar to PT injury in acute kidney injury mouse models. This was further illustrated by the presence of KIM-1 (kidney injury molecule-1), SOX-9, and Ki67-positive cells in the PT of male Ehhadh KO kidneys. Male Ehhadh KO kidneys had metabolite changes consistent with peroxisomal dysfunction as well as an elevation in glycosphingolipids levels. Orchiectomy of Ehhadh KO mice reversed kidney enlargement and decreased the number of KIM-1 positive cells. We reveal a pronounced sexual dimorphism in the expression of peroxisomal FAO proteins in mouse kidney, underlining a role of androgens in the kidney phenotype of Ehhadh KO mice.

ConclusionsOur data highlight the importance of EHHADH and peroxisomal metabolism in male kidney physiology and reveal peroxisomal FAO as a sexual dimorphic metabolic pathway in mouse kidneys.


title: SPIRO-the automated Petri plate imaging platform designed by biologists, for biologists

abstract:
The imaging of plant seedlings, fungal mycelia and bacterial colonies grown on Petri plates is common in phenotyping assays, and is typically done manually despite the procedures being time-consuming and laborious. The main reason for this is the still limited availability of existing automated phenotyping tools and facilities. Additionally, constructing a custom-made automated solution is a daunting task for most research groups specializing in biology.

Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photos of up to four vertically oriented Petri plates in a single experiment. SPIRO was designed for biologists by biologists; thus, its assembly does not require experience in engineering or programming and its operation is sufficiently intuitive to be carried out without training. SPIRO has a small footprint optimal for fitting into standard incubators for plants and microbes, and is equipped with an LED light source for imaging in the dark, thus allowing acquisition of photos under optimal growth conditions. SPIROs web-based user interface allows setting up experiments and downloading data remotely, without interfering with samples growth. SPIROs excellent image quality is optimal for automated image processing, which we demonstrate with two semi-automated assays for analysis of commonly used phenotypic traits: seed germination and root growth.

Moreover, the robot can be easily customized for a specific use, as all information about SPIRO, including the models for 3D-printed structural components, control software, and scripts for image analysis, are released under permissive open-source licenses.


title: SPIRO - the automated Petri plate imaging platform designed by biologists, for biologists


title: Causal inference on microbiome-metabolome relations via in silico in vivo association pattern analyses

abstract: The effects of the microbiome on the hosts metabolism are core to understanding the role of the microbiome in health and disease. Herein, we develop the paradigm of in silico in vivo association pattern analyses, entailing a methodology to combine microbiome metabolome association studies with in silico constraint-based microbial community modelling. By dissecting confounding and causal paths, we show that in silico in vivo association pattern analyses allows for causal inference on microbiome-metabolome relations in observational data. Then, we demonstrate the feasibility and validity of our approach on a published multi-omics dataset (n=346), demonstrating causal microbiome-metabolite relations for 43 out of 53 metabolites from faeces. Finally, we utilise the identified in silico in vivo association pattern to estimate the microbial component of the faecal metabolome, revealing that the retrieved metabolite prediction scores correlate with the measured metabolite concentrations, and they also reflect the multivariate structure of the faecal metabolome. Concluding, we integrate with hypothesis free screening association studies and knowledge-based in silico modelling two major paradigms of systems biology, generating a promising new paradigm for causal inference in metabolic host-microbe interactions.

category: systems biology


title: Altered Effective Connectivity in Sensorimotor Cortices: a Novel Signature of Severity and Clinical Course in Depression

abstract:
Functional neuroimaging research on depression has traditionally targeted neural networks associated with the psychological aspects of depression. In this study, instead, we focus on alterations of sensorimotor function in depression. We used resting-state functional MRI data and Dynamic Causal Modeling (DCM) to assess the hypothesis that depression is associated with aberrant effective connectivity within and between key regions in the sensorimotor hierarchy. Using hierarchical modeling of between-subject effects in DCM with Parametric Empirical Bayes we first established the architecture of effective connectivity in sensorimotor cortices. We found that in (interoceptive and exteroceptive) sensory cortices across participants, the backward connections are predominantly inhibitory whereas the forward connections are mainly excitatory in nature. In motor cortices these parities were reversed. With increasing depression severity, these patterns are depreciated in exteroceptive and motor cortices and augmented in the interoceptive cortex: an observation that speaks to depressive symptomatology. We established the robustness of these results in a leave-one-out cross validation analysis and by reproducing the main results in a follow-up dataset. Interestingly, with (non-pharmacological) treatment, depression associated changes in backward and forward effective connectivity partially reverted to group mean levels. Overall, altered effective connectivity in sensorimotor cortices emerges as a promising and quantifiable candidate marker of depression severity and treatment response.

Significance StatementResearch into neurobiology of depression primarily focuses on its complex psychological aspects. Here, we propose an alternative approach and target sensorimotor alterations - a prominent but often neglected feature of depression. We demonstrated using resting-state fMRI data and computational modelling that top-down and bottom-up information flow in sensory and motor cortices is altered with increasing depression severity in a way that is consistent with depression symptoms. Depression associated changes were found to be consistent across sessions, amenable to treatment and of effect size sufficiently large to predict whether somebody has mild or severe depression. These results pave the way for a new avenue of research into the neural underpinnings of mental health conditions.


title: Bump attractor dynamics underlying stimulus integration in perceptual estimation tasks

abstract: Perceptual decision and continuous stimulus estimation tasks involve making judgments based on accumulated sensory evidence. Network models of evidence integration usually rely on competition between neural populations each encoding a discrete categorical choice and do not maintain information that is necessary for a continuous perceptual judgement. Here, we show that a continuous attractor network can integrate a circular stimulus feature and track the stimulus average in the phase of its activity bump. We show analytically that the network can compute the running average of the stimulus almost optimally, and that the nonlinear internal dynamics affect the temporal weighting of sensory evidence. Whether the network shows early (primacy), uniform or late (recency) weighting depends on the relative strength of the stimuli compared to the bumps amplitude and initial state. The global excitatory drive, a single model parameter, modulates the specific relation between internal dynamics and sensory inputs. We show that this can account for the heterogeneity of temporal weighting profiles and reaction times observed in humans integrating a stream of oriented stimulus frames. Our findings point to continuous attractor dynamics as a plausible mechanism underlying stimulus integration in perceptual estimation tasks.


title: Changing in the hierarchical organization of local information dynamics during motor decision in the premotor cortex of primates

abstract: Despite recent works have investigated functional and effective cortical networks in animal models, the dynamical information transfer among functional modules underneath cognitive control is still largely unknown. Here, we addressed the issue using Transfer Entropy and graph theory methods on neural activities recorded from a multielectrode array in the dorsal premotor cortex of rhesus monkeys. We focused our analysis on the decision time of a Stop-signal (countermanding) task. When comparing trials with successful inhibition to those with generated movement the local network resulted organized in four classes of modules hierarchically arranged and differently partaking in information transfer. Interestingly, the hierarchical organization of modules changed during the task, being different for generated movements and cancelled ones. Our results suggest that motor decisions are based on a topological re-organization of the premotor functional network.


title: Hierarchical organization changes of local information dynamics during motor decision in the premotor cortex of primates


title: Open Data Commons for Preclinical Traumatic Brain Injury Research: Empowering Data Sharing and Big Data Analytics

abstract: Traumatic brain injury (TBI) is a major unsolved public health problem worldwide with considerable preclinical research dedicated to recapitulating clinical TBI, deciphering the underlying pathophysiology, and developing therapeutics. However, the heterogeneity of clinical TBI and correspondingly in preclinical studies have made translation from bench to bedside difficult. Here, we present the potential of data sharing, data aggregation, and multivariate analytics to integrate heterogeneity and empower researchers. We introduce the Open Data Commons for Traumatic Brain Injury (ODC-TBI.org) as a user-centered web platform and cloudbased repository focused on preclinical TBI research that enables data citation with persistent identifiers, promotes data element harmonization, and follows FAIR data sharing principles. Importantly, the ODC-TBI implements data sharing at the level of individual subjects, thus enabling data reuse for granular big data analytics and data-hungry machine learning approaches. We provide use cases applying descriptive analytics and unsupervised machine learning on pooled ODC-TBI data. Descriptive statistics included subject-level data for 11 published papers (N = 1250 subjects) representing six distinct TBI models across mice and rats (implementing controlled cortical impact, closed head injury, fluid percussion injury, and CHIMERA TBI modalities). We performed principal component analysis (PCA) on cohorts of animals combined through the ODC-TBI to identify persistent inflammatory patterns across different experimental designs. Our workflow ultimately improved the sensitivity of our analyses in uncovering patterns of pro- vs anti-inflammation and oxidative stress without the multiple testing problems of univariate analyses. As the practice of open data becomes increasingly required by the scientific community, ODC-TBI provides a foundation that creates new scientific opportunities for researchers and their work, facilitates multi-dataset and multidimensional analytics, and drives collaboration across molecular and computational biologists to bridge preclinical research to the clinic.


title: Retinal factors of visual sensitivity in the human fovea

abstract: Humans direct their gaze towards visual objects of interest such that the retinal images of fixated objects fall onto the fovea, a small anatomically and physiologically specialized region of the retina displaying highest visual fidelity. One striking anatomical feature of the fovea is its non-uniform cellular topography, with a steep decline of cone photoreceptor density and outer segment length with increasing distance from its center. We here assessed in how far the specific cellular organization of the foveola is reflected in visual function. Increment sensitivity to small spot visual stimuli (1 x 1 arcmin, 543 nm light) was recorded psychophysically in 4 human participants at 17 locations placed concentric within a 0.2-degree diameter around the preferred retinal locus of fixation with adaptive optics scanning laser ophthalmoscopy based microstimulation. While cone density as well as maximum outer segment length differed significantly among the four tested participants, the range of observed threshold was similar, yielding an average increment threshold of 3.3 {+/-} 0.2 log10 photons at the cornea. Thresholds were correlated with retinal eccentricity, as well as cone density and outer segment length. Biophysical simulation allowed to develop a model of foveal sensitivity based on these parameters, explaining at least 37% of the observed threshold variability. Based on high reproducibility in replicate testing, the residual variability is assumed to be caused by individual cone and bipolar cell weighting at the specific target locations.


title: Spontaneous Eye Blink Rate during the Working Memory Delay Period Predicts Task Performance

abstract: Spontaneous eye blink rate (sEBR) has been linked to attention and memory, specifically working memory (WM). sEBR is also related to striatal dopamine (DA) activity with schizophrenia and Parkinsons disease showing increases and decreases respectively in sEBR. A weakness of past studies of sEBR and WM is that correlations have been reported using blink rates taken at baseline either before or after performance of the tasks used to assess WM. The goal of the present study was to understand how fluctuations in sEBR during different phases of a visual WM task predict task accuracy. In two experiments, with recordings of sEBR collected inside and outside of a magnetic resonance imaging bore, we observed sEBR to be positively correlated with WM task accuracy during the WM delay period. We also found task-related modulation of sEBR, including higher sEBR during the delay period compared to rest, and lower sEBR during task phases (e.g., stimulus encoding) that place demands on visual attention. These results provide further evidence that sEBR could be an important predictor of WM task performance with the changes during the delay period suggesting a role in WM maintenance. The relationship of sEBR to DA activity and WM maintenance is discussed.


title: Spontaneous Eye Blink Rate during the Working Memory Delay Period Predicts Task Accuracy


title: Time series analysis of trial-to-trial variability of MEG power spectrum during rest state, unattented listening and frequency-modulated tones classification

abstract:
The nonstationarity of EEG/MEG signals is important for understanding the functioning of human brain. From the previous research we know that even very short, i.e. 250--500ms MEG signals are variance-nonstationary. The covariance of stochastic process is mathematically associated with its spectral density, therefore we investigate how the spectrum of such nonstationary signals varies in time.

We analyze the data from 148-channel MEG, that represent rest state, unattented listening and frequency-modulated tones classification. We transform short-time MEG signals to the frequency domain using the FFT algorithm and for the dominant frequencies 8--12 Hz we prepare the time series representing their trial-to-trial variability. Then, we test them for level- and trend-stationarity, unit root, heteroscedasticity and gaussianity and based on their properties we propose the ARMA-modelling for their description.

The analyzed time series have the weakly stationary properties independently of the functional state of brain and localization. Only their small percentage, mostly related to the cognitive task, still presents nonstationarity. The obtained mathematical models show that the spectral density of analyzed signals depends on only 2--3 previous trials.

The presented method has limitations related to FFT resolution and univariate models, but it is not computationally complicated and allows to obtain a low-complex stochastic models of the EEG/MEG spectrum variability.

Although the physiological short-time MEG signals are in principle nonstationary in time domain, its power spectrum at the dominant frequencies varies as weakly stationary stochastic process. Described technique has the possible applications in prediction of the EEG/MEG spectral properties in theoretical and clinical neuroscience.


title: Analysis of M4 transmembrane domains in NMDA receptor function: a negative allosteric modulation site at the GluN1 M4 is determining the efficiency of neurosteroid modulation

abstract:
Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels that play a crucial role in excitatory synaptic transmission in the central nervous system. Each subunit contributes with three transmembrane domains (M1, M3, and M4) and a pore loop (M2) forming the channel pore. Recent studies suggest that the architecture of all eukaryotic iGluRs derives from a common prokaryotic ancestral receptor that lacks M4 and consists only of the transmembrane domain segments M1-M3. Although a crucial contribution of M4 to the assembly and trafficking of iGluRs is suspected, the role of this additionally evolved domain in receptor function remains controversial. Here, we investigated how deletions and mutations of M4 in members of the NMDA receptor subfamily, the conventional heteromeric GluN1/GluN2 and glycine-gated GluN1/GluN3 NMDA receptors, affect expression and function in Xenopus oocytes. We show that deletion of M4 in the GluN1, GluN2, or GluN3 subunit, despite retained receptor assembly and cell surface expression, results in nonfunctional membrane receptors. Coexpression of the corresponding M4 domains as an isolated peptide in M4-deleted receptors rescued receptor function of GluN1/GluN2A NMDARs without altering the affinity of glutamate or glycine. Substitution of non-conserved residues and insertion of interhelical disulfide bridges confirmed the proximity of positions M813 and F817 in M4 of GluN1 to residues of the TMs of neighboring subunits. Electrophysiological analyses of agonist-induced receptor function and its modulation by the neurosteroid pregnenolone sulfate (PS) at mutations of the GluN1-M4/GluN2/3-TM interface indicate a crucial role of interdomain interactions in the functional coupling of M4 to the nuclear receptor and the modulatory effect of PS. Our results show that although the M4 domains in NMDA receptors are not important for receptor assembly and surface expression, residues at the subunit interface are substantially involved in M4 recognition to the core receptor and regulation of PS efficacy. Because mutations in the M4 of GluN1 specifically resulted in loss of PS-induced inhibition of NMDA receptor currents, our results point to distinct roles of M4s in NMDA receptor modulation and highlight the importance of evolutionarily newly evolved M4s for selective in vivo modulation of glutamate- and glycine-activated NMDA receptors by steroids.

Highlights- The role of the M4 transmembrane domain in the assembly and function of ionotropic glutamate receptors remains controversial
- Here we show that deletion of M4 in glutamate-gated GluN1/GluN2A and glycine-gated GluN1/GluN3A receptors results in nonfunctional NMDA receptors with retained surface expression
- The functional loss in M4-deleted GluN1/GluN2A receptors is rescued without affecting agonist affinity by a M4 transmembrane domain of the respective subunit expressed as an isolated peptide
- Specific interactions in the M4 interfaces with the M1 and M3 domain of the adjacent subunit are required for the recognition of the isolated M4 and the functional rescue
- Finally, the M4 domain-interfaces of GluN1 determine the negative modulatory effect of pregnenolone sulfate in glutamate-gated GluN1/GluN2A and glycine-gated GluN1/GluN3A NMDA receptors


title: The inflammasome-activated cytokine IL-1 is targeted for ubiquitylation and proteasomal degradation to limit its inflammatory potential

abstract: Interleukin-1{beta} (IL-1{beta}) is activated by inflammasome-associated caspase-1 in rare autoinflammatory conditions and in wide-spread diseases. Therefore, IL-1{beta} activity must be fine-tuned to enable antimicrobial responses whilst limiting collateral damage. Here we report that precursor IL-1{beta} is rapidly turned over by the proteasome and this correlates with its decoration by K11-, K63- and K48-linked ubiquitin chains. The ubiquitylation of IL-1{beta} is not just a degradation signal triggered by inflammasome priming and activating stimuli, but also limits IL-1{beta} cleavage by caspase-1. We further demonstrate that IL-1{beta} K133 is modified by ubiquitin and forms a salt bridge with IL-1{beta} D129. Loss of IL-1{beta} K133 ubiquitylation, or disruption of the K133:D129 electrostatic interaction, stabilizes IL-1{beta}. Accordingly, IL-1{beta}K133R/K133R mice display increased precursor IL-1{beta} upon inflammasome priming and increased bioactive IL-1{beta}, both in vitro and following LPS injection in vivo. These findings reveal new mechanisms for limiting IL-1{beta} activity and safeguarding against damaging inflammation.

category: immunology


title: Tissue environment, not ontogeny, defines intestinal intraepithelial T lymphocytes

abstract: Tissue-resident intestinal intraepithelial T lymphocytes (T-IEL) patrol the gut and have important roles in regulating intestinal homeostasis. T-IEL include both induced T-IEL, derived from systemic antigen-experienced lymphocytes, and natural IEL, which are developmentally targeted to the intestine. While the processes driving T-IEL development have been elucidated, the precise roles of the different subsets and the processes driving activation and regulation of these cells remain unclear. To gain functional insights into these enigmatic cells, we used high-resolution, quantitative mass spectrometry to investigate the proteomic landscape of the main T-IEL populations in the gut. Comparing the proteomes of induced T-IEL and natural T-IEL subsets, with naive CD8+ T cells from lymph nodes exposes the dominant effect of the gut environment over ontogeny on T-IEL phenotypes. Analyses of protein copy numbers of >7000 proteins in T-IEL reveal skewing of the cell surface repertoire towards epithelial interactions and checkpoint receptors; strong suppression of the metabolic machinery indicating a high energy barrier to functional activation; and changes in T cell antigen receptor signalling pathways reminiscent of chronically activated T cells. These novel findings illustrate how multiple input signals need to be integrated to regulate T-IEL function.


title: Responding kinetic of B-cell receptor repertoire to the Toll-like receptor 7/8 stimulation in non-human primates

abstract: TLR7 and 8 regulate B cell immunity, but the precise details of the mechanism are still unclear. Here, we studied the kinetics of both heavy and light chains (IgKL) of B-cell receptor (BCR) repertoire responding to the TLR7/8 stimulation in two geniuses of non-human primates (NHPs), African green monkeys (AGMs) and rhesus macaques (RMs). We evaluated the activation of lymphocytes by flow cytometry, and studied characteristics of BCR repertoire in terms of gene usage, repertoire diversity, and the number of lineages. Although AGMs had a weaker activation than RMs, and a different responding kinetic, both AGMs and RMs presented an increased IgKL repertoire diversity and lineages expansion. It suggested that the responding time rather than initiation of TLR7/8-induced IgKL repertoire response related to B cell activation. Expanded IgKL lineages with frequency from 0.001% to 1% had an elevated mutation rate and expanded IgH lineages used more IgA/G/E, suggesting that the TLR7/8 stimulation expanded low-frequent but high-mutated lineages. Besides, most of expanded IgKL lineages were {lambda} isotype. In conclusion, TLR7/8 selectively expands IgKL lineages with a high mutation rate, low frequency, and {lambda} isotype. The selective effect of TLR7/8 on BCR repertoire allows TLR7/8 agonists to be adjuvant for selectively accelerating antibody maturation.


title: Amotosalen is a bacterial multidrug efflux pump substrate potentially affecting its pathogen inactivation activity

abstract:
Pathogen inactivation is a strategy to improve the safety of transfusion products. The Cerus Intercept technology makes use of a psoralen compound called amotosalen in combination with UVA light to inactivate bacteria, viruses and protozoa. Psoralens have structural similarity to bacterial multidrug-efflux pump substrates. As these efflux pumps are often overexpressed in multidrug-resistant pathogens and with recent reported outbreaks of transfusion-associated sepsis with Acinetobacter, we tested whether contemporary drug-resistant pathogens might show resistance to amotosalen and other psoralens based on multidrug efflux mechanisms through microbiological, biophysical and molecular modeling analysis. The main efflux systems in Enterobacterales and Acinetobacter baumannii, tripartite RND (resistance-nodulation-cell division) systems which span the inner and outer membranes of Gram-negative pathogens and expel antibiotics from the bacterial cytoplasm into the extracellular space, were specifically examined. We found that amotosalen was an efflux substrate for the TolC-dependent RND efflux pumps in E. coli and the AdeABC efflux pump from Acinetobacter baumannii, and that minimal inhibitory concentrations for contemporary bacterial isolates in vitro approached and exceeded the concentration of amotosalen used in the approved platelet and plasma inactivation procedures. These findings suggest that otherwise safe and effective inactivation methods should be further studied to exclude possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.

ImportancePathogen inactivation is a strategy to enhance the safety of transfused blood products. We identify the compound, amotosalen, widely used for pathogen inactivation, as a bacterial multidrug efflux substrate. Specifically, experiments suggest that amotosalen is pumped out of bacteria by the major TolC-dependent RND efflux pumps in E. coli and the AdeABC efflux pump in Acinetobacter baumannii. Such efflux pumps are often overexpressed in multidrug-resistant pathogens. Importantly, the minimal inhibitory concentrations for contemporary multidrug-resistant Enterobacterales, Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia spp., and Stenotrophomonas maltophilia isolates approached or exceeded the amotosalen concentration used in approved platelet and plasma inactivation procedures, potentially as a result of efflux pump activity. Although there are important differences in methodology between our experiments and blood product pathogen inactivation, these findings suggest that otherwise safe and effective inactivation methods should be further studied to exclude possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.