title: ECD of PepT1 interacts with TM1 to facilitate substrate transport

abstract: Mammalian peptide transporters, PepT1 and PepT2, mediate uptake of a wide variety of di- and tri-peptides and are essential for the absorption of dietary peptides in the digestive tract and the recovery of peptides in renal filtrate. PepT also mediates absorption of many drugs and prodrugs to enhance their bioavailability. PepT has 12 transmembrane (TM) helices that fold into two domains, the N-terminal domain (NTD, TM1-6) and C-terminal domain (CTD, TM7-12), and a large extracellular domain (ECD) of [~]200 amino acids between TM9 and TM10. It is known that peptide transport involves large motions of the N- and C-domains, but the role of ECD remains unclear. Here we report the structure of PepT1 from Equus caballus (horse) determined by cryo-electron microscopy. The structure shows that ECD interacts with TM1 and bridges the N- and C-domains. Deletion of the ECD or mutations to the TM1-ECD interface both impair the transport activity. These results demonstrate a role of ECD in structure and function of PepT1 and enhance our understanding of the mechanism of transport in PepT1.


title: A Standardized Framework for Taxonomy Analysis in Bacterial Kingdom based on Protein Domain

abstract:
The taxonomy research on the bacterial kingdom is important and can be usually conducted by the analysis on conserved genes, 16s rRNA, protein domain, and so on. Comparatively, the protein domains maintain a most direct relationship with phenotypes. In this paper, based on the protein domain, we propose a 3-step framework to standardize the classification process of bacteria. Different model candidates are involved and discussed in each step. By comparing the classification results with existing taxonomy, we select the most appropriate candidate to improve the framework, and furthermore, discuss their biological significance. Finally, we put forward taxonomy suggestions based on the best classification results.

ImportanceWe standardize a 3-step framework to carry out bacterial taxonomy research based on protein domain. Furthermore, we filter out the best solution in each step that can together generate the most appropriate classification result, and at the same time, we discuss the biological significant it indicates. Finally, we propose suggestions on NCBI bacterial taxonomy based on the classification results.

category: bioinformatics


title: A detailed map of mitochondrial repeat-mediated recombination in Arabidopsis msh1 mutants using long-read sequencing

abstract:
The abundant repeats in plant mitochondrial genomes can cause rapid genome rearrangements and are also a major obstacle in short-read sequencing studies. Nuclear-encoded proteins such as MSH1 are known to suppress the generation of repeat-associated mitochondrial genome variants, but our understanding of these mechanisms has been constrained by the limitations of short-read technologies. Here, we used highly accurate long-read sequencing (PacBio HiFi) to characterize mitochondrial and plastid genome variants in Arabidopsis thaliana msh1 mutant individuals. The HiFi reads successfully quantified parental and crossover recombination products of both large and small repeats, providing a global view of recombination dynamics. We found that recombination breakpoints were distributed relatively evenly across the length of repeated sequences and observed extensive non-crossover recombination (gene conversion) between pairs of imperfect repeats in the mitochondrial genome of msh1 mutants. Comparison of long-read assemblies of seven other Arabidopsis thaliana accessions showed that mitochondrial genome structural divergence between accessions paralleled the repeat-mediated dynamics observed in msh1 mutants. The Arabidopsis plastid genome generally lacks small repeats and exhibited a very different pattern of variant accumulation in msh1 mutants compared with the mitochondrial genome. Our data illustrate the power of HiFi technology in studying repeat-mediated recombination in plant mitochondrial genomes and improved the sequence resolution for recombinational processes suppressed by MSH1.

SignificancePlant mitochondrial genomes undergo rapid rearrangements while maintaining protein genes. Long-read sequencing provides a detailed and quantitative view of mitochondrial repeat-mediated recombination suppressed by MSH1, advancing our understanding of plant mitochondrial genome dynamics.


title: Development of a dedicated Golden Gate Assembly platform (RtGGA) for Rhodotorula toruloides

abstract: Rhodotorula toruloides is a potential chassis to microbial cell factories as this yeast can metabolise different substrates into a diverse range of natural products, but the lack of efficient synthetic biology tools hinders its applicability. In this study, the modular, versatile and efficient Golden Gate DNA assembly system was adapted to the first basidiomycete, an oleaginous yeast R. toruloides (RtGGA). R. toruloides CCT 0783 was sequenced, and used for the RtGGA design. The DNA fragments were assembled with predesigned 4-nt overhangs and a library of standardized parts was created containing promoters, genes, terminators, insertional regions, and resistance genes. The library was combined to create cassettes for the characterization of promoters strength and to overexpress the carotenoid production pathway. A variety of reagents, plasmids, and strategies were used and the RtGGA proved to be robust. The RtGGA was used to build three versions of the carotenoid overexpression cassette were built by using different promoter combinations. The cassettes were transformed into R. toruloides and the three new strains were characterized. Total carotenoid concentration increased by 41%. The dedicated GGA platform fills a gap in the advanced genome engineering toolkit for R. toruloides, enabling the efficient design of complex metabolic pathways.

category: synthetic biology


title: Inducible CRISPR/Cas9 allows for multiplexed and rapidly segregated single target genome editing in Synechocystis sp. PCC 6803

abstract: Establishing various synthetic biology tools is crucial for the development of cyanobacteria for biotechnology use, especially tools that allow for precise and markerless genome editing in a time-efficient manner. Here we describe a riboswitch-inducible CRISPR/Cas9 system, contained on one single replicative vector, for the model cyanobacteria Synechocystis sp. PCC 6803. A theophylline-responsive riboswitch allowed tight control of Cas9 expression, which enabled reliable transformation of the CRISPR/Cas9 vector into Synechocystis. Induction of the CRISPR/Cas9 mediated various types of genomic edits, specifically deletions and insertions of varying size. The editing efficiency varied depending on the target and intended edit; smaller edits overall performed better, reaching e.g. 100% for insertion of a FLAG-tag onto rbcL. Importantly, the single-vector CRISPR/Cas9 system described herein was also shown to mediate multiplexed editing of up to three targets in parallel in Synechocystis. All single-target and several double-target mutants were also fully segregated after the first round of induction, adding to the usefulness of this system. Further, a vector curing system that is separately induced by nickel and contained on the CRISPR/Cas9 vector itself, improved curing efficiencies by roughly 4-fold, enabling the final mutants to become truly markerless.


title: A parametric logistic equation with light flux and media concentration for cultivation planning of microalgae

abstract: Microalgae are considered to be promising producers of bioactive chemicals, feeds, and fuels from carbon dioxide by photosynthesis. Thus, the prediction of microalgal growth profiles is important for the planning of cost-effective and sustainable cultivation-harvest cycles. This paper proposes a mathematical model capable of predicting the effect of light flux into culture and medium concentration on the growth profiles of microalgae by incorporating these growth-limiting factors into the logistic equation. The specific form of the equation is derived based on the experimentally measured growth profiles of Monoraphidium sp., a microalgal strain isolated by the authors, under 16 conditions consisting of the combinations of incident light fluxes into culture and initial medium concentrations. Using a cross-validation method, it is shown that the proposed model has the ability to predict necessary incident light flux into culture and initial medium concentration for harvesting target biomass at target time. Finally, the model-guided cultivation planning is performed and is evaluated by comparing the result with the experimental data.

category: systems biology


title: A parametric logistic equation with light flux and medium concentration for cultivation planning of microalgae


title: A vector reward prediction error model explains dopaminergic heterogeneity

abstract: The hypothesis that midbrain dopamine (DA) neurons broadcast an error signal for the prediction of reward (reward prediction error, RPE) is among the great successes of computational neuroscience1-3. However, recent results contradict a core aspect of this theory: that the neurons uniformly convey a scalar, global signal. Instead, when animals are placed in a high-dimensional environment, DA neurons in the ventral tegmental area (VTA) display substantial heterogeneity in the features to which they respond, while also having more consistent RPE-like responses at the time of reward. Here we introduce a new "Vector RPE" model that explains these findings, by positing that DA neurons report individual RPEs for a subset of a population vector code for an animals state (moment-to-moment situation). To investigate this claim, we train a deep reinforcement learning model on a navigation and decision-making task, and compare the Vector RPE derived from the network to population recordings from DA neurons during the same task. The Vector RPE model recapitulates the key features of the neural data: specifically, heterogeneous coding of task variables during the navigation and decision-making period, but uniform reward responses. The model also makes new predictions about the nature of the responses, which we validate. Our work provides a path to reconcile new observations of DA neuron heterogeneity with classic ideas about RPE coding, while also providing a new perspective on how the brain performs reinforcement learning in high dimensional environments.

category: neuroscience


title: Kv7 channel antagonists block glycine receptors

abstract: XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone) is currently the most widely used and specific antagonist of the Kv7 (KCNQ) family of K+ channels. We report an unexpected antagonistic effect of this drug on ionotropic glycine receptors. In recordings of synaptic transmission in two brainstem nuclei (the medial nucleus of the trapezoid body and the dorsal cochlear nucleus), 10 M XE991, a concentration typical for Kv7 studies in brain tissue, inhibited evoked glycinergic inhibitory postsynaptic currents (IPSCs) without altering paired-pulse ratio, and also reduced the amplitude of glycinergic miniature IPSCs. These results are indicative of a direct effect of the drug on postsynaptic glycine receptors. XE991 also produced dose-dependent block of the response to exogenously applied glycine, to a degree comparable to the block of synaptic transmission. Moreover, the drug inhibited homomeric glycine receptors expressed on presynaptic membrane of the calyx of Held. The degree of block was independent of glycine concentration, suggesting an allosteric interaction. The effects of XE991 on glycine responses are not likely to reflect block of the glycine-activated Cl- channels themselves, because block was voltage independent, and because GABA-activated Cl- currents were resistant to XE991 at concentrations up to 100 {micro}M. Linopirdine, but not retigabine, also antagonized glycine receptor currents. Given the prevalence of glycine receptor signaling in the brain, these observations should be taken into account in studies of the roles of Kv7 channels in neural circuit function and disease.


title: Single cell atlas of the neonatal small intestine with necrotizing enterocolitis

abstract: Necrotizing enterocolitis (NEC) is a gastrointestinal complication of premature infants with high rates of morbidity and mortality. A comprehensive view of the cellular changes and aberrant interactions that underlie this disease is lacking. Here, we combine single cell RNA sequencing, T Cell Receptor beta (TCR{beta}) analysis, bulk transcriptomics, and imaging to characterize cell identities, interactions and zonal changes in NEC. We find that inflammatory macrophages are abundant in NEC and that T cells exhibit increased expression of inflammatory genes and cytokines accompanied by an increase in TCR{beta} clonal expansion. Fibroblasts and endothelial cells increase in proportion and exhibit a switch to an activated pro-inflammatory state. Villus tip epithelial cell identity is substantially reduced in NEC and the remaining epithelial cells up-regulate pro-inflammatory genes. We establish a detailed map of aberrant epithelial-mesenchymal-immune interactions that may be driving inflammation in NEC mucosa. Our analyses highlight the cellular changes underlying NEC disease pathogenesis and identify potential targets for biomarker discovery and therapeutics.

category: immunology


title: A bivalent CMV vaccine formulated with human compatible TLR9 agonist CpG1018 elicits potent cellular and humoral immunity in HLA expressing mice

abstract: There is now convincing evidence that the successful development of an effective CMV vaccine will require improved formulation and adjuvant selection that is capable of inducing both humoral and cellular immune responses. Here, we have designed a novel bivalent subunit vaccine formulation based on CMV-encoded oligomeric glycoprotein B (gB) and polyepitope protein in combination with human compatible TLR9 agonist CpG1018. The polyepitope protein includes multiple minimal HLA class I-restricted CD8+ T cell epitopes from different antigens of CMV. This subunit vaccine generated durable anti-viral antibodies, CMV-specific CD4+ and CD8+ T cell responses in multiple HLA expressing mice. Antibody responses included broad TH1 isotypes (IgG2a, IgG2b and IgG3) and potently neutralized CMV infection in fibroblasts and epithelial cells. Furthermore, polyfunctional antigen-specific T cell immunity and antiviral antibody responses showed long-term memory maintenance. These observations argue that this novel vaccine strategy, if applied to humans, could facilitate the generation of robust humoral and cellular immune responses which may be more effective in preventing CMV-associated complications in various clinical settings.


title: Distinct gene expression by expanded clones of quiescent memory CD4+ T cells harboring intact latent HIV-1 proviruses

abstract:
Antiretroviral therapy controls but does not cure HIV-1 infection due to a reservoir of rare CD4+ T cells harboring latent proviruses. Little is known about the transcriptional program of latent cells. Here we report a novel strategy to enrich clones of latent cells carrying intact, replication- competent HIV-1 proviruses from blood based on their expression of unique T cell receptors. Latent cell enrichment enabled single cell transcriptomic analysis of 1,050 CD4+ T cells belonging to expanded clones harboring intact HIV-1 proviruses from 6 different individuals. The analysis revealed that most of these cells are T effector memory cells that are enriched for expression of HLA-DR, HLA-DP, CD74, CCL5, Granzymes A and K, cystatin F, LYAR and DUSP2. We conclude that expanded clones of latent cells carrying intact HIV-1 proviruses persist preferentially in a distinct CD4+ T cell population opening new possibilities for eradication.

Graphical Abstract

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title: Intranasal Immunization with a Proteosome-Adjuvanted SARS-CoV2 Spike Protein-Based Vaccine is Immunogenic and Efficacious in Mice & Hamsters

abstract: With the persistence of the SARS-CoV-2 pandemic and the emergence of novel variants, the development of novel vaccine formulations with enhanced immunogenicity profiles could help reduce disease burden in the future. Intranasally delivered vaccines offer a new modality to prevent SARS-CoV-2 infections through the induction of protective immune responses at the mucosal surface where viral entry occurs. Herein, we evaluated a novel protein subunit vaccine formulation containing a resistin-trimerized prefusion Spike antigen (SmT1v3) and a proteosome-based mucosal adjuvant (BDX301) formulated to enable intranasal immunization. In mice, the formulation induced robust antigen-specific IgG and IgA titers, in the blood and lungs, respectively. In addition, the formulations were highly efficacious in a hamster challenge model, reducing viral load and body weight loss. In both models, the serum antibodies had strong neutralizing activity, preventing the cellular binding of the viral Spike protein based on the ancestral reference strain, the Beta (B.1.351) and Delta (B.1.617.2) variants of concern. As such, this intranasal vaccine formulation warrants further development as a novel SARS-CoV-2 vaccine.


title: Immunological memory to Common Cold Coronaviruses assessed longitudinally over a three-year period

abstract: Understanding immune memory to Common Cold Coronaviruses (CCCs) is relevant for assessing its potential impact on the outcomes of SARS-CoV-2 infection, and for the prospects of pan-corona vaccines development. We performed a longitudinal analysis, of pre-pandemic samples collected from 2016-2019. CD4+ T cells and antibody responses specific for CCC and to other respiratory viruses, and chronic or ubiquitous pathogens were assessed. CCC-specific memory CD4+ T cells were detected in most subjects, and their frequencies were comparable to those for other common antigens. Notably, responses to CCC and other antigens such as influenza and Tetanus Toxoid (TT) were sustained over time. CCC-specific CD4+ T cell responses were also associated with low numbers of HLA-DR+CD38+ cells and their magnitude did not correlate with yearly changes in the prevalence of CCC infections. Similarly, spike RBD-specific IgG responses for CCC were stable throughout the sampling period. Finally, high CD4+ T cell reactivity to CCC, but not antibody responses, was associated with high pre-existing SARS-CoV-2 immunity. Overall, these results suggest that the steady and sustained CCC responses observed in the study cohort are likely due to a relatively stable pool of CCC-specific memory CD4+ T cells instead of fast decaying responses and frequent reinfections.


title: Feeding Growing Mushrooms

abstract:
A number of experiments were done to further our understanding of the substrate utilization in button mushroom crops (Agaricus bisporus). An analysis of the degradation of dry matter of the substrate during a crop cycle revealed that for pin formation the upper layer is used, for the production of flush one all layers are involved and for flush two mainly the lower layer is used. A reduction in substrate depth leads to a decrease in yield/m2 but an apparent increase in yield per ton of substrate but with a lower mushroom quality. A short daily interruption of the connection between the casing soil with all the substrate results in a delay of the first flush. Repetitive interruption of the connection with all or only part of the substrate leads to a shift in yield from flush one to flush two but the total yield remains unchanged. The mycelial biomass in the substrate increases from filling up to pinning, a steeper increase during flush one, levelling off during flush two, indicating that in the period of venting and up to/including flush one, enzymes are secreted by growing hyphae generating nutrients to feed a fixed amount of mushroom biomass for two flushes. A sidewise extension of the substrate (without casing soil, thus not producing mushrooms) showed that the substrate at a distance more than 20-50 cm away from the casing soil does not contribute to feeding mushrooms in the first two flushes. The observations are discussed with respect to relevant previous research.

ImportanceButton mushrooms (Agaricus bisporus) are cultivated on a substrate prepared of composted wheat straw, chicken and/or horst manure and gypsum. This manuscript describes a number of experiments indicating that in a harvesting strategy towards quality mushrooms there is a fixed amount of nutrients prepared/available to feed two flushes. The increase of mycelial biomass in the substrate after venting and especially during the development of flush one indicates that likely a branching of mycelium occurs during this period, leading to a substantial number of growing hyphal tips excreting lignocellulolytic enzymes that degrade substrate and make nutrients available during flush one which are utilised in flush one and two. The presently used substrate seems to have reached its limits and there are hardly opportunities for further improvement. Research on alternative, more energy dense, substrates are needed to improve the efficiency of the present production system.

category: microbiology


title: Modelling the synergistic effect of bacteriophage and antibiotics on bacteria: killers and drivers of resistance evolution

abstract:
Bacteriophage (phage) are bacterial predators which can also spread antimicrobial resistance (AMR) genes between bacteria by generalised transduction. Phage are often present alongside antibiotics in the environment, yet evidence of their joint killing effect on bacteria is conflicted, and the dynamics of transduction in such systems are unknown. Here, we combine in vitro data and mathematical modelling to identify conditions where phage and antibiotics act in synergy to remove bacteria or drive AMR evolution.

We adapt a published model of phage-bacteria dynamics, including transduction, to add the pharmacodynamics of erythromycin and tetracycline, parameterised from new in vitro data. We simulate a system where two strains of Staphylococcus aureus are present at stationary phase, each carrying either an erythromycin or tetracycline resistance gene, and where multidrug-resistant bacteria can be generated by transduction only. We determine rates of bacterial clearance and multidrug-resistant bacteria appearance, when either or both antibiotics and phage are present at varying timings and concentrations.

Although phage and antibiotics act in synergy to kill bacteria, by reducing bacterial growth antibiotics reduce phage production. A low concentration of phage introduced shortly after antibiotics fails to replicate and exert a strong killing pressure on bacteria, instead generating multidrug-resistant bacteria by transduction which are then selected for by the antibiotics. Multidrug-resistant bacteria numbers were highest when antibiotics and phage were introduced simultaneously.

The interaction between phage and antibiotics leads to a trade-off between a slower clearing rate of bacteria (if antibiotics are added before phage), and a higher risk of multidrug-resistance evolution (if phage are added before antibiotics), exacerbated by low concentrations of phage or antibiotics. Our results form hypotheses to guide future experimental and clinical work on the impact of phage on AMR evolution, notably for studies of phage therapy which should investigate varying timings and concentrations of phage and antibiotics.


title: Cell-specific regulation of gene expression using splicing-dependent frameshifting.

abstract: Precise and reliable cell-specific gene delivery remains technically challenging. Here we report a splicing-based approach for controlling gene expression whereby separate translational reading frames are coupled to the inclusion or exclusion of cell-specific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA sequencing datasets and filtering by cell specificity, sequence conservation, and local intron length. This method, which we denote splicing-linked expression design (SLED), can be combined in a Boolean manner with existing techniques such as minipromoters and viral capsids. SLED vectors can leverage the strong expression of constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. We generated SLED vectors to selectively target all neurons, photoreceptors, or excitatory neurons, and demonstrated that specificity was retained in vivo when delivered using AAVs. We further demonstrated the utility of SLED by creating what would otherwise be unobtainable research tools, specifically a GluA2 flip/flop reporter and a dual excitatory/inhibitory neuronal calcium indicator. Finally, we show the translational potential of SLED by rescuing photoreceptor degeneration in Prph2rds/rds mice and by developing an oncolytic vector that can selectively induce apoptosis in SF3B1 mutant cancer cells. The flexibility of SLED technology enables new avenues for basic and translational research.

category: molecular biology


title: Characterizing circadian connectome of O. tenuiflorum using an integrated network theoretic framework

abstract: Ocimum tenuiflorum (Tulsi) is among the most valuable medicinal plants as almost every part of this herb and the essential oil it produces possess pharmaceutical properties that have been utilized since ancient times to cure a large number of diseases. Like in other plants, circadian clock in Tulsi regulate important physiological processes like growth, development, defence etc. by anticipating environmental cues. In the present work, identification and characterization of core circadian clock and clock associated proteins in Tulsi genome is reported. We mined 24 core clock (CC) proteins in 56 template plant genomes to build their hidden Markov models (HMMs). These HMMs were used to identify 24 core clock proteins in O. tenuiflorum which were further subjected to functional annotation. A hybrid network theoretic methodology comprising of random walk with restart (RWR) and graphlet degree vectors (GDV) was implemented to explore the local topology of the interologous, Tulsi protein interaction network (TulsiPIN) and mine CC associated raw candidate proteins. Statistical and biological significance of the raw candidates was determined using permutation and enrichment tests. A total of 70 putative CC associated proteins were identified which were further subjected to functional annotation.


title: The conserved transcriptional program of metazoan male germ cells uncovers ancient origins of human infertility

abstract: Germ cells provide the cellular basis for sexual reproduction in multicellular animals. In males, germ cells differentiate into sperm, one of the most morphologically diverse eukaryotic cell types. Owing both to this remarkable diversity and to the rapid evolution of reproduction-related genes, the transcriptional program of male germ cells is widely regarded as divergent across species1,2. However, the possibility that these cells retain a distinctive evolutionarily-conserved genetic basis remains largely unexplored. Here we show, using phylostratigraphy, that the complex male germ cell transcriptome has an old evolutionary origin shared between vertebrate and invertebrate species. Through multilayer network analysis of the human, mouse and fruit fly meiotic transcriptome, we establish that old genes serve as a genetic scaffold from which complexity has evolved, and identify a core set of 79 ancient functional interactions at the heart of male germ cell identity. By silencing a cohort of 920 candidate genes likely to affect the acquisition and maintenance of this identity, we uncover 164 previously unknown spermatogenesis genes. Integrating this information with whole-exome sequencing data from azoospermic men reveals three novel genetic causes of human male infertility shared between species that have diverged for more than 600 million years. Our results redefine the importance of evolutionary history on human reproductive disease and illustrate the translational potential of a new synergy between comparative network biology and medical genetics. We anticipate that our open-access and easily-adaptable interdisciplinary research platform will be harnessed in the context of other cell types and diseases.


category: evolutionary biology


title: Antiviral and Neuroprotective Abilities of Influenza Virus Infection in Tractable Brain Organoids

abstract: Human pluripotent stem cell (hPSC)-derived brain organoids offer an unprecedented opportunity for various applications as an in vitro model, such as modeling virus infection and drug screening. In this study, we present an experimental brain organoid platform for modeling infection with multiple viruses (e.g., influenza virus or enterovirus). Brain organoids challenged by influenza viruses (H1N1-WSN and H3N2-HKT68) had decreased overall organoid size, similar to ZIKA virus infection, while enteroviruses (EV68 and EV71) infected brain organoids displayed the opposite result. Then, we studied the molecular events in WSN-infected organoids, and we found that WSN could widely infect multiple cell types, and preferentially infected MAP2+ neurons compared to SOX2+ neural stem cells (NSCs) and GFAP+ astrocytes in brain organoids, and induced apoptosis of NSCs and neurons, but not astrocytes. The inflammatory responses in organoids observed to occur (Tumor necrosis factor alpha, interferon gamma, and interleukin 6) after WSN infection may further facilitate brain damage. Furthermore, transcriptional profiling revealed several upregulated genes (CSAG3 and OAS2) and downregulated genes (CDC20B, KCNJ13, OTX2-AS1, CROCC2, and F5) after WSN infection for 24 hpi and 96 hpi, implicating antiviral drugs development responses to WSN. Finally, we explored neurotrophic factors (e.g., BDNF, GDNF, and NT3) and PYC-12 as antiviral and neuroprotective reagents, which could significantly suppress virus infection, apoptosis, and inflammatory responses. Collectively, we established a tractable experimental model system to investigate the impact and mechanism of virus infection on human brain development, and provide a platform for rapidly screening therapeutic compounds, advancing the development of antiviral strategies.

category: developmental biology


title: The Fox gene repertoire in the annelid Owenia fusiformis reveals multiple expansions of the foxQ2 class in Spiralia

abstract:
Fox genes are a large and conserved family of transcription factors involved in many key biological processes, including embryogenesis and body patterning. Although the role of Fox genes has been studied in an array of model systems, comprehensive comparative studies in Spiralia--a large clade of invertebrate animals including molluscs and annelids--are scarce but much needed to better understand the evolutionary history of this gene family. Here, we reconstruct and functionally characterise the Fox gene complement in the annelid Owenia fusiformis, a slow evolving species and member of the sister group to all remaining annelids. The genome of O. fusiformis contains at least a single ortholog of each of the 23 Fox gene classes that are ancestral to Bilateria, except for foxE and foxI. Temporal and spatial expression dynamics reveal a conserved role of Fox genes in gut formation (foxA), mesoderm patterning (foxF, foxL1, foxC, foxH) and cilia formation (foxJ1) in Annelida and Spiralia. Moreover, we uncover an ancestral expansion of foxQ2 genes in Spiralia, represented by 11 paralogs in O. fusiformis. Notably, although all foxQ2 copies have apical expression in O. fusiformis, they show variable spatial domains and staggered temporal activation, which suggest cooperation and sub-functionalisation among foxQ2 genes for the development of apical fates in this annelid. Altogether, our study informs the evolution and developmental roles of Fox genes in Annelida and Spiralia generally, providing the basis to explore how regulatory changes in Fox gene expression might have contributed to developmental and morphological diversification in Spiralia.

Significance statementThe role of Fox genes, a group of DNA-binding proteins required for the formation of many animal organs, is poorly understood in invertebrate groups such as molluscs and annelids. Here, by studying the genome and embryogenesis of the annelid Owenia fusiformis, we demonstrate that Fox genes are involved in the development of the gut, muscles, cilia, and nervous system. Importantly, we find that a group of Fox genes (referred to as foxQ2) expressed in the anterior end of most animals has more copies in annelids and molluscs than in other invertebrate groups like insects and sea stars. Together, our findings clarify the evolution of Fox genes and their contribution to the diversity of forms and organs found in marine invertebrates.


title: A novel quantitative trait locus implicates Msh3 in the propensity for genome-wide short tandem repeat expansions in mice

abstract: Short tandem repeats (STRs) are a class of rapidly mutating genetic elements characterized by repeated units of 1 or more nucleotides. We leveraged whole genome sequencing data for 152 recombinant inbred (RI) strains from the BXD family derived from C57BL/6J and DBA/2J mice to study the effects of genetic background on genome-wide patterns of new mutations at STRs. We defined quantitative phenotypes describing the numbers and types of germline STR mutations in each strain and identified a locus on chromosome 13 associated with the propensity of STRs to expand. Several dozen genes lie in the QTL region, including Msh3, a known modifier of STR stability at pathogenic repeat expansions in mice and humans. Detailed analysis of the locus revealed a cluster of variants near the 5 end of Msh3, including multiple protein-coding variants within the DNA mismatch recognition domain of MSH3, and a retrotransposon insertion overlapping an annotated exon. Additionally, gene expression analysis demonstrates co-localization of this QTL with expression QTLs for multiple nearby genes, including Msh3. Our results suggest a novel role for Msh3 in regulating genome-wide patterns of germline STR mutations and demonstrate that inherited genetic variation can contribute to variability in accumulation of new mutations across individuals.

category: genomics


title: Integrative experimental/computational approach establishes active cellular protrusion as the primary driving force of phagocytic spreading by immune cells

abstract:
The dynamic interplay between cell adhesion and protrusion is a critical determinant of many forms of cell motility. When modeling cell spreading on adhesive surfaces, traditional mathematical treatments often consider passive cell adhesion as the primary, if not exclusive, mechanistic driving force of this cellular motion. To better assess the contribution of active cytoskeletal protrusion to immune-cell spreading during phagocytosis, we here develop a computational framework that allows us to optionally investigate both purely adhesive spreading ("Brownian zipper hypothesis") as well as protrusion-dominated spreading ("protrusive zipper hypothesis"). We model the cell as an axisymmetric body of highly viscous fluid surrounded by a cortex with uniform surface tension and incorporate as potential driving forces of cell spreading an attractive stress due to receptor-ligand binding and an outward normal stress representing cytoskeletal protrusion, both acting on the cell boundary. We leverage various model predictions against the results of a directly related experimental companion study of human neutrophil phagocytic spreading on substrates coated with different densities of antibodies. We find that the concept of adhesion-driven spreading is incompatible with experimental results such as the independence of the cell-spreading speed on the density of immobilized antibodies. In contrast, the protrusive zipper model agrees well with experimental findings and, when adapted to simulate cell spreading on discrete adhesion sites, it also reproduces the observed positive correlation between antibody density and maximum cell-substrate contact area. Together, our integrative experimental/computational approach shows that phagocytic spreading is driven by cellular protrusion, and that the extent of spreading is limited by the density of adhesion sites.

Author SummaryTo accomplish many routine biological tasks, cells must rapidly spread over different types of surfaces. Here, we examine the biophysical underpinnings of immune cell spreading during phagocytosis, the process by which white blood cells such as neutrophils engulf pathogens or other foreign objects. Our computational framework models the case in which a human neutrophil spreads over a flat surface coated with antibodies, which we also test experimentally in a companion paper. Our primary purpose is to assess whether phagocytic spreading is actively driven by protrusive forces exerted by the cell, or passively by adhesive forces acting between receptors in the cell membrane and antibodies on the surface. By directly comparing our model predictions to experimental results, we demonstrate that phagocytic spreading is primarily driven by protrusion, but the extent of spreading is still limited by the availability of binding sites. Our findings improve the fundamental understanding of phagocytosis and may also pave the way for future investigations of the balance between adhesion and protrusion in other forms of cell spreading, such as wound healing or cancer cell migration.

category: biophysics


title: Increased core body temperature exacerbates defective protein prenylation in mouse avatars of mevalonate kinase deficiency

abstract: Mevalonate kinase deficiency (MKD) is caused by biallelic loss-of-function mutations in MVK, leading to recurrent fevers and systemic inflammation. We describe new mouse avatars of MKD bearing p.Val377Ile (the commonest variant) or deletions in Mvk. Compound heterozygous mice recapitulated the biochemical phenotype of MKD, with build-up of unprenylated GTPases and increased plasma mevalonic acid. Mice with different deficiencies in mevalonate kinase revealed new insights into the genotype-phenotype relationship and mirrored the variability in the prenylation defect in human MKD, with p.V377I homozygous mice having a milder phenotype than compound heterozygous animals. The inflammatory response to LPS was enhanced in compound heterozygous mice in vivo and elevated serum interleukin-1{beta} was abrogated by NLRP3 inflammasome inhibition. Increased temperature dramatically but reversibly exacerbated the deficit in the mevalonate pathway and defective prenylation in vitro and in vivo, highlighting increased body temperature as a likely trigger of inflammatory flares and an additional potential target for future therapeutic approaches.

category: physiology


title: Evaluation of entomopathogenic nematodes against red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

abstract: The entomopathogenic nematodes play a pivotal role as bio-control agents of different species of insect pests including red palm weevil. In current investigations, infective capabilities of four species of entomopathogenic nematodes including Hetrerorhabditis bacteriophora, Steinernema feltiae, Steinernema glesri and Steinernema carpocapsae were evaluated against larvae, pupae and adult red palm weevil, under laboratory and field conditions. The pathogenic potential of selected nematode species was assessed based on dissection and adult emergence of weevils. Results indicated that S. carpocapsae and H. bacteriophora with 94.68 and 92.68% infection were found most effective EPN species against red palm weevil larvae. Focusing the adult emergence, aforementioned EPNs were found most pathogenic when pupae of red palm weevil were treated and resulted into 83.60 and 80.20% infested pupae. It was noted that the adult emergence is the better option for the evaluation of pathogenic potential of EPN compared to dissection of insect. The S. carpocapsae was found most effective against 6th instar larvae of red palm weevil and caused 100% mortality at 340 hours after treatment; whereas; S. glesri and S. feltiae were found least pathogenic and caused 70 and 76% mortality. All the evaluated nematode species were found highly infective under field conditions. The S. carpocapsae was found most pathogenic causing 83.60% mortality of red palm weevil. Overall; the tested nematodes were found most effective against larvae followed by adult weevils. The tested nematode species were found least effective against pupae of red palm weevil. Based on current findings, it is concluded that the tested species of nematodes can be used as sustainable option for the management of red palm weevil.

category: ecology


title: Sights and sounds of dolphins, Tursiops truncatus, preying on native fish of San Diego Bay and offshore in the Pacific Ocean

abstract: For the first time, dolphins wearing video cameras were observed capturing and eating live native fish. While freely swimming in San Diego Bay, one dolphin caught 69 resident fish, 64 demersal, 5 near surface, while the other caught 40, 36 demersal and 4 near the surface. Two other dolphins were observed capturing 135 live native fish in a sea water pool. Two additional dolphins were observed feeding opportunistically during open water sessions in the Pacific Ocean. Notably, one of these dolphins was observed to consume 8 yellow-bellied sea snakes (Hydrophis platurus). Searching dolphins clicked at intervals of 20 to 50 ms. On approaching prey, click intervals shorten into a terminal buzz and then a squeal. Squeals were bursts of clicks that varied in duration, peak frequency, and amplitude. Squeals continued as the dolphin seized, manipulated and swallowed the prey. If fish escaped, the dolphin continued the chase and sonar clicks were heard less often than the continuous terminal buzz and squeal. During captures, the dolphins lips flared to reveal nearly all of the teeth. The throat expanded outward. Fish continued escape swimming even as they entered the dolphins mouth, yet the dolphin appeared to suck the fish right down.

category: animal behavior and cognition


title: What drives wolf preference towards wild ungulates? Insights from a multi-prey system in the Slovak Carpathians

abstract: The wolf is a generalist-opportunistic predator that displays diverse and remarkably adaptable feeding strategies across its range with local adaptations to certain prey species depending on their availability and vulnerability. The multi-prey system of the Slovak Carpathians supports important portion of the European wolf population; however, it has been markedly understudied. We evaluated winter diet composition and prey selection of Slovak wolves based on 321 scat samples collected between September - February within four different study areas during 2015 - 2017. The winter diet of wolves in the Slovak Carpathians was characterized by a 98% occurrence of wild large-sized and medium-sized ungulates with red deer occurring in wolf scats most often, consistent with their highest density among other wild ungulates. However, by comparing the consumption with availability of wild prey, we found that wolves in fact selected for wild boar especially in areas with higher altitudinal range, while selected for red deer in areas with low altitudinal range where this prey species was more spatially predictable. Although wolves showed the potential to switch between red deer and wild boar when their density increases, we found that this variation can be rather linked to changing prey vulnerability, which is dependent on particular environmental conditions at local scale such as topography and snow accumulation. The present study provides valuable insights into the winter foraging ecology of Slovak wolves in a multi-prey system of the Carpathians and allows for practical implications in the management of the rapidly increasing populations of wild ungulates across Europe.

category: zoology


title: Presence or absence of a prefrontal sulcus is linked to reasoning performance during child development

abstract: The relationship between structural variability in late-developing association cortices like the lateral prefrontal cortex (LPFC) and the development of higher-order cognitive skills is not well understood. Recent findings show that the morphology of LPFC sulci predicts reasoning performance; this work led to the observation of substantial individual variability in the morphology of one of these sulci, the para-intermediate frontal sulcus (pimfs). Here, we sought to characterize this variability and assess its behavioral significance. To this end, we identified the pimfs in a developmental cohort of 72 participants, ages 6-18. When controlling for age, the presence or absence of the ventral component of the pimfs was associated with reasoning, as was the total surface area of pimfs. These findings show that multiple features of sulci can support the development of complex cognitive abilities and highlight the importance of considering individual differences in local morphology when exploring the neurodevelopmental basis of cognition.


title: Fluctuation-driven plasticity allows for flexible rewiring of neuronal assemblies

abstract: Synaptic connections in neuronal circuits are modulated by pre- and post-synaptic spiking activity. Heuristic models of this process of synaptic plasticity can provide excellent fits to results from in-vitro experiments in which pre- and post-synaptic spiking is varied in a controlled fashion. However, the plasticity rules inferred from fitting such data are inevitably unstable, in that given constant pre- and post-synaptic activity the synapse will either fully potentiate or depress. This instability can be held in check by adding additional mechanisms, such as homeostasis. Here we consider an alternative scenario in which the plasticity rule itself is stable. When this is the case, net potentiation or depression only occur when pre- and post-synaptic activity vary in time, e.g. when driven by time-varying inputs. We study how the features of such inputs shape the recurrent synaptic connections in models of neuronal circuits. In the case of oscillatory inputs, the resulting structure is strongly affected by the phase relationship between drive to different neurons. In large networks, distributed phases tend to lead to hierarchical clustering. Our results may be of relevance for understanding the effect of sensory-driven inputs, which are by nature time-varying, on synaptic plasticity, and hence on learning and memory.


title: Cellular and Circuit Organization of the Locus Coeruleus of Adult Mice

abstract: The locus coeruleus (LC) houses the vast majority of noradrenergic neurons in the brain and regulates many fundamental functions. While efferent projections of the LC have been extensively investigated, little is known about its local circuit organization. Here, we performed large-scale multi-patch recordings of LC noradrenergic neurons to profile their morpho-electric properties while simultaneously examining their interactions. LC neurons were diverse and could be classified into two major morpho-electric types. While chemical transmission was not detected, these neurons electrically connected at a rate comparable to many brain regions. Most connections form between dendrites and were restricted to narrowly spaced pairs or small clusters of neurons of the same type. Furthermore, more than two coupled pairs were often identified from individual recording sets that follow a train-like organizational pattern. The assembly of LC neurons follows a spatial and cell type-specific wiring principle that may be imposed by a unique train-like rule.


title: Fertilizer reduction with bio-organic fertilizer to regulate the root soil microbial community structure to improve Baby Chinese cabbage yield

abstract:
Using high-throughput sequencing, this study aimed to explore the response of soil microbial community and Baby Chinese cabbage yield to the reduction of chemical fertilizers combined with bio-organic fertilizer in the Gansu plateau, China. Our experiments consisted of conventional fertilizer (CK), 30% chemical fertilizer reduction + 6,000 kg bio-organic fertilizer (T1), 30% chemical fertilizer reduction + 9,000 kg bio-organic fertilizer (T2), 40% chemical fertilizer reduction + 6,000 kg bio-organic fertilizer (T3), and 40% chemical fertilizer reduction + 9,000 kg bio-organic fertilizer (T4). Compared with CK, soil microbial diversity and richness were higher for all treatments with added bio-organic fertilizer. PCoA showed that the bacterial and fungal communities in T2 and T4 were similar to each other. Redundancy and Spearmans correlation analyses of microbial communities and soil physicochemical properties revealed that reductions in chemical fertilizer rate combined with bio-organic fertilizer had a stronger impact on the fungal than the bacterial community. They also increased the relative abundance of the dominant bacterial and fungal phyla. Baby Chinese cabbage yield was relatively higher under the combined bio-organic fertilizer plus reduced chemical fertilizer rate with T2 showing the highest yield. Therefore, this approach is feasible for sustainable agricultural, cost-effective and profitable crop production.

ImportanceO_LIBio-organic + moderately reduced chemical fertilizer raised Chinese cabbage yield
C_LIO_LIBio-organic + chemical fertilizer was more efficacious than either one alone
C_LIO_LIPresence of bio-organic fertilizer enhanced overall rhizosphere physicochemistry
C_LIO_LIBio-organic fertilizer improved beneficial bacterial & fungal abundance & diversity
C_LIO_LIFertilizer combination sustainably & cost-effectively improves crop & soil quality
C_LI


title: The GEF-GAP pair Solo and DLC3 balances Rho activity during endocytic transport

abstract: The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How defined pairs of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) come together to locally balance GTPase activation cycles in this process is nevertheless largely unclear. By performing a microscopy-based RNAi screen we here identify the RhoGEF protein Solo as a counterpart of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that the interplay between Solo and DLC3 controls not only the activity but also total protein levels of RhoB. Together, the results of our study uncover the first functionally connected RhoGAP-RhoGEF pair at endomembranes and place the Solo-DLC3 axis at the core of endocytic trafficking.

category: cell biology


title: Efficient Gene Knockout in Salivary Gland Epithelial Organoid Cultures

abstract:
We have developed methods to achieve efficient CRISPR-Cas9 mediated gene knockout in ex vivo mouse embryonic salivary epithelial organoids. Salivary epithelial organoids provide a valuable model for characterizing cell signaling, differentiation, and epithelial morphogenesis, but research has been limited by a paucity of efficient gene perturbation methods. Here, we demonstrate highly efficient gene perturbation by transient transduction of guide RNA-expressing lentiviruses into Cas9-expressing salivary epithelial buds isolated from Cas9 transgenic mice. We first show that salivary epithelial buds can be cultured in low-concentration, non-solidified Matrigel suspensions in 96-well plates, which greatly increases sample throughput compared to conventional cultures embedded in solidified Matrigel. We then describe an efficient workflow to produce experiment-ready, high-titer lentiviruses within 1 week after molecular cloning. To track transduced cells, we designed the lentiviral vector to co-express a nuclear fluorescent reporter with the guide RNA. We routinely achieve 50-80% transduction efficiency as indicated by the fluorescent reporter. Importantly, we detected robust loss of targeted protein products when testing 8 guide RNAs for 3 different genes. Moreover, targeting the {beta}1 integrin gene (Itgb1) inhibited branching morphogenesis, which supports the importance of cell-matrix adhesion in driving branching morphogenesis. In summary, we have established a lentivirus-based method that can efficiently perturb genes of interest in salivary epithelial organoids, which will greatly facilitate studies of specific gene functions using this system.

One-sentence description of our articleWe present a CRISPR-Cas9 mediated gene perturbation system in mouse embryonic salivary epithelial organoid cultures and use it to demonstrate a critical role for {beta}1 integrin in branching morphogenesis.

category: genetics


title: Aging the Brain: Multi-Region Methylation Principal Component Based Clock in the Context of Alzheimer's Disease

abstract: Alzheimers disease (AD) risk increases exponentially with age and is associated with multiple molecular hallmarks of aging, one of which is epigenetic alterations. Epigenetic age predictors based on 5 cytosine methylation (DNAm) have previously suggested that biological age acceleration may occur in AD brain tissue. To further investigate brain epigenetic aging in AD, we generated a novel age predictor termed PCBrainAge that was trained solely in cortical samples. This predictor utilizes a combination of principal components analysis and regularized regression, which reduces technical noise and greatly improves test-retest reliability. For further testing, we generated DNAm data from multiple brain regions in a sample from the Religious Orders Study and Rush Memory & Aging Project. PCBrainAge captures meaningful heterogeneity of aging, calculated according to an individuals age acceleration beyond expectation. Its acceleration demonstrates stronger associations with clinical AD dementia, pathologic AD, and APOE {varepsilon}4 carrier status compared to extant epigenetic age predictors. It does so across multiple cortical and subcortical regions. Overall, PCBrainAge is useful for investigating heterogeneity in brain aging, as well as epigenetic alterations underlying AD risk and resilience.

category: bioinformatics


title: Therapeutic targeting of differentiation state-dependent metabolic vulnerabilities in DIPG

abstract: H3K27M diffuse intrinsic pontine gliomas (DIPG) exhibit cellular heterogeneity comprising less-differentiated, stem-like glioma cells that resemble oligodendrocyte precursors (OPC) and more differentiated astrocyte (AC)-like cells. H3K27M DIPG stem-like cells exhibit tumor-seeding capabilities in vivo, a feature lost or greatly diminished in the more differentiated AC-like cells. In this study, we established isogenic in vitro models of DIPG that closely recapitulated the OPC-like and AC-like phenotypes of DIPG cells. Using these tools, we performed transcriptomics, metabolomics, and bioenergetic profiling to identify metabolic programs operative in the different cellular states. From this, we defined new strategies to selectively target metabolic vulnerabilities within the specific tumor populations. Namely, we showed that the AC-like cells exhibited a more mesenchymal phenotype and were thus sensitized to ferroptotic cell death. In contrast, OPC-like cells upregulated cholesterol metabolism and mitochondrial oxidative phosphorylation (OXPHOS) and were accordingly more sensitive to statins and OXPHOS inhibitors. Additionally, statins and OXPHOS inhibitors showed efficacy and extended survival in preclinical orthotopic models established with stem-like H3K27M DIPG cells. Together, this study demonstrates that cellular subtypes within DIPGs harbor distinct metabolic vulnerabilities that can be uniquely and selectively targeted for therapeutic gain.

category: cancer biology


title: Measurements of damage and repair in aging mice and humans reveals that robustness and resilience decrease with age, operate over broad timescales, and are affected differently by interventions

abstract: As an organism ages, its health-state is determined by a balance between the processes of damage and repair. Measuring these processes requires longitudinal data. We extract damage and repair rates from repeated observations of health deficits in mice and humans to explore the contribution of robustness and resilience, which respectively decrease damage rates and increase repair rates, to aging health. We find a conserved decline with age in robustness and resilience in mice and humans, implying that both contribute to worsening health in aging. A decline in robustness, however, has a greater effect than a decline in resilience on accelerating damage accumulation with age, and a greater association with reduced survival. We also find that deficits are damaged and repaired over a wide range of timescales ranging from the shortest measurement scales towards organismal lifetime timescales. We explore the effect of systemic interventions that have been shown to improve health, including the angiotensin-converting enzyme inhibitor enalapril and voluntary exercise for mice, and household wealth for humans. We find that these interventions affect both damage and repair rates - but in different proportions for different interventions. These findings have implications for how health in aging, and interventions targeting health, are conceptualized and assessed.

category: systems biology


title: TUNNELING NANOTUBES CONNECT BLOOD-BRAIN BARRIER CELLS: ROLE OF PERICYTES IN BARRIER PRESERVATION DURING ISCHEMIA

abstract:
Crosstalk mechanisms between pericytes, endothelial cells, and astrocytes preserve integrity and function of the blood-brain-barrier (BBB) under physiological conditions. Long intercellular channels allowing the transfer of small molecules and organelles between distant cells called tunneling nanotubes (TNT) represent a potential substrate for energy and matter exchanges between the tripartite cellular compartments of the BBB. However, the role of TNT across BBB cells under physiological conditions and in the course of BBB dysfunction is unknown.

In this work, we analyzed the TNTs role in the functional dialogue between human brain endothelial cells, and brain pericytes co-cultured with human astrocytes under normal conditions or after exposure to ischemia/reperfusion, a condition in which BBB breakdown occurs, and pericytes participate in the BBB repair. Using live time-lapse fluorescence microscopy and laser-scanning confocal microscopy, we found that astrocytes form long TNT with pericytes and endothelial cells and receive functional mitochondria from both cell types through this mechanism. The mitochondrial transfer also occurred in multicellular assembloids of human BBB that reproduce the three-dimensional architecture of the BBB. Under conditions of ischemia/reperfusion, TNT formation is upregulated, and astrocytes exposed to oxygen-glucose deprivation were rescued from apoptosis by healthy pericytes through TNT-mediated transfer of functional mitochondria, an effect that was virtually abolished in the presence of TNT-destroying drugs. The results establish a functional role of TNT in the crosstalk between BBB cells and demonstrate that TNT-mediated mitochondrial transfer from pericytes rescues astrocytes from ischemia/reperfusion-induced apoptosis. Our data confirm that the pericytes might play a pivotal role in preserving the structural and functional integrity of BBB under physiological conditions and participate in BBB repair in brain diseases.

category: neuroscience


title: Speeded Inference Game: Opening a new chapter in the assessment of error awareness

abstract:
Influential theories on error processing assume that when we conduct errors adaptive processes are triggered to improve our behaviour and prevent errors in the future. These processes appear to be more effective after participants have detected an error. Therefore, the assessment of error awareness allowing a differential analysis of detected and undetected errors in the context of cognitive control and behavioural adjustments has gained more and more attention in the past decades. A common methodological challenge posed on all studies investigating error detection is that the number of undetected errors is usually relatively low.

Here, we introduce a gamified experimental task that uses an adaptive algorithm to generate a robust and stable amount of errors with a high rate of undetected errors. Further, we were able to identify error types, which interestingly differed in terms of their detection rate. Moreover, the game-like appearance of the novel experimental task led to highly motivated participants. The results of the first study were replicated and extended by a second behavioural study. Notably, in study 2, a change in task design specifically modulated error detection, while these changes did not affect the total error rate.

Potential applications of the open-source code will be discussed. With this newly developed paradigm, we wish to lay the ground for future research to understand better (neural) processes associated with error awareness.


title: Intelligence and Visual Mismatch Negativity: Is Pre-Attentive Visual Discrimination Related to General Cognitive Ability?

abstract:
Intelligence captures the cognitive ability level of an individual person and electroencephalography (EEG) has been used for decades to identify neurocognitive processes related to intelligence. The mismatch negativity (MMN) is a component of the event-related brain potential (ERP) that is elicited when, within a series of frequent standard stimuli, rare deviant stimuli are presented. As stimuli are typically presented outside the focus of attention, the MMN is suggested to capture automatic pre-attentive discrimination processes, and mixed results have been reported in relation to intelligence. However, the MMN and its relation to intelligence has only been studied in the auditory domain, thus preventing any conclusion about the involvement of automatic discrimination processes in humans dominant sensory modality, i.e., vision. Here we provide a first test of this question. Electroencephalography was recorded from 50 healthy participants during a passive visual oddball task that presented simple sequence violations as well as deviations within a more complex hidden pattern. Signed area amplitudes and fractional area latencies of the visual mismatch negativity (vMMN) were calculated with and without Laplacian transformation. Neither vMMN amplitudes nor vMMN latencies were significantly related to intelligence (Ravens Advanced Progressive Matrices, RAPM) and Bayes Factors suggest moderate evidence for the absence of associations. Our study presents the first test of whether neural indices of pre-attentive visual discrimination processes are related to intelligence. We discuss critical differences between the auditory and visual MMN and conclude with a discussion of limitations and recommendations for further research in this evolving field.

HIGHLIGHTS- Testing whether intelligence is related to automatic visual discrimination
- Visual mismatch negativity (vMMN) as a neural indicator of pre-attentive processing
- Passive visual oddball task presents simple and complex rule violations
- No association between intelligence and vMMN amplitudes or latencies
- Fundamental differences between auditory and visual MMN?


title: Bioinformatic analyses of plasmid resistome changes in pOXA-48

abstract:
Infections caused by carbapenem resistant Enterobacteriales (CPE) represent a significant threat in clinical settings. blaOXA-48 is one of the most frequent carbapenemase genes among Enterobacteriales. The blaOXA-48 is typically encoded on the prototypical IncL conjugative pOXA-48 plasmid. The pOXA-48 plasmid encodes only the blaOXA-48 resistance gene. However, aminoglycoside and extended spectrum {beta}-lactamase (ESBL) resistance genes have also been detected on the same pOXA-48 plasmid backbone. These pOXA-48 plasmids encoding additional antimicrobial resistance (AMR) genes have been associated with both poor patient outcome and increased minimal inhibitory concentrations (MICs) to antibiotics including broad-spectrum cephalosporins.

The blaOXA-48 gene was sourced from the pOXA-48 reference plasmid and set as a query using the BLASTn tool. Non-duplicate blaOXA-48 containing plasmids were downloaded, incompatibility typed and annotated for resistance genes using ResFinder 4.0. Bioinformatic analyses identified three distinct variants of the pOXA-48 plasmid encoding 4, 5, and 6 antimicrobial resistance genes. All plasmids encoded the ESBL blaCTX-M-14b, blaOXA-48 and either 2, 3 or 4 aminoglycoside resistance genes, in addition to conjugative transfer machinery. Plasmid variants 1 and 3 encoded aminoglycoside genes bracketed between IS26 and ISEc63 insertion elements, forming a potential transposon. The potential transposon structure had resemblance to the Tn5393 transposon (accession: M96392), including both aph(3)-Ib, aph(6)-Id genes, and a Tn3 resolvase. The IS element ISEcp1 lies upstream of blaCTX-M-14b. All three plasmid variants appear related. Notably, all pOXA-48 plasmid variants were identified in multiple countries. In particular, variant 1 including 6 AMR genes was detected in 7 unique countries.

Plasmids encoding additional AMR genes were associated with clinical/surveillance samples suggesting antibiotic pressure in clinical settings may promote changes in the resistome of pOXA-48. Acquisition of pOXA-48 resistant plasmids carrying additional AMR genes beyond blaOXA-48 can change the resistome of susceptible isolates in a single-step, rendering previously susceptible strains refractory to almost all available treatment options.


title: Earliest evidence for frugivory and seed dispersal by birds

abstract: The Early Cretaceous diversification of birds was a major event in the history of terrestrial ecosystems, occurring during the earliest phase of the Cretaceous Terrestrial Revolution. Frugivorous birds play an important role in seed dispersal today, and may have done so since their origins. However, evidence of this has been lacking. Jeholornis is one of the earliest-diverging birds, only slightly more derived than Archaeopteryx, but its cranial anatomy has been poorly understood, obscuring diet-related functional interpretations. Originally hypothesised to be granivorous based on seeds preserved as gut contents, this interpretation has become controversial. We conducted high-resolution synchrotron tomography on an exquisitely preserved new skull of Jeholornis, revealing remarkable cranial plesiomorphies combined with a specialised rostrum. We use this to provide a near-complete cranial reconstruction of Jeholornis, and exclude the possibility that Jeholornis was granivorous, based on morphometric analyses of the mandible (3D) and cranium (2D), and comparisons with the 3D alimentary contents of extant birds. We show that Jeholornis was at least seasonally frugivorous, providing the earliest evidence for fruit consumption in birds, and indicating that seed dispersal was present from early in the avian radiation. As highly-mobile seed dispersers, early frugivorous birds could expand the scope for biotic dispersal in plants, and may explain, in part, the subsequent evolutionary expansion of fruits, indicating a potential role of bird-plant interactions in the Cretaceous Terrestrial Revolution.

category: paleontology


title: Insights of the role of estrogen in obesity from two models of ERα deletion

abstract: Sex hormones play a pivotal role in physiology and disease. Estrogen, the female sex hormone, has been long implicated in having protective roles against obesity. However, the direct impact of estrogens in white adipose tissue (WAT) function and growth are not understood. Here, we show that deletion of estrogen receptor alpha (ER) from adipocytes using Adiponectin-cre does not affect adipose mass in male or female mice under normal or high-fat diet (HFD) conditions. However, loss of ER in adipocyte precursor cells (APs) via PdgfR-cre leads to exacerbated obesity upon HFD feeding in both male and female mice, with subcutaneous adipose (SWAT)-specific expansion in male mice. Further characterization of these mice revealed infertility and increased plasma levels of sex hormones, including estradiol in female mice and androgens in male mice. These findings compromise the study of estrogen signaling within the adipocyte lineage using the PdgfR-cre strain. However, AP transplant studies demonstrate that the increased AP hyperplasia in male SWAT upon PdgfR-cre-mediated ablation of ER is not driven by AP-intrinsic mechanisms, but are rather mediated by off-target effects. These data highlight the inherent difficulties in studying models that disrupt the intricate balance of sex hormones. Thus, better approaches are needed to study the cellular and molecular mechanisms of sex hormones in obesity and disease.


title: EEG evidence that morally relevant autobiographical memories can be suppressed

abstract: Remembering unpleasant events can trigger negative feelings. Fortunately, research indicates that unwanted memories can be suppressed to prevent them from intruding into awareness, improving our mental state. The current scientific understanding of memory suppression is, however, based mostly on simpler memories such as associations between words or pictures, which may not reflect how people control unpleasant memory intrusions in everyday life. Here, we investigated the neural and behavioural dynamics of suppressing personal and emotional autobiographical memories using a modified version of the Think/No-Think task. We asked participants to suppress memories of their own past immoral actions, which were hypothesised to be both highly intrusive and motivating to suppress. We report novel evidence from behavioural, ERP and EEG oscillation measures that autobiographical memory retrieval can be suppressed and suggest that autobiographical suppression recruits similar neurocognitive mechanisms as suppression of simple laboratory associations. Suppression did fail sometimes, and EEG oscillations indicated that such memory intrusions occurred from lapses in sustained control. Importantly however, participants improved at limiting intrusions with repeated practice. Furthermore, both behavioural and EEG evidence indicated that intentional suppression may be more difficult for memories of our morally wrong actions than memories of our morally right actions. The findings elucidate the neurocognitive correlates of autobiographical retrieval suppression and have implications for theories of morally motivated memory control.


title: Magnetoencephalography can reveal deep brain network activities linked to memory processes

abstract: Recording from deep neural structures such as hippocampus non-invasively and yet with high temporal resolution remains a major challenge for human neuroscience. Although it has been proposed that deep neuronal activity might be recordable during cognitive tasks using magnetoencephalography (MEG), this remains to be demonstrated as the contribution of deep structures to MEG recordings may be too small to be detected or might be eclipsed by the activity of large-scale neocortical networks. In the present study, we disentangled mesial activity and large-scale networks from the MEG signals thanks to blind source separation (BSS). We then validated the MEG BSS components using intracerebral EEG signals recorded simultaneously in patients during their presurgical evaluation of epilepsy. In the MEG signals obtained during a memory task involving the recognition of old and new images, we identified with BSS a putative mesial component, which was present in all patients and all control subjects. The time course of the component selectively correlated with SEEG signals recorded from hippocampus and rhinal cortex, thus confirming its mesial origin. This finding complements previous studies with epileptic activity and opens new possibilities for using MEG to study deep brain structures in cognition and in brain disorders.


title: Direct fluorescent labeling of NF186 and NaV1.6 in living primary neurons using bioorthogonal click chemistry

abstract: The axon initial segment (AIS) is a highly specialized neuronal compartment that regulates the generation of action potentials and maintenance of neuronal polarity. Despite its importance, live imaging of the AIS is challenging due to the limited number of suitable labeling methods. To overcome this limitation, we established a novel approach for live labeling of the AIS using unnatural amino acids (UAAs) and bioorthogonal click chemistry. The small size of the UAAs and the possibility of introducing them virtually anywhere into the target proteins make this method particularly suitable for live labeling and imaging of complex and spatially restricted proteins. With this approach, we labeled two large AIS components, the 186 kDa isoform of neurofascin (NF186) and the 260 kDa voltage-gated sodium channel (NaV1.6), and performed widefield and confocal microscopy in fixed and living neurons. Moreover, we demonstrated the applicability of this method by studying the localization of two epilepsy-causing NaV1.6 variants with a loss-of-function effect. Finally, to further improve the efficiency of the UAA incorporation, we developed adeno-associated viral (AAV) vectors for click labeling in primary neurons. The use of AAV vectors will facilitate the transfer of UAA-based click labeling technology to more complex biological systems, such as organotypic slice cultures, organoids, and animal models.


title: Highly efficient generation of blastocyst-like structures from mouse totipotent blastomere-like cells

abstract: Mammalian embryogenesis begins with a totipotent zygote. Early embryogenesis can be recapitulated by aggregated extended pluripotent stem cells (EPSCs) in a 3D culture system. However, the efficiency of generating blastoids is low and whether other reported totipotent stem cells retained a similar capacity remains unknown. Here we show that spliceosomal repressed totipotent blastomere-like cells (TBLCs) form blastocyst-like structures when aggregated in 3D microwells with around 80% efficiency. TBLC-blastoids resemble blastocyst in morphology and cell-lineage allocation, show similar transcriptional profile with natural blastocyst and contain more TE cells and fewer undefined intermediate cells compared to blastoids from EPSCs. Moreover, TBLC-blastoids can develop beyond the implantation stage in vitro and induce decidualization after transferred into uterus. In summary, we supply an alternative cell type to generate ameliorated blastoids highly efficiently for studying early mouse embryogenesis.


title: Environmental thresholds in the functional mycobiome of global drylands

abstract: Fungi are major drivers of ecosystem functions. Increases in aridity are known to negatively impact fungal communities in dryland ecosystems globally, however, much less is known on the potential influence of other environmental drivers. To fill this knowledge gap, we reanalyzed fungal data from 912 soil samples, providing the largest and most complete fungal community dataset from global drylands. We used machine learning tools to examine geographical patterns in community composition and spatial, edaphic, and climatic factors driving them. Further, we determined critical thresholds of community turnover along those gradients. Our analysis identifies UV index, climate seasonality, and sand content as the most important environmental predictors of community shifts, harbouring greatest association with the richness of putative plant pathogens and saprobes. Important nonlinear relationships existed with each of these fungal guilds, with increases in UV and temperature seasonality above 7.5 and 900 SD, respectively, being associated with an increased probability of plant pathogens and unspecified saprotrophs occurrence. Conversely, these environmental parameters had a negative relationship with litter and soil saprotrophs richness. Consequently, these functional groups might be differentially sensitive to environmental changes, which might result in an inevitable disturbance of current plant-soil dynamics in drylands.


title: Symbiosis with rhizobia limits range expansion only in polyploid legumes

abstract:
O_LIBoth mutualism and polyploidy are thought to influence invasion success in plants but few studies have tested their joint effects. Mutualism can limit range expansion when plants cannot find a compatible partner in a novel habitat, or facilitate range expansion when mutualism increases a plants niche breadth. Polyploids are also expected to have greater niche breadth because of greater self-compatibility and phenotypic plasticity, increasing invasion success.
C_LIO_LIFor 839 legume species, we compiled data from published sources to estimate ploidy, symbiotic status with rhizobia, specificity on rhizobia, and the number of introduced ranges.
C_LIO_LIWe found that diploid species have had limited spread around the globe regardless of whether they are symbiotic or how many partners of rhizobia they can host. Polyploids, in contrast, have been successfully introduced to many new ranges, but interactions with rhizobia constrain their range expansion. In a hidden state model of trait evolution, we also found evidence of a high rate of re-diploidization in symbiotic legume lineages, suggesting that symbiosis and ploidy may interact at macroevolutionary scales.
C_LIO_LIOverall, our results suggest that symbiosis with rhizobia affects range expansion only in polyploid legumes.
C_LI


title: Spatiotemporal changes in genetic diversity and structure of a recent fish invasion in eastern North America

abstract: Introduced and geographically expanding populations experience similar eco-evolutionary challenges, including founder events, genetic bottlenecks, and novel environments. Theory predicts that reduced genetic diversity resulting from such genetic phenomena limits the colonization success of introduced populations. We examined an invasive population of a Eurasian freshwater fish, Tench (Tinca tinca), that has been expanding geographically in eastern North America for three decades. Using genomic data, we evaluated evidence for single versus multiple introductions and the connectivity of the population across the entire range in which it has been spreading. Tench exhibited low levels of genetic diversity, a lack of marked population subdivision across time and space, and evidence of a recent genetic bottleneck. These results suggest that the invasion stemmed from a single introduction, consistent with the reported invasion history. Furthermore, the large genetic neighbourhood size and weak within-population genetic substructure suggest high connectivity across the invaded range, despite the large area occupied, and no evidence of substantial diminution of genetic diversity from the invasion core to the margins. As eradicating the species within a ~112 km radius would be necessary to prevent recolonization, eradicating Tench is likely not feasible at watershed--and possibly local--scales. Management should instead focus on reducing abundance in priority conservation areas to mitigate adverse impacts. Our study supports the argument that introduced populations can thrive despite recent bottlenecks and low levels of genetic diversity, and it suggests that landscape heterogeneity and population demographics can generate variability in spatial patterns of genetic diversity within a single range expansion.


title: FunHoP analysis reveals upregulation of mitochondrial genes in prostate cancer

abstract: Mitochondrial activity in cancer cells has been central to cancer research since Otto Warburg first published his thesis on the topic in 1956. Although Warburg proposed that oxidative phosphorylation in the tricarboxylic acid (TCA) cycle was perturbed in cancer, later research has shown that oxidative phosphorylation is activated in most cancers, including prostate cancer (PCa). However, more detailed knowledge on mitochondrial metabolism and metabolic pathways in cancers is still lacking. In this study we expand our previously developed method for analyzing functional homologous proteins (FunHoP), which can provide a more detailed view of metabolic pathways. FunHoP uses results from differential expression analysis of RNA-Seq data to improve pathway analysis. By adding information on subcellular localization based on experimental data and computational predictions we can use FunHoP to differentiate between mitochondrial and non-mitochondrial processes in cancerous and normal prostate cell lines. Our results show that mitochondrial pathways are upregulated in PCa and that splitting metabolic pathways into mitochondrial and non-mitochondrial counterparts using FunHoP adds to the interpretation of the metabolic properties of PCa cells.


title: A reduced-memory multi-layer perceptron withsystematic network weights generated and trainedthrough distribution hyper-parameters

abstract: A multi-layer perceptron (MLP) consists of a number of forward-connected weights (Wijk) from each feeding layer node (nij) to the many initially equivalent nodes (ni+1,k) in the next layer. Exact a priori order and search space of these weights (Wijk) is random and prone to redundancy, irreproducibility and non-optimality. We demonstrate that a weight subspace (Wijk for each i and j), generated systematically using a statistical distribution with predefined breakpoints and Genetic algorithm-trained hyper-parameters substantially reduces the computational complexity of an MLP and produces comparable or better performance than similarly trained equivalent models with fully defined weights. This distribution based neural network (DBNN) provides a novel framework to create very large neural network models with currently prohibitive memory requirements.


title: Protein-protein interaction map in pre-eclampsia through the interaction of hub genes, transcription factors and microRNAs

abstract: Pre-eclampsia causes complications in pregnancy and characterized by uremia, proteinuria and hypertension in unattended cases. Blood biomarkers for pre-eclampsia are lacking. In this study, microarray gene expression data from peripheral blood of pre-eclampsia women was analyzed. In our study we developed a combined network approach for hub node prediction regulated by transcription factors and microRNAs corresponding to pre-eclampsia. Differentially expressed genes (DEGs) interaction map was constructed using STRING database. JUN, RPL35, NDUFB2, ATP5I, UQCRQ, COX7C, and FN1 were predicted as potential novel hub genes. Pathway analysis showed metabolic pathways, cytokine signaling in the immune system, Wnt, and MAPK signaling pathways involvement in pre-eclampsia. Regulatory network analysis showed that transcription factors JUN and STAT1 were connected with hub nodes, and microRNAs (miRNAs) like hsa-miR-26b-5p and hsa-miR-155-5p. In conclusion, the expression pattern of hub genes, analyzed deciphers a molecular signature for understanding the pathophysiology of pre-eclampsia and prediction of biomarkers for diagnosis.


title: PRL1 and CK2 collaborate to time morning and evening phase in short photoperiods

abstract: The TIMELESS (TIM) phosphatase, Phosphatase of Regenerating Liver-1 (PRL-1), is required for setting behavioral phase especially under short photoperiod conditions. Yet the circadian clock neurons and underlying mechanisms important for these effects are unknown. Here we demonstrate that PRL-1 function in PDF neurons is necessary and sufficient for timing morning and evening phase under short photoperiod (6:18) conditions. In standard (12:12) photoperiod, we find that PRL-1 rescue or blocking PRL-1 knockdown in PDF neurons restores wild-type morning phase. Expression of the TIM kinase shaggy (sgg) in PDF neurons suppresses in an additive manner PRL-1 period lengthening and delayed morning phase further underscoring the role of PDF neurons. In contrast to knockdown in all clock neurons, knockdown of PRL-1 selectively in PDF clock neurons does not alter the timing of morning or evening phase under standard photoperiod conditions, suggesting PRL-1 function in non-PDF neurons may also be important. In contrast to SGG effects, we find that phase delay of evening onset due to expression of a dominant negative version of the TIM kinase CK2 (CK2alphaTik) requires PRL-1. Given that TIM is a substrate of both the PRL-1 phosphatase and CK2 kinase, we hypothesize that PRL-1 dephosphorylation at specific sites may be necessary for subsequent CK2 phosphorylation to time behavior.


title: Regional variation in cholinergic terminal activity determines the non-uniform occurrence of cortical slow-wave activity during REM sleep

abstract: Sleep consists of two basic stages: non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. NREM sleep is characterized by slow high-amplitude cortical EEG signals, while REM sleep is characterized desynchronized cortical rhythms. While, until recently, it has been widely believed that cortical activity during REM sleep is globally desynchronized, recent electrophysiological studies showed slow waves (SW) in some cortical areas during REM sleep. Electrophysiological techniques, however, have been unable to resolve the regional structure of these activities, due to relatively sparse sampling. We mapped functional gradients in cortical activity during REM sleep using mesoscale imaging in mice, and observed local SW patterns occurring mainly in somatomotor and auditory cortical regions, with minimum presence within the default mode network. The role of the cholinergic system in local desynchronization during REM sleep was also explored by calcium imaging of cholinergic terminal activity within the mouse cortex. Terminal activity was weaker in regions exhibiting SW activity more frequently during REM sleep. We also analyzed Allen Mouse Brain Connectivity dataset and found that these regions have weaker cholinergic projections from the basal forebrain.


title: Machine learning-aided multidimensional phenotyping of Parkinson's disease patient stem cell-derived midbrain dopaminergic neurons

abstract: Combining multiple Parkinsons disease (PD) relevant cellular phenotypes might increase the accuracy of midbrain dopaminergic (mDA) in vitro models. We differentiated patient-derived induced pluripotent stem cells (iPSCs) with a LRRK2 G2019S mutation, isogenic control and genetically unrelated iPSCs into mDA neurons. Using automated fluorescence microscopy in 384-well plate format, we identified elevated levels of -synuclein and Serine 129 phosphorylation (pS129), reduced dendritic complexity, and mitochondrial dysfunction. Next, we measured additional image-based phenotypes and used machine learning (ML) to accurately classify mDA neurons according to their genotype. Additionally, we show that chemical compound treatments, targeting LRRK2 kinase activity or -synuclein levels, are detectable when using ML classification based on multiple image-based phenotypes. We validated our approach using a second isogenic patient derived SNCA gene triplication mDA neuronal model. This phenotyping and classification strategy improves the exploitability of mDA neurons for disease modelling and the identification of novel PD drug targets.


title: Retinal neuroblast migration and laminar organization requires the cytoskeletal-interacting protein Mllt11

abstract:
BackgroundThe vertebrate retina is an organized laminar structure comprised of distinct cell types populating three nuclear layers. During development, each retinal cell type follows a stereotypical temporal order of genesis, differentiation, and migration, giving rise to its stratified organization. Once born, the precise positioning of cells along the apico-basal (radial) axis of the retina is critical for subsequent connections to form, relying on orchestrated migratory processes. While these processes are critical for visual function to arise, the regulators of cellular migration and retinal lamination remain largely unexplored.

ResultsWe report a role for a microtubule-interacting protein, Mllt11 (Myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 Fused Gene From Chromosome 1q) in mammalian retinal cell migration during retinogenesis. We show that Mllt11 loss-of-function in mouse retinal neuroblasts affected the migration of ganglion and amacrine cells into the ganglion cell layer, and led to their ectopic accumulation in the inner plexiform layer.

ConclusionsWe demonstrate that Mllt11 plays a critical role in the migration and lamination of neurons in the retina, and its loss impacted formation of the basal-most retinal layers.


title: APOEϵ4 and exercise interact to influence systemic and cerebral risk factors for dementia

abstract:
INTRODUCTIONAPOE{varepsilon}4 is the strongest genetic risk factor for Alzheimers disease and related dementias (ADRDs) affecting many different pathways that lead to cognitive decline. Exercise is one of the most widely proposed prevention, and intervention strategies to mitigate risk and symptomology of ADRDs. Importantly, exercise and APOE{varepsilon}4 affect similar processes on the body and brain. While both APOE{varepsilon}4, and exercise have been studied extensively, their interactive effects are not well understood.

METHODSTo address this, male and female APOE{varepsilon}3/{varepsilon}3, APOE{varepsilon}3/{varepsilon}4 and APOE{varepsilon}4/{varepsilon}4 mice ran voluntarily from wean (1mo) to midlife (12mo). Longitudinal and cross-sectional phenotyping was performed on the periphery and the brain, on markers of risk for dementia such as weight, body composition, circulating cholesterol composition, activities of daily living, energy expenditure, and cortical and hippocampal transcriptional profiling.

RESULTSData revealed chronic running decreased age-dependent weight gain, lean and fat mass, and serum LDL concentration dependent on APOE genotype. Additionally, murine activities of daily living and energy expenditure were significantly influenced by an interaction between APOE genotype and running in both sexes. Transcriptional profiling of the cortex and hippocampus predicted that APOE genotype and running interact to affect numerous biological processes including vascular integrity, synaptic/neuronal health, cell motility, and mitochondrial metabolism, in a sex-specific manner.

DISCUSSIONThese data provide compelling evidence that APOE genotype should be considered for population-based strategies that incorporate exercise to prevent ADRDs.


title: High Calcium Transport by Polycystin-2 (TRPP2) Induces Channel Clustering and Coordinated Oscillatory Behavior

abstract:
The regulation by Ca2+ of Ca2+-permeable ion channels represents an important mechanism in the control of cell function. Polycystin-2 (PC2, TRPP2), a member of the TRP channel family (Transient Potential Receptor), is a Ca2+ permeable non-selective cation channel. Previous studies from our laboratory demonstrated that physiological concentrations of Ca2+ do not regulate in vitro translated PC2 (PC2iv) channel activity. However, the issue as to PC2s Ca2+ permeability and regulation remain ill-defined. In this study, we assessed Ca2+ transport by PC2iv, in a lipid bilayer reconstitution system in the presence of a high Ca2+ gradient (CaCl2 100 mM cis, CaCl2 10 mM trans). PC2iv channel reconstitution was conducted in the presence of either 3:7 or 7:3 1-palmitoyl-2-oleoyl-choline (POPC) and ethanolamine (POPE) lipid mixtures. Reconstituted PC2iv showed spontaneous Ca2+ currents, in both lipid mixtures with a maximum conductance of 63 {+/-} 13 pS (n = 19) and 105 pS {+/-} 9.8 (n = 9), respectively. In both cases, experimental data were best fitted with the Goldman-Hodgkin-Katz equation, showing a reversal potential (Vrev ~ -27 mV) consistent with strict Ca2+ selectivity. The R742X mutated PC2 (PC2R742X), lacking the carboxy terminal domain of the channel showed no differences with wild type PC2. Interestingly, spontaneous Ca2+ current oscillations were observed whenever PC2-containing samples were reconstituted in the 3:7, but not 7:3 POPC:POPE lipid mixture. The amplitude and frequency of the oscillations were highly dependent on the applied voltage, the imposed Ca2+ gradient, and the presence of high Ca2+, which induced PC2 channel clustering as observed by atomic force microscopy (AFM). We also used the QuB suite to kinetically model the PC2 channel Ca2+ oscillations based on the presence of subconductance states in the channel. The encompassed data provide new evidence to support a high Ca2+ permeability by PC2, and a novel regulatory feedback mechanism dependent on the presence of Ca2+ and phospholipids on its function.

Statement of SignificanceThe regulation by Ca2+ of Ca2+-permeable ion channels represents an important mechanism in the control of cell function. The Transient Potential Receptor channel Polycystin-2 (TRPP2, PC2), is a Ca2+ permeable non-selective cation channel. Ca2+ transport by PC2 has largely been inferred by changes in reversal potential. This study provides experimental evidence on the Ca2+-transporting capabilities of PC2 in high Ca2+ that is modulated by lipids and generates a novel phenomenon of oscillatory currents by channel clustering and multiple subconductance behavior. PC2 can be self-regulated by feedback mechanisms, which are independent of external regulatory proteins. This oscillatory behavior, previously unknown for a single channel species, depend on the presence of Ca2+ interaction sites as have been postulated for the channel protein.


title: Design of an islet-on-a-chip device reveals glucose-stimulated respiration is substrate limited by glycolytic flux through PKM2

abstract:
1.Pancreatic islets respond metabolically to glucose by closing KATP channels resulting in Ca2+-influx and insulin secretion. Previous work showed that the importance of glycolytic flux in triggering insulin secretion. However, it is unclear whether the triggered ( first phase) secretion is further amplified by Ca2+-stimulation of mitochondrial NADH production and/or oxidative phosphorylation (OxPhos). To tease apart the metabolism of first phase glucose-stimulated insulin secretion, we designed an islet-on-a-chip microfluidic device to simultaneously measure O2-consumption rate (OCR) and Ca2+-activity of individual islets with high temporal resolution. We used finite element analysis to optimize the placement of sensor in optically clear microwells on a thin glass coverslip. The microfluidic channels were subsequently fabricated using O2-impermeable plastic to limit outside-in diffusion and push islets in the device against the microsensor. We validated our device using living mouse islets and well-established modulators of respiration. By inhibiting glycolysis and mitochondrial pyruvate transport, we showed islet OxPhos is limited by substrate-NADH rather than ADP in low and high glucose. We subsequently imaged glucose-stimulated OCR and Ca2+-influx simultaneously to reveal a biphasic respiratory response that is determined by glycolytic flux through pyruvate kinase (PKM2) and independent of Ca2+. Ultimately, we validated our device and successfully used it to show islet OxPhos is determined by glycolytic flux and not amplified by Ca2+-activity.

SignificanceLoss of the first phase glucose-stimulated insulin secretion is one of the earliest signs of Type 2 diabetes (T2D). Revealing the mechanisms that trigger and amplify first phase insulin secretion from pancreatic islets could lead to strategies to mitigate the onset of T2D. In this study, we designed an islet-on-a-chip microfluidic device to measure O2-consumption rate and Ca2+ activity of individual islets dynamically through first phase secretion (< 5 min). Our data show first phase islet respiration is limited by glycolytic production of pyruvate independent of Ca2+-influx.

category: bioengineering


title: Protonation-dependent alternating access of a spinster transporter, an emerging family of broad-specificity efflux pumps

abstract: Spinster (Spns) lipid transporters are critical for transporting sphingosine-1-phosphate (S1P) across cellular membranes. In humans, Spns2 functions as the main S1P transporter in endothelial cells, making it a potential drug target for modulating S1P signaling. We employed an integrated approach in lipid membranes to identify unknown conformational states of a bacterial Spns from Hyphomonas neptunium (HnSpns) and to define its proton- and substrate-coupled conformational dynamics. Our systematic study reveals conserved residues critical for protonation steps and their regulation, and how sequential protonation of these proton switches coordinates the conformational transitions in the context of a noncanonical ligand-dependent alternating access. A conserved periplasmic salt bridge (Asp60TM2:Arg289TM7) keeps the transporter in a closed conformation, while proton-dependent conformational dynamics are significantly enhanced on the periplasmic side, providing a pathway for ligand exchange. Furthermore, our resistance assays reveal substrate polyspecificity and HnSpns multidrug resistance (MDR) activity that underscore the previously unknown role of Spns proteins in MDR, beyond their activity in sphingolipid transport and signaling.


title: Proton-driven alternating access in a spinster transporter, an emerging family of broad-specificity efflux pumps