title: Larger lesion volume in people with multiple sclerosis is associated with increased transition energies between brain states and decreased entropy of brain activity

abstract:
BackgroundNeuronal loss, demyelination, and inflammation in the brains of people with multiple sclerosis (pwMS) can cause impairment or disability across a range of domains. However, the mapping between severity or location of this brain tissue damage and an individuals impairment remains challenging. Advanced neuroimaging techniques may enable us to better understand the neuropathological or compensatory mechanisms in MS and quantify the link between the brains functional activity and its structural backbone. One recently developed approach uses network control theory to characterize the brains energetic landscape using an individuals structural connectome and brain activity dynamics. While brain activity dynamics have previously been analyzed in MS cohorts, investigating how structural network damage, one of the hallmarks of MS, relates to shifts in activity dynamics has not yet been performed.

AimsWe apply brain activity dynamics analysis and network control theory (NCT) to functional time series and structural connectomes in healthy controls (HC) and pwMS with and without disability and compare the three groups resulting metrics. We also assessed the entropy of the three groups brain activity time series. Our hypotheses were that pwMS with disability would have increased brain state transition energies compared to pwMS without disability and controls, and that greater energies would be related to greater lesion burden and decreased entropy of brain activity.

Materials and MethodsNinety-nine pwMS and 19 HC were included in our study; individuals underwent MRI, including anatomical, diffusion and resting-state functional scans. Expanded Disability Status Scale (EDSS) was used to assess disability; 66 pwMS with EDSS<2 were considered as not having disability. Commonly recurring brain states were identified using k-means clustering of the regional brain activity time series. NCT was used to compute the energy required to transition between each pair of states or to remain in the same state, taking into account an individuals structural connectome. The entropy of brain activity time series was quantified and correlated with overall transition energy and lesion volume. Finally, logistic regression with ridge regularization was used to classify the groups using transition energies and demographics/clinical information and the models feature importance assessed.

ResultsSix brain states were identified, including pairs of states with high and low visual (VIS+/-), ventral attention (VAN)+/- and somatomotor (SOM+/-) network activity. Global entropy was negatively correlated with lesion volume (r=-0.20, p=0.03) as was global transition energy (r=-0.18, p=0.04) in pwMS. Smaller transition energies were associated with pwMS without disability, while larger transition energies were associated with pwMS with disability.

DiscussionWe conjecture that functional compensation of brain activity in pwMS without disability results in decreased transition energies between states compared to controls, but, as this mechanism diminishes, transition energies increase and disability occurs. We also conjecture that larger volumes of MS-related lesions result in greater energetic demands to transition between brain states which then leads to lower overall entropy of brain activity.


title: Single-cell immune profiling reveals novel thymus-seeding populations, T cell commitment, and multi-lineage development in the human thymus

abstract: T cell development in the mouse thymus has been studied rather extensively; in contrast, strikingly little is known regarding T cell development in the human thymus. To close this knowledge gap, we used a combination of single-cell techniques and functional assays to perform deep immune profiling of human T cell development, focusing on the initial stages of pre-lineage commitment. We identified three thymus-seeding progenitor populations that also have counterparts in the bone marrow. In addition, we found that the human thymus physiologically supports the development of monocytes, dendritic cells, and NK cells, as well as limited development of B cells. These results are an important step towards monitoring and guiding regenerative therapies in patients following hematopoietic stem cell transplantation.

category: immunology


title: Tyrosine kinase inhibitors trigger lysosomal damage-associated cell lysis to activate the NLRP3 inflammasome

abstract:
Inflammasomes are intracellular protein complexes that control proteolytic maturation and secretion of inflammatory interleukin-1 (IL-1) family cytokines and are thus important in host defense. While some inflammasomes are activated simply by binding to pathogen-derived molecules, others, including those nucleated by NLRP3 and NLRP1, have more complex activation mechanisms that are not fully understood. We screened a library of small molecules to identify new inflammasome activators that might shed light on activation mechanisms. In addition to validating dipeptidyl peptidase (DPP) inhibitors as NLRP1 activators, we find that clinical tyrosine kinase inhibitors (TKIs) including imatinib and masitinib activate the NLRP3 inflammasome. Mechanistically, these TKIs cause lysosomal swelling and damage, leading to cathepsin-mediated destabilization of myeloid cell membranes and cell lysis. This is accompanied by potassium (K+) efflux, which activates NLRP3. Both lytic cell death and NLRP3 activation but not lysosomal damage induced by TKIs are prevented by the cytoprotectant high molecular weight polyethylene glycol (PEG). Our study establishes a screening method that can be expanded for inflammasome research and immunostimulatory drug development, and provides new insight into immunological off-targets that may contribute to efficacy or adverse effects of TKIs.

One Sentence SummaryA functional small molecule screen identifies imatinib, masitinib and other tyrosine kinase inhibitors that destabilize myeloid cell lysosomes, leading to cell lysis and K+ efflux-dependent NLRP3 inflammasome activation.


title: Structural insights into the assembly and activation of IL-27 signalling complex

abstract: Interleukin 27 (IL-27) is a heterodimeric cytokine that elicits potent immuno-suppressive responses. Comprised of EBI3 and p28 subunits, IL-27 binds GP130 and IL-27R receptor chains to activate the JAK/STAT signalling cascade. However, how these receptors recognize IL-27 and form a complex capable of phosphorylating JAK proteins remains unclear. Here, we used cryo electron microscopy (cryoEM) to solve the structure of the IL-27 receptor recognition complex. Our data show how IL-27 serves as a bridge connecting IL-27R with GP130 to initiate signalling. While both receptors weakly bind the p28 component of the heterodimeric cytokine, EBI3 stabilizes the complex by binding a positively charged surface of IL-27R. We find that assembly of the IL-27 receptor recognition complex is distinct from both IL-12 and IL-6 cytokine families and provides a mechanistic blueprint for tuning IL-27 pleiotropic actions.


title: Towards phage therapy for acne vulgaris: Topical application in a mouse model

abstract:
Acne vulgaris is a common neutrophile-driven inflammatory skin disorder in which Cutibacterium acnes (C. acnes) bacteria play a significant role. Until now, antibiotics have been widely used to treat acne vulgaris, with the inevitable increase in bacterial antibiotic resistance. Phage therapy is a promising solution to the rising problem of antibiotic-resistant bacteria, utilizing viruses that specifically lyse bacteria.

Here, we explored the feasibility of phage therapy against C. acnes. By combining eight novel phages we had isolated, together with commonly used antibiotics, we achieved 100% eradication of clinically isolated C. acnes strains. Using topical phage therapy in an acne mouse model resulted in significantly superior clinical scores, as well as a reduction in neutrophil infiltration compared to the control group. These results demonstrate the potential of phage therapy in acne vulgaris treatment, especially when antibiotic-resistant strains are involved.

category: microbiology


title: SpheroidAnalyseR - an online platform for analysing data from 3D spheroids or organoids grown in 96-well plates

abstract:
Spheroids and organoids are increasingly popular three-dimensional (3D) cell culture models. Spheroid models are more physiologically relevant to a tumour compared to two-dimensional (2D) cultures and organoids are a simplified version of an organ with similar composition. Both can be used in cell migration studies, disease modelling and drug discovery. A drawback of these models is, however, the lack of appropriate analytical tools for high throughput imaging and analysis over a time course. To address this, we have developed an R Shiny app called SpheroidAnalyseR: a simple, fast, effective open-source app that allows the analysis of spheroid or organoid size data generated in a 96-well format. SpheroidAnalyseR processes and analyses datasets of image measurements that can be obtained via a bespoke software, described herein, that automates spheroid imaging and quantification using the Nikon A1R Confocal Laser Scanning Microscope. However, templates are provided to enable users to input spheroid image measurements obtained by user-preferred methods. SpheroidAnalyseR facilitates outlier identification and removal followed by graphical visualisation of spheroid measurements across multiple predefined parameters such as time, cell-type and treatment(s). Spheroid imaging and analysis can, thus, be reduced from hours to minutes, removing the requirement for substantial manual data manipulation in a spreadsheet application. The combination of spheroid generation in 96-well ultra-low attachment microplates, imaging using our bespoke software, and analysis using SpheroidAnalyseR toolkit allows high throughput, longitudinal quantification of 3D spheroid growth whilst minimising user input and significantly improving the efficiency and reproducibility of data analysis. Our bespoke imaging software is available from https://github.com/GliomaGenomics. SpheroidAnalyseR is available at https://spheroidanalyser7.azurewebsites.net, and the source code found at https://github.com/GliomaGenomics.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/481039v1_ufig1.gif" ALT="Figure 1">
View larger version (40K):
org.highwire.dtl.DTLVardef@1f779fborg.highwire.dtl.DTLVardef@698103org.highwire.dtl.DTLVardef@94f252org.highwire.dtl.DTLVardef@b1180e_HPS_FORMAT_FIGEXP  M_FIG Graphical abstract - SpheroidAnalyseR main steps.

A summary of the process followed within the SpheroidAnalyseR app. Dotted boxes are the steps required to produce the input data for SpheroidAnalyseR. Solid boxes show the 5 main steps of SpheroidAnalyseR: Data input, Preview, Outlier removal, Merging and Plotting.

C_FIG

category: cell biology


title: Integrated epigenome and transcriptome analysis of normal and arrested meiotic initiation during mouse spermatogenesis

abstract: The transition from mitotic to meiotic cell cycles is a transcriptional event that entails the activation of genes important for meiosis and requires germline-specific retinoic acid (RA) signaling target, Stra8. To identify novel transcription factors underlying mammalian meiotic initiation, we conducted integrative snATAC-seq and scRNA-seq analysis using wild-type and Stra8-deficient mouse testicular cells to map the chromatin accessibility and gene expression landscapes of normal and genetically arrested meiotic initiation. Our results identified a cluster of putative inhibitory transcription factors for meiotic initiation, which we consider "meiotic inhibitors". STRA8 binds to the regulatory sequences of these meiotic inhibitors and represses their expression upon meiotic initiation. In Stra8-deficient cells that suffer meiotic initiation arrest, the chromatin accessibility of these meiotic inhibitors is increased, concurrent with their uncontrolled and sustained expression. Among these meiotic inhibitors include KLF4, MAX, and MAZ. Importantly, by analyzing the single cell transcriptomes of human testes, our data show that these putative meiotic inhibitor genes are upregulated in early germ cells from patients with spermatogenic failure. Our study suggests that proper repression of meiotic inhibitors is essential for both mouse and human spermatogenesis.

category: developmental biology


title: A Chromodomain Mutation Identifies Separable Roles for C. elegans MRG-1 in Germline and Somatic Development

abstract: The packaging of DNA into chromatin strongly influences gene regulation. Post-translational modifications of histones, and the proteins that bind to them, alter the accessibility of chromatin and contribute to the activation and repression of genes. The human MRG15 (MORF4- Related Gene on chromosome 15) protein is a conserved chromodomain-containing protein that binds to methylated lysine 36 on histone H3 (H3K36me) and plays important roles in development, genome integrity, and gene regulation. MRG15 affects transcriptional regulation through its interactions with both histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes. MRG-1, its C. elegans homolog, has similarly been shown to have important roles in genomic integrity and development, and has also been shown to co- purify with HDAC complexes. Like MRG15, MRG-1 is predicted to bind to H3K36me through its chromodomain, yet despite mrg-1 mutants displaying developmental and germline phenotypes that overlap with H3K36 methyltransferase mutants, the role of the MRG-1 chromodomain has never been characterized. In this study, we examined meiotic cells lacking H3K36me3 to compare to mrg-1 mutant germ cell phenotypes, and mutated key residues in the MRG-1 chromodomain (CD) to assess its function. The CD mutations cause embryonic lethality but few post-embryonic germline defects, in contrast to mrg-1 deletion mutants which are viable but sterile. The CD mutations therefore disrupt somatic development despite the apparent absence of a requirement for MRG-1 protein in embryogenesis. Furthermore, the CD mutants exhibit a dominant RNAi resistance phenotype that is not seen in other mrg-1 mutant alleles. This suggests that the function of MRG-1, and the chromatin modifying complexes with which it interacts, includes tissue-specific interactions involving different requirements for a functional chromodomain. We propose that the CD mutation disrupts proper guidance of complexes within which it acts, and this guidance defect results in improper HDAC and/or HAT regulation causing an indirect defect in RNAi machinery expression or targeting.

category: genetics


title: Systematic Evaluation of Different R-loop Mapping Methods: Achieving Consensus, Resolving Discrepancies and Uncovering Distinct Types of RNA:DNA Hybrids

abstract: R-loop, a three-stranded nucleic acid structure, has been recognized to play pivotal roles in critical physiological and pathological processes. Multiple technologies have been developed to profile R-loops genome-wide, but the existing data suffer from major discrepancies on determining genuine R-loop localization and its biological functions. Here, we experimentally and computationally evaluate eight representative R-loop mapping technologies, and reveal inherent biases and artifacts of individual technologies as key sources of discrepancies. Analyzing signals detected with different R-loop mapping strategies, we note that genuine R-loops predominately form at gene promoter regions, whereas most signals in gene body likely result from structured RNAs as part of repeat-containing transcripts. Interestingly, our analysis also uncovers two classes of R-loops: The first class consists of typical R-loops where the single-stranded DNA binding protein RPA binds both the template and non-template strands. By contrast, the second class appears independent of Pol II-mediated transcription and is characterized by RPA binding only in the template strand. These two different classes of RNA:DNA hybrids in the genome suggest distinct biochemical activities involved in their formation and regulation. In sum, our findings will guide future use of suitable technology for specific experimental purposes and the interpretation of R-loop functions.

category: genomics


title: Complete sequences of the velvet worm slime proteins reveal that slime formation is enabled by disulfide bonds and intrinsically disordered regions

abstract: The slime of velvet worms (Onychophora) is a strong and fully biodegradable protein material, which upon ejection undergoes a fast liquid-to-solid transition to ensnare prey. However, the molecular mechanisms of slime self-assembly are still not well understood, notably because the primary structures of slime proteins are yet unknown. Combining transcriptomic and proteomic studies, we have obtained the complete primary sequences of slime proteins and identified key features for slime self-assembly. The high molecular weight slime proteins contain Cys residues at the N- and C-termini that mediate the formation of multi-protein complexes via disulfide bonding. Low complexity domains in the N-termini were also identified and their propensity for liquid-liquid phase separation established, which may play a central role for slime biofabrication. Using solid-state nuclear magnetic resonance, rigid and flexible domains of the slime proteins were mapped to specific peptide domains. The complete sequencing of major slime proteins is an important step towards sustainable fabrication of polymers inspired by the velvet worm slime.

category: bioengineering


title: Protein profiling of WERI RB1 and etoposide resistant WERI ETOR reveals new insights into topoisomerase inhibitor resistance in retinoblastoma

abstract: Chemotherapy resistance is one of the reasons for eye loss in patients with retinoblastoma (RB). RB chemotherapy resistance has been studied in different cell culture models such as WERI RB1. In addition, chemotherapy resistant RB subclones like the etoposide resistant WERI ETOR cell line have been established to improve the understanding of chemotherapy resistance in RB. The objective of this study was to characterize cell line models of an etoposide sensitive WERI RB1 and its etoposide resistant subclone WERI ETOR by proteomic analysis. Subsequently, quantitative proteomic data served for correlation analysis with known drug perturbation profiles. Methodically, WERI RB1 and WERI ETOR were cultured and prepared for quantitative mass spectrometry (MS). This was carried out in a data-independent acquisition (DIA) mode (Sequential Window Acquisition of All Theoretical Mass Spectra, SWATH-MS). The raw SWATH files were processed using neural networks in a library free mode along with machine learning algorithms. Pathway enrichment was performed using the REACTOME pathway resource and correlated to the Molecular Signature Database (MSigDB) hallmark gene set collections for functional annotation. Furthermore, a drug connectivity analysis using the L1000 database was used to correlate the mechanism-of-action (MOA) for different anticancer reagents to WERI RB1/WERI ETOR signatures. A total of 4,756 proteins were identified across all samples, showing a distinct clustering between the groups. Of these proteins, 64 were significantly altered (q < 0.05 & log2FC |>2|, 22% higher in WERI ETOR). Pathway analysis revealed an enriched metabolic pathway for "retinoid metabolism and transport" in WERI ETOR and for "sphingolipid de novo biosynthesis" in WERI RB1. In addition, this study revealed similar protein signatures of topoisomerase inhibitors in WERI ETOR as well as ATPase inhibitors, acetylcholine receptor antagonists and vascular endothelial growth factor receptor (VEGFR) inhibitors in WERI RB1. In this study, WERI RB1 and WERI ETOR were analyzed as a cell line model for chemotherapy resistance in RB using data-independent MS. The global proteome identified activation of "sphingolipid de novo biosynthesis" in WERI RB1 and revealed future potential treatment options for etoposide resistance in RB.

category: cancer biology


title: Alternative c-MYC mRNA transcripts as an additional tool for c-Myc2 and c-MycS production in BL60 tumors.

abstract: While studying c-Myc protein expression in several Burkitt lymphoma cell lines and in lymph nodes from a mouse model bearing a translocated c-MYC gene from the human BL line IARC-BL60, we surprisingly discovered a complex electrophoretic profile. Indeed, the BL60 cell line carrying the t(8;22) c-MYC translocation exhibits a simple pattern, with a single c-Myc2 isoform. Analysis of the c-MYC transcripts expressed by tumor lymph nodes in the mouse{lambda} c-MYC (Avy/a) showed for the first time five transcripts associated with t(8;22) c-MYC translocation. The five transcripts were correlated with the production of c-Myc2 and c-MycS, and loss of c-Myc1. The contribution of these transcripts to the oncogenic activation of the t(8;22) c-MYC is discussed.


title: Deep learning accurately quantifies plasma cell percentages on CD138-stained bone marrow samples

abstract:
BackgroundThe diagnosis of plasma cell neoplasms requires accurate, and ideally precise, percentages. This plasma cell percentage is often determined by visual estimation of CD138-stained bone marrow biopsies and clot sections. While not necessarily inaccurate, estimates are by definition imprecise. For this study, we hypothesized that deep learning can be used to improve precision.

MethodsWe trained a semantic segmentation-based convolutional neural network (CNN) using annotations of CD138+ and CD138- cells provided by one pathologist on small image patches of bone marrow and validated the CNN on an independent test set of images patches using annotations from two pathologists and a non-deep-learning commercial software. Once satisfied with performance, we scaled-up the CNN to evaluate whole slide images (WSIs), and deployed the system as a workflow friendly web application to measure plasma cell percentages using snapshots taken from microscope cameras.

ResultsOn validation image patches, we found that the intraclass correlation coefficients for plasma cell percentages between the CNN and pathologist #1, a non-deep learning commercial software and pathologist #1, and pathologists #1 and #2 were 0.975, 0.892, and 0.994, respectively. The overall results show that CNN labels were almost as accurate pathologist labels at a cell-by-cell level. On WSIs from 10 clinical cases, the CNN continued to perform well, and identified two cases where the sign-out pathologist overestimated plasma cell percentages.

ConclusionsThe high labeling accuracy of the CNN supports its eventually application as a computational second-opinion tool for the measurement of plasma cell percentages in clinical practice.

category: pathology


title: Controlling microbial co-culture based on substrate pulsing can lead to stability through differential fitness advantages

abstract: Microbial consortia are exciting platforms for the bioproduction of complex metabolic products. However, the functional properties of microbial communities remain challenging to control, given the complex interactions between the co-cultured organisms. Microbial communities are invariably heterogeneous, possessing different phenotypic states compartmentalised in each microorganism. Furthermore, each strain can switch to alternative phenotypic states exhibiting different metabolic and fitness potentials. These transitions are related to the biological behaviour exhibited by cellular systems, leading to phenotypic diversification and fitness evolution processes. In this work, Escherichia coli and Saccharomyces cerevisiae were co-cultured with different feeding profiles designed to generate transitory environmental conditions and metabolic shifts, leading to the co-existence of the two microbial strains in continuous cultures. Intermittent feeding profiles allowed to generate temporal niches, providing fitness advantages to each strain, further ensuring co-culture stability. Single-strain cultures were used for inferring the growth and metabolic parameters for each strain. These parameters were then used to design a simplified cybernetic model for the co-culture, which simulated the consortiums performance under continuous and intermittent feeding profiles at various frequencies, feed step times and dilution rates. Two discontinuous feeding profiles were selected for co-culture experiments. Models and experiments pointed out that the intermittent process conditions allowed to produce alternating periodic conditions promoting the growth of E. coli and S. cerevisiae, enabling temporal niche fitness advantages for both strains. E. coli response was found to be less prone to substrate co-utilisation due to its greater catabolic repression features, while S. cerevisiae exhibited more flexibility regarding simultaneous carbon source utilisation. Experiments pointed out that the given intermittent feeding profiles could dynamically stabilise the co-existence of the two strains during long-lasting continuous cultivations. Furthermore, these specific frequencies and feeding profiles affected cellular interaction and community composition.

category: systems biology


title: Interplay between external inputs and recurrent dynamics during movement preparation and execution in a network model of motor cortex

abstract: The primary motor cortex has been shown to coordinate movement preparation and execution through computations in approximately orthogonal subspaces. The underlying network mechanisms, and in particular the roles played by external and recurrent connectivity, are central open questions that need to be answered to understand the neural substrates of motor control. We develop a recurrent neural network model that recapitulates the temporal evolution of single-unit activity recorded from M1 of a macaque monkey during an instructed delayed-reach task. We explore the hypothesis that the observed dynamics of neural covariation with the direction of motion emerges from a synaptic connectivity structure that depends on the preferred directions of neurons in both preparatory and movement-related epochs. We constrain the strength both of synaptic connectivity and of external input parameters by using the data as well as an external input minimization cost. Our analysis suggests that the observed patterns of covariance are shaped by external inputs that are tuned to neurons preferred directions during movement preparation, and they are dominated by strong direction-specific recurrent connectivity during movement execution, in agreement with recent experimental findings on the relationship between motor-cortical and motor-thalamic activity, both before and during movement execution. We also demonstrate that the manner in which single-neuron tuning properties rearrange over time can explain the level of orthogonality of preparatory and movement-related subspaces. We predict that the level of orthogonality is small enough to prevent premature movement initiation during movement preparation; however, it is not zero, which allows the network to encode a stable direction of motion at the population level without direction-specific external inputs during movement execution.

category: neuroscience


title: Self-organization, quality control, and preclinical studies of human iPSC-derived retinal sheets for tissue-transplantation therapy

abstract: Three-dimensional retinal organoids (3D-retinas) are a promising graft source for transplantation therapy. We previously developed self-organizing culture for 3D-retina generation from human pluripotent stem cells (hPSCs). Here we present a quality control method and preclinical studies for tissue-sheet transplantation. Self-organizing hPSCs differentiated into both retinal and off-target tissues. Gene expression analyses identified the major off-target tissues as eye-related, cortex-like, and spinal cord-like tissues. For quality control, we developed a qPCR-based test in which each hPSC- derived neuroepithelium was dissected into two tissue-sheets: inner-central sheet for transplantation and outer-peripheral sheet for qPCR to ensure retinal tissue selection. During qPCR, tissue-sheets were stored for 3-4 days using a newly developed preservation method. In a rat tumorigenicity study, no transplant-related adverse events were observed. In retinal degeneration model rats, retinal transplants differentiated into mature photoreceptors and exhibited light responses in electrophysiology assays. These results demonstrate our rationale toward self-organizing retinal sheet transplantation therapy.


title: An NKX-COUP-TFII genomic code for mucosal vascular addressins and organ morphogenesis

abstract: Immunoglobulin family and carbohydrate vascular addressins encoded by Madcam1 and St6gal1 control lymphocyte homing into intestinal tissues, regulating immunity and inflammation. The addressins are developmentally programmed to decorate endothelial cells lining gut post-capillary and high endothelial venules, providing a prototypical example of organ- and segment-specific endothelial specialization. We identify conserved NKX-COUP-TFII composite elements (NCCE) in regulatory regions of Madcam1 and St6gal1 that bind intestinal homeodomain protein NKX2-3 cooperatively with venous nuclear receptor COUP-TFII to activate transcription. The Madcam1 element also integrates repressive signals from arterial/capillary Notch effectors. Pan-endothelial COUP-TFII overexpression induces ectopic addressin expression in NKX2-3+ capillaries, while NKX2-3 deficiency abrogates expression by HEV. Phylogenetically conserved NCCE are enriched in genes involved in neuron migration and morphogenesis of the heart, kidney, pancreas and other organs. Our results define a genomic address code for targeted expression of mucosal vascular addressins and implicate NCCE in fundamental processes in cell specification and development.


title: Chemical exploration of a highly selective scaffold with activity against intracellular Mycobacterium tuberculosis

abstract: We previously identified a phenylthiourea series with activity against intracellular Mycobacterium tuberculosis using a high throughput, high content assay. We conducted a catalog structure-activity relationship study with a collection of 35 analogs. We identified several thiourea derivatives with excellent potency against intracellular bacteria and good selectivity over eukaryotic cells. Compounds had much lower activity against extracellular bacteria which was not increased by using cholesterol as the sole carbon source. Compounds were equally active against strains with mutations in QcrB or MmpL3, thereby excluding common, promiscuous targets as the mode of action. The phenylthiourea series represents a good starting point for further exploration to develop novel anti-tubercular agents.


title: Detection of specific uncultured bacteriophages by fluorescence in situ hybridisation in pig microbiome

abstract: Microbial communities have huge impacts on their ecosystems and local environments spanning from marine and soil communities to the mammalian gut. Bacteriophages (phages) are important drivers of population control and diversity in the community, but our understanding of complex microbial communities is halted by biased detection techniques. Metagenomics have provided a method of novel phage discovery independent of in vitro culturing techniques and have revealed a large proportion of understudied phages. Here, five large phage genomes, that were previously assembled in silico from pig faecal metagenomes, are detected and observed directly in their natural environment using a modified phageFISH approach, and combined with methods to decrease bias against large phages. These phages are uncultured with unknown hosts. The specific phages were detected by PCR and fluorescent in situ hybridisation in their original faecal samples as well as across other faecal samples. Co-localisation of bacterial signals and phage signals allowed detection of the different stages of phage life cycle. All phages displayed examples of early infection, advanced infection, burst, and free phages. To our knowledge, this is the first detection of jumbophages in faeces, which were investigated independently of culture, host identification, and size, and based solely on the genome sequence. This approach opens up opportunities for characterisation of novel in silico phages in vivo from a broad range of gut microbiomes.


title: Effective infection with dengue virus in experimental neonate and adult mice through intranasal route

abstract:
As a member of the family Flaviviridae, the dengue virus is transmitted to humans through mosquito vectors. The virus causes dengue fever with flu-like symptoms, life-threatening dengue hemorrhagic fever, and dengue shock syndrome that affects four billion people in 128 countries. It can also be transmitted through atypical routes, such as needle stick injury, vertical transmission, and the receipt of contaminated blood or organs. Although sporadic cases cannot be classified as atypical transmission routes and raise respiratory exposure concerns, the risks remain unclear. Here, we analyzed the respiratory infectivity of the dengue virus in BALB/c suckling and A6 adult mice using the dengue virus serotype 2 and intranasal inoculation. The results showed that the clinical symptoms of intranasally infected mice included excitement, emaciation, malaise, and death, and histopathological changes and viremia dynamics were observed in multiple tissues, including the brain, liver, and spleen. Notably, the DENV serotype 2 showed clear brain tropism after intranasal infection, and viral loads peaked at seven days post-inoculation. Furthermore, the virus was isolated from mouse brains, and the sequence homology between the viral stock and dengue virus isolates was 99.99%. Similar results were observed in adult IFN-/{beta} receptor-deficient mice. Taken together, our results suggest that the DENV serotype 2 can infect suckling mice and adult immune-deficient mice via the nasal route. The results of our study broaden our perception of atypical dengue transmission routes and may help explain dengue virus infections that occur without the presence of mosquito vectors.

ImportanceDengue is transmitted mainly via mosquito vectors. Flu-like dengue fever or life-threatening severe dengue threatens four billion people in 128 countries. Non-vector dengue cases have occurred occasionally since 1990. These cases cannot be classified as atypical transmission routes and are suspected to have arisen via sexual or respiratory transmission. Recently, the sexual transmission of dengue has been confirmed in humans. The respiratory infectivity of other members of the Flaviviridae family, such as Zika and Japanese encephalitis viruses, has been determined under experimental conditions. The potential for respiratory infection with the dengue virus has not yet been confirmed. In this study, we analyzed the respiratory infectivity of the dengue virus in suckling mice and adult immunodeficient mice via intranasal inoculation, thereby exploring the potential for respiratory transmission in cases with non-vector dengue infection, especially for patients who were exposed to dengue virus contaminants.


title: Resolving drug selection and migration in an inbred South American Plasmodium falciparum population with identity-by-descent analysis

abstract: The human malaria parasite Plasmodium falciparum is globally widespread, but its prevalence varies significantly between and even within countries. Most population genetic studies in P. falciparum focus on regions of high transmission where parasite populations are large and genetically diverse, such as sub-Saharan Africa. Understanding population dynamics in low transmission settings, however, is of particular importance as these are often where drug resistance first evolves. Here, we use the Pacific Coast of Colombia and Ecuador as a model for understanding the population structure and evolution of Plasmodium parasites in small populations harboring low genetic diversity. The combination of low transmission and a high proportion of monoclonal infections means there are few outcrossing events and clonal lineages persist for long periods of time. Yet despite this, the population is evolutionarily labile and has successfully adapted to multiple drug regimes. Using 166 newly sequenced whole genomes, we measure relatedness between parasites, calculated as identity by descent (IBD), and find 17 distinct but highly related clonal lineages, six of which have persisted in the region for at least a decade. This inbred population structure is captured in more detail with IBD than with other common population structure analyses like PCA, ADMIXTURE, and distance-based trees. We additionally use patterns of intra-chromosomal IBD and an analysis of haplotypic variation to explore the role of recombination in spreading drug resistance mutations throughout the region. Two genes associated with chloroquine resistance, crt and aat1, show evidence of hard selective sweeps, while selection appears soft and/or incomplete at three other key resistance loci (dhps, mdr1, and dhfr). Overall, this work highlights the strength of IBD analyses for studying parasite population structure and resistance evolution in regions of low transmission, and emphasizes that drug resistance can evolve and spread in extremely small populations, as will occur in any region nearing malaria elimination.

category: evolutionary biology


title: Evaluating Impacts of Syntenic Block Detection Strategies on Rearrangement Phylogeny Using Mycobacterium tuberculosis Isolates

abstract: AO_SCPLOWBSTRACTC_SCPLOWPhylogenetic inference based on genomic structural variations, that manipulate the gene order and content of whole chromosomes, promises to inform a more comprehensive understanding of evolution. The first challenge in using such data, the incompleteness of available de novo assemblies, is easing as long read technologies enable (near-)complete genome assembly, but methodological challenges remain. To obtain the input to rearrangement-based inference methods, we need to detect syntenic blocks of orthologous sequences, a task that can be accomplished in many ways, none of which are obviously preferable. In this paper, we use 94 reference quality genomes of primarily Mycobacterium tuberculosis (Mtb) isolates as a benchmark to evaluate these methods. The clonal nature of Mtb evolution, the manageable genome sizes, along with substantial levels of structural variation make this an ideal benchmarking dataset. We test several methods for detecting homology and obtaining syntenic blocks, and two methods for inferring phylogenies, comparing them to the standard method that uses substitutions for inferring the tree. We find that not only the choice of methods but also their parameters can impact results, especially among branches with lower support. In particular, a method based on an encoding of adjacencies applied to Cactus-defined blocks was fully compatible with the highly supported branches of the substitution-based tree. Thus, we were able to combine the two trees to obtain a supertree with high resolution utilizing both SNPs and rearrangements. Furthermore, we observed that the results were much less affected by the choice of the tree inference method than by the method used to determine the underlying syntenic blocks. Overall, our results indicate that accurate trees can be inferred using genome rearrangements, but the choice of the methods for inferring the homology matters and requires care.


title: Reproducibility metrics for CRISPR screens

abstract: CRISPR screens are used extensively to systematically interrogate the phenotype-to-genotype problem. In contrast to early CRISPR screens, which defined core cell fitness genes, most current efforts now aim to identify context-specific phenotypes that differentiate a cell line, genetic background or condition of interest, such as a drug treatment. While CRISPR-related technologies have shown great promise and a fast pace of innovation, a better understanding of standards and methods for quality assessment of CRISPR screen results is crucial to guide technology development and application. Specifically, many commonly used metrics for quantifying screen quality do not accurately measure the reproducibility of context-specific hits. We highlight the importance of reporting reproducibility statistics that directly relate to the purpose of the screen and suggest the use of metrics that are sensitive to context-specific signal.


title: Accurate proteome-wide measurement of methionine oxidation in aging mouse brains

abstract: The oxidation of methionine side chains has emerged as an important posttranslational modification of proteins. A diverse array of low-throughput and targeted studies have suggested that the oxidation of methionine residues in select proteins can have diverse impacts on cell physiology, ranging from detrimental effects on protein structure and stability to functional roles in cell signaling. Despite its importance, the large-scale investigation of methionine oxidation in a complex matrix, such as the cellular proteome, has been historically hampered by technical limitations. Herein we report a methodology, Methionine Oxidation by Blocking (MobB), that allows for accurate and precise quantification of low levels of methionine oxidation typically observed in vivo. To demonstrate the utility of this methodology, we applied MobB to the brain tissues of young (6 m.o.) and old (20 m.o.) mice and identified over 280 novel sites for in vivo methionine oxidation. We further demonstrated that oxidation stoichiometries for specific methionine residues are highly consistent between individual animals and methionine sulfoxides are enriched in clusters of functionally related gene products including membrane and extracellular proteins. However, we did not detect significant changes in methionine oxidation in brains of old mice. Our results suggest that under normal in vivo conditions methionine oxidation is a biologically regulated process rather than a result of stochastic chemical damage.

category: biochemistry


title: Heme overdrive rewires pan-cancer cell metabolism

abstract:
All cancers reprogram their metabolism. In this study, we report that all cancer cells employ imbalanced and dynamic heme metabolic pathways essential for their oncogenic growth and coined the term  heme overdrive. Three key characteristics define heme overdrive, i.e., cancer universality, cancer essentiality and cancer specificity. While heme overdrive is absent in diverse differentiated cells or somatic stem cells, it is present in hundreds of cancers, and is delineated in patient-derived tumor progenitor cells by single cell RNAseq. The only exception to heme overdrive in normal tissues is identified in preimplantation human embryos, where heme overdrive likely links to embryonic omnipotency. Among the major drivers are proteins involved in biosynthesis of heme intermediates (hydroxymethylbilane synthase (HMBS) and uroporphyrinogen decarboxylase (UROD)) and heme trafficking (FLVCR1). CRISPR/Cas9 editing to engineer leukemia cells with impaired heme biosynthesis steps confirmed our whole genomic data analyses that heme overdrive is linked to oncogenic states and cellular differentiation. Finally, we devised a novel "bait- and-kill" strategy to target this cancer metabolic vulnerability.

One-Sentence SummaryHeme overdrive re-programs cellular metabolism across diverse types of cancer.


title: Progressive maturation of the root apical meristem in Arabidopsis thaliana lateral roots

abstract:
Meristems are stem cells niches that support the formation of all plant organs and are either set during embryogenesis and maintained throughout the plant life or specified de novo, post-embryonically. The embryo-derived root apical meristem is organized around a group of infrequently dividing cells, the quiescent centre, that maintains the stem cells, organizes growth along two axes and owing to its resistance to 3ic stress can replace damaged stem cells. In most cases, lateral roots post-embryonically branch off the primary and establish a new root meristem which organization is identical to the primary root one. The cellular and molecular processes underpinning the emergence of new stem cell niches are not well known. Here, we characterize the de novo establishment of the root apical meristem in lateral roots. While the position of the new stem cell niche is set early during morphogenesis, its cellular layout, unique gene expression profile and mitotic quiescence are only acquired after emergence concomitant to the establishment of two diverging growth axis. Our results show that the intertwined attributes of the mature root stem cell niche are progressively acquired during lateral root formation, and support a model in which the position of the stem cell niche emerges from the establishment of diverging growth axis.

HighlightAnalyze of the ontogeny of the quiescent center during lateral root ontogeny reveal its late formation and supports that its emergence results from the establishment of two diverging growth axis.

category: plant biology


title: Differential binding of salicylic acid, phenolic acid derivatives and co- factors determines the roles of Arabidopsis NPR1 to NPR4 in plant immunity

abstract:
SummaryO_LIGenetic studies have demonstrated that NPR1 is the key positive regulator of salicylic acid (SA)-induced PR-1 gene induction and systemic acquired resistance (SAR). In Arabidopsis, family members NPR1 to NPR4 share domain architecture.
C_LIO_LIYeast hybrid assays were used to explore biochemical capabilities of NPR1 to NPR4.
C_LIO_LIAll NPR1 to NPR4 are responsive to SA. SA perception proceeds via the conserved arginine embedded in a C-terminal LENRV-like motif. Clade 2 proteins NPR3 and NPR4 perceive SA directly, while clade 1 members NPR1 and NPR2 require interaction with partner proteins NIMIN1/NIMIN2 and TGA factors, respectively, to enable SA sensing. Intriguingly, NPR3 is considerably more sensitive to the synthetic analog 3,5-dichloroanthranilic acid than to SA, and all NPR1 to NPR4 are able to sense the microbial metabolite 6-methyl SA.
C_LIO_LIWe suggest that the plant{acute}s ability to track SA and phenolic acid derivatives through NPR proteins has evolved to support diverse defense signaling outputs that are activated in parallel by agonists which may be of microbial or plant origin. In this line, NPR1-NIMIN2/NIMIN1 complex is the prime receptor for SA synthesized by plants in response to microbial attack, while NPR3 induces defense different from SAR primarily via unrecognized signal molecules.
C_LI


title: Single cell analysis of gene expression in the substantia nigra pars compacta of a pesticide-induced mouse model of Parkinson's disease

abstract: Exposure to pesticides in humans increases the risk of Parkinsons disease (PD), but the mechanisms remain poorly understood. To elucidate these pathways, we dosed C57BL/6J mice with a combination of the pesticides maneb and paraquat (MNPQ). Behavioral analysis revealed motor deficits consistent with PD. Single cell RNA sequencing of substantia nigra pars compacta revealed both cell-type specific genes and genes expressed differentially between pesticide and control, including Fam241b, Emx2os, Bivm, Gm1439, Prdm15 and Rai2. Neurons had the largest number of significant differentially expressed genes, but comparable numbers were found in astrocytes and less so in oligodendrocytes. In addition, network analysis revealed enrichment in functions related to the extracellular matrix. These findings emphasize the importance of support cells in pesticide-induced PD and refocus our attention away from neurons as the sole agent of this disorder.


title: ERROR MODELLED GENE EXPRESSION ANALYSIS (EMOGEA) PROVIDES A SUPERIOR OVERVIEW OF TIME COURSE RNA-SEQ MEASUREMENTS AND LOW COUNT GENE EXPRESSION

abstract:
Serial RNA-seq studies of bulk samples are widespread and provide an opportunity for improved understanding of gene regulation during e.g., development or response to an incremental dose of a pharmacotherapeutic. In addition, the widely popular single cell RNA-seq (scRNA-seq) data implicitly exhibit serial characteristics because measured gene expression values recapitulate cellular transitions. Unfortunately serial RNA-seq data continue to be analyzed by methods that ignore this ordinal structure and yield results that are difficult to interpret. Here, we present Error Modelled Gene Expression Analysis (EMOGEA), a principled framework for analyzing RNA-seq data that incorporates measurement uncertainty in the analysis, while introducing a special formulation for modelling data that are acquired as a function of time or other continuous variable. By incorporating uncertainties in the analysis, EMOGEA is specifically suited for RNA-seq studies in which low-count transcripts with small fold-changes lead to significant biological effects. Such transcripts include signaling mRNAs and non-coding RNAs (ncRNA) that are known to exhibit low levels of expression. Through this approach, missing values are handled by associating with them disproportionately large uncertainties which makes it particularly useful for single cell RNA-seq data. We demonstrate the utility of this framework by extracting a cascade of gene expression waves from a well-designed RNA-seq study of zebrafish embryogenesis and, a scRNA-seq study of mouse pre-implantation and provide unique biological insights into the regulation of genes in each wave. For non-ordinal measurements, we show that EMOGEA has a much higher rate of true positive calls and a vanishingly small rate for false negative discoveries compared to common approaches. Finally, we provide an R package (https://github.com/itikadi/EMOGEA) that is self-contained and easy to use.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/481000v1_figG1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@18f3f99org.highwire.dtl.DTLVardef@1989635org.highwire.dtl.DTLVardef@ad964borg.highwire.dtl.DTLVardef@6650be_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Graphical representation of EMOGEA indicating the incorporation of measurement errors in modeling RNA-seq data to generate superior results in exploratory analysis, differential gene expression analyses and, scRNA-seq and Time Course analyses.

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category: bioinformatics


title: Neuromodulation due to propofol affects anesthetic oscillatory coupling

abstract:
The anesthetic propofol produces prominent oscillatory signatures on the EEG. Despite the strong correlation between oscillations and the anesthetic state, the fundamental mechanisms of this unconsciousness remain unknown. On the EEG, propofol elicits alpha oscillations (8-14 Hz), slow oscillations (0.5-2.0 Hz), and dose-dependent phase-amplitude coupling (PAC) between these rhythms. A low enough dose causes "trough-max" PAC, where alpha oscillation amplitude is consistently maximal during slow troughs; this occurs at the same time as arousable unconsciousness. A high enough dose causes consistent "peak-max" PAC, where alpha amplitude is maximal during the slow peak, at the same time as unarousable unconsciousness. Much of the anesthetic state is dominated by a mixture of both states. Using thalamocortical Hodgkin-Huxley simulations, we show that, in addition to propofol effects on GABAA synapses and thalamocortical H-currents, propofol-induced changes to neuromodulation may generate LFP oscillations and their dose-dependent coupling. We show this for acetylcholine specifically, though other neuromodulators may produce the same effects. We find that LFP- and EEG-relevant synapses of local thalamocortical circuits stochastically display either trough-max or peak-max PAC on any given slow cycle. Trough-max PAC signals are present only in thalamocortical synaptic currents, and not identifiable via membrane potentials alone. PAC preference depends critically on the neuromodulatory state, which is dose-dependent: high doses are associated with statistically more peak-max than trough-max, and vice-versa. This is caused by increased cortical synchronization at higher doses. Our results have important consequences for analyzing LFP/EEG data, in that local network trough-or peak-max may only be seen on a cycle-by-cycle basis, and not when averaging. We hypothesize that this increased cortical synchronization leads to an inability to process signals in a flexible manner needed for awake cognition.

New & NoteworthyWe simulate biophysical neural networks to investigate how the anesthetic propofol enables alpha and slow oscillations to emerge and interact. Direct effects of propofol on inhibition and indirect effects on acetylcholine level are necessary for dose-dependent emergence and coupling of these rhythms. Local groups of anesthetized cells behave with more complexity than global EEG would suggest. Higher doses are associated with more cortical synchrony, which may underlie the reduced ability to respond to stimuli.


title: The virtual aging brain: a model-driven explanation for cognitive decline in older subjects

abstract: Healthy aging is accompanied by heterogeneous decline of cognitive abilities among individuals, especially during senescence. The mechanisms of this variability are not understood, but have been associated with the reorganization of white matter fiber tracts and the functional co-activations of brain regions. Here, we built a causal inference framework to provide mechanistic insight into the link between structural connectivity and brain function, informed by brain imaging data and network modeling. By applying various degrees of interhemispheric degradation of structural connectivity, we were not only able to reproduce the age-related decline in interhemispheric functional communication and the associated dynamical flexibility, but we obtained an increase of global modulation of structural connectivity over the brain function during senescence. Notably, the increase in modulation between structural connectivity and brian function was higher in magnitude and steeper in its increase in older adults with poor cognitive performance. We independently validated the causal hypothesis of our framework via a Bayesian approach based on deep-learning. The current results might be the first mechanistic demonstration of dedifferentiation and scaffolding during aging leading to cognitive decline demonstrated in a large cohort.


title: The impact of familiarity on cortical taste coding

abstract: The role of the gustatory region of the insular cortex in mediating associative taste learning, such as conditioned taste aversion, has been well studied. However, while associative learning plays a role in some taste behaviors, such as avoidance of toxins, taste stimuli are often encountered by animals in their natural environment without explicit consequences. This type of inconsequential experience with sensory stimuli has been studied in other sensory systems, generally with the finding that neuronal responses habituate with repeated sensory exposure. The present study sought to determine the effect of taste familiarity on population taste coding in mouse gustatory cortex (GC). Using microendoscope calcium imaging, we studied the taste responses of visually identifiable neurons over five days of taste experience, during which animals could freely choose to consume taste stimuli. We found that the number of active cells in insular cortex, as well as the number of cells characterized as taste-responsive, significantly decreased as animals became familiar with taste stimuli. Moreover, the magnitude of taste-evoked excited responses increased, and inhibited responses decreased with experience. By tracking individual neurons over time, we focused on taste coding in a subpopulation of "stable" neurons that were present on all days of the taste familiarity paradigm. The population-level response across these stable cells was distinct when taste stimuli were novel but became more intercorrelated among those taste stimuli mice willingly consumed as the stimuli became familiar. Overall, these results highlight the effects of familiarity on taste responses in gustatory cortex.


title: Human regulatory T-cells locally differentiate and are functionally heterogeneous within the inflamed arthritic joint

abstract:
ObjectiveTregs are crucial for immune regulation, and environment-driven adaptation of effector (e)Tregs is essential for local functioning. However, the extent of human Treg heterogeneity in inflammatory settings is unclear.

MethodsWe combined single-cell RNA- and TCR-sequencing on Tregs derived from 4-6 patients with juvenile idiopathic arthritis (JIA) to investigate the functional heterogeneity of human synovial fluid (SF)-derived Tregs from inflamed joints. Confirmation and suppressive function of the identified Treg clusters was assessed by flow cytometry.

ResultsFour Treg clusters were identified; incoming, activated eTregs with either a dominant suppressive or cytotoxic profile, and GPR56+CD161+CXCL13+ Tregs. Pseudotime analysis showed differentiation towards either classical eTreg profiles or GPR56+CD161+CXCL13+ Tregs supported by TCR data. Despite its most differentiated phenotype GPR56+CD161+CXCL13+ Tregs were shown to be suppressive. Furthermore, BATF was identified as an overarching eTreg regulator, with the novel Treg-associated regulon BHLHE40 driving differentiation towards GPR56+CD161+CXCL13+ Tregs, and JAZF1 towards classical eTregs.

ConclusionOur study reveals a heterogeneous population of Tregs at the site of inflammation in JIA. SF Treg differentiate to a classical eTreg profile with a more dominant suppressive or cytotoxic profile that share a similar TCR repertoire, or towards GPR56+CD161+CXCL13+ Tregs with a more distinct TCR repertoire. Genes characterizing GPR56+CD161+CXCL13+ Tregs were also mirrored in other T-cell subsets in both the tumor and autoimmune setting. Finally, the identified key regulators driving SF Treg adaptation may be interesting targets for autoimmunity or tumor interventions.


title: Active haptic training improves somatosensory-motor feedback control in the motor skills of experts

abstract: A common problem experts experience is the challenge of overcoming the ceiling effects of highly trained sensorimotor skills. Recent studies have demonstrated that the amount of practice fails to explain interindividual variation in experts motor expertise, implicating hidden bottlenecks that prevent experts from overcoming this ceiling. Here, we show that somatosensory function in motion is one that defines further improvement of the sensorimotor skills of expert pianists. We found that even in expert pianists, specialized training that was designed to enhance somatosensory perception in motion improved sensorimotor skills in which feedback, but not feedforward, control plays a predominant role. Furthermore, training increased reliance on afferent information in motion relative to predictive information that could originate from internal models of movement. Together, these findings suggest that enhancement of somatosensory processing in motion improves the somatosensory-motor feedback loop and thereby enabling experts to surpass the ceiling that initially limited their motor expertise.


title: Reduced sensorimotor beta dynamics could represent a "slowed movement state" in healthy individuals

abstract: Beta oscillations (~13-30 Hz) recorded from the sensorimotor cortex have canonical amplitude changes during movement. Specifically, a movement-related beta decrease (MRBD) occurs before movement, and a post-movement beta rebound (PMBR) follows. We investigated how the MRBD and PMBR vary with movement speed. Individuals performed a task with blocks that generated longer reaction times (RTs) and shorter RTs (Slow and Fast blocks, respectively) while scalp-electroencephalography (EEG) was recorded. The timing of task events before movement was also modulated to generate blocks with certain and uncertain timing (Fixed and Varied blocks, respectively). Beta modulation was reduced in Slow blocks compared to Fast blocks (i.e., a less negative MRBD and less positive PMBR). For the movement certainty manipulation, we saw mixed behavioral and EEG results. Our primary findings align with previous work which has shown reduced movement-related beta modulation in patients with Parkinsons disease. We propose that a "slowed movement state", whether it is experimentally induced or a manifestation of Parkinsons disease bradykinesia, is represented through reduced beta dynamics. Altogether, the MRBD and PMBR may represent motor speed on a continuum with Parkinsons disease as an extreme example of slowed movement.


title: Inducing synthetic lethality for selective targeting of acute myeloid leukemia cells harboring STAG2 mutations

abstract: Targeted therapies exploiting selective vulnerabilities of malignant cells are highly desired for clinical applications. The cohesin protein complex comprises of RAD21, SMC3, SMC1A as well as a fourth subunit that consists of either STAG1 or STAG2 and is essential for proper chromosomal segregation during mitosis. STAG2 loss-of-function mutations are recurrent driver events in acute myeloid leukemia (AML) and appear relatively early during leukemogenesis. Studies in cell lines have shown that STAG2 deficient cells are uniquely vulnerable to STAG1 perturbation, and this vulnerability could thus be exploited to selectively eliminate STAG2 null AML cells. Here we show that partial perturbation of STAG1 is well tolerated by normal human hematopoietic stem cells and does not affect their functionality. By contrast, STAG1 knockdown is lethal to STAG2 null human HSCs by inducing major mitotic defects. Moreover, STAG1 knockdown induced synthetic lethality in primary human AML cells harboring a STAG2 mutation and completely abrogated leukemia progression in xenograft models. Overall, our study provides proof-of-concept demonstration of a synthetic lethal approach to selectively target primary human cancer cells with STAG2 mutations


title: Right atrial mechanism contributes to atrial fibrillation in a canine model of pulmonary arterial hypertension

abstract:
ObjectivesThe aim of this study was to investigate proarrhythmic substrates of atrial fibrillation (AF) in a canine model of dehydromonophylline (DMCT)-induced pulmonary arterial hypertension (PAH).

MethodsAll cannines (n=12) underwent baseline echocardiographic and hemodynamic examinations, 7 of which were injected with DMCT (3.0mg/kg) to induce PAH via jugular vein cannulation. The control beagles (n=5) were given the same dose of normal saline. Then, both groups were monitored by insertable cardiac monitors. Hemodynamic, echocardiographic, electrophysiological and histological examinations were performed 8 weeks later.

ResultsIn PAH group, 2 died after the injection (mortality 28.6%). Thus, 10 beagles (PAH group: 5, control group: 5) underwent all the examinations. The pulmonary artery pressure increased significantly while the right atrium (RA) and right ventricle expanded slightly. Spontaneous AF episodes were recorded in all PAH canines 1 week after the injection. The AF burden increased rapidly from 1 week (7.6{+/-}1.8%) and remained high after 2-3 weeks (32.0{+/-}4.9% at 8 weeks). Compared with the control group, the PAH group had abbreviated effective refractory periods (ERPs), increased atrial ERP dispersion, and slower conduction velocities. Notably, AF susceptibility and atrial remodeling in RA was more significant those in LA, such as increased WOV(39.0{+/-}6.5ms vs. 28.0{+/-}5.7ms, P=0.022), enlarged low voltage regions (7.66{+/-}0.46% vs. 4.40{+/-}0.55%, P<0.0001) and fibrosis (8.22{+/-}0.61% vs. 4.93{+/-}0.60%, P <0.0001).

ConclusionsDMCT-induced canine PAH model increased the incidence of spontaneous and induced AF. The electrophysiological and structural remodeling of the RA facilitated the AF genesis.

category: physiology


title: Representational dynamics of memories for real-life events

abstract: The continuous flow of experience that characterizes real-life events is not recorded as such in episodic memory. The unfolding of events is represented as a succession of moments of prior experience (referred to as "experience units") that are separated by temporal discontinuities, such that events are temporally compressed when remembered. To unravel the neural basis of this temporal structure of episodic memories, we recorded a series of real-life events using wearable camera technology and then used fMRI to investigate brain activity during the temporal unfolding of these events in memory. We found that, compared to the representation of static scenes in memory, dynamically unfolding memory representations were associated with greater activation of posterior brain regions that support episodic recollection (i.e., the precuneus, posterior cingulate cortex, retrosplenial cortex, and angular gyrus). Strikingly, by analyzing the autocorrelation of brain activity patterns at successive time points throughout the retrieval period, we found that these brain regions showed higher temporal dynamics when remembering the unfolding of events compared to static scenes. Furthermore, these effects were more pronounced when recalling events that included a higher density of experience units (i.e., actions compared to spatial displacements). These results reveal the key role of the posterior medial network in representing the dynamic unfolding of event segments that constitute episodic memories.


title: Exploring the link between Parkinson's disease and Diabetes Mellitus in Drosophila

abstract: Parkinsons disease (PD) is the second most common neurodegenerative disease. Diabetes mellitus (DM) is a metabolic disease characterized by high levels of glucose in blood. Recent epidemiological studies are highlighting the link between both diseases; it is even considered that DM might be a risk factor for PD. To further investigate the likely relation of these diseases, we have used a Drosophila PD model based on inactivation of the DJ-1{beta} gene (ortholog of human DJ-1), and diet-induced Drosophila and mouse T2DM models, together with human neuron-like cells. T2DM models were obtained by feeding flies with a high sugar containing medium, and mice with a high fat diet. Our results showed that both fly models exhibit common phenotypes such as alterations in carbohydrate homeostasis, mitochondrial dysfunction or motor defects, among others. In addition, we demonstrated that T2DM might be a risk factor of developing PD since our diet-induced fly and mouse T2DM models present DA neurodegeneration, a hallmark of PD. We have also confirmed that neurodegeneration is caused by increased glucose levels, which has detrimental effects in human neuron-like cells by triggering apoptosis and leading to cell death. Besides, the observed phenotypes were exacerbated in DJ-1{beta} mutants cultured in the high sugared medium, indicating that DJ-1 might have a role in carbohydrate homeostasis. Finally, we have confirmed that metformin, an antidiabetic drug, is a potential candidate for PD treatment and that it could prevent PD onset in T2DM model flies. This result supports antidiabetic drugs as promising PD therapeutics.


title: Ethanol exposure alters Alzheimer's-related pathology, behavior, and metabolism

abstract: Alcohol use disorder is a risk factor for Alzheimers disease (AD), yet the mechanisms underlying this relationship are poorly understood. Here, we employed the APP/PS1 mouse model of A{beta} pathology to examine the effects of a 10 week moderate ethanol drinking paradigm on AD-related phenotypes. Ethanol drinking led to a circadian shift in feeding behavior and glucose intolerance in APPswe/PSEN1dE9 (APP/PS1), but not wild-type or ethanol-naive APP/PS1 mice. Ethanol exposure also selectively disrupted nest building, a metric of self-care, and decreased brain mass in APP/PS1 mice. While there was a trend towards increased cortical A{beta} deposition in APP/PS1 mice, ethanol exposure shifted the distribution of plaque size and numbers, increasing the number of smaller plaques. Lastly, we used in vivo microdialysis to determine if acute ethanol directly affected hippocampal interstitial fluid (ISF) A{beta} levels in unanesthetized APP/PS1 mice. A{beta} levels were bidirectionally modulated by acute ethanol - decreasing as brain ethanol levels rose and increasing above baseline during withdrawal. Collectively, these studies provide the first evidence that acute ethanol alters ISF A{beta} levels and suggest that even modest levels of chronic ethanol consumption interact with A{beta} to exacerbate AD-related pathology, behavioral dysfunction, and metabolic deficits.


title: Moderate ethanol consumption interacts with Aβ to exacerbate Alzheimer's-like pathological, behavioral, and metabolic deficits in mice


title: Long noncoding RNA MEG3 activates neuronal necroptosis in Alzheimer's disease

abstract: Abstract/SummaryNeuronal cell loss is a defining feature of Alzheimers disease (AD), but it remains unclear how neurons die and how this relates to other defining characteristics of the disease1. Existing in vivo AD models only partially recapitulate the neuropathology of AD with very mild or no neuronal cell loss. Here we demonstrate that human neurons xenografted in mouse brain exposed to amyloid pathology develop sarkosyl-insoluble tau filaments, positive Gallyas silver staining, release phosphorylated tau (P-tau181) into the blood, and display considerable neuronal cell loss, providing a model for the induction of full Tau pathology by simple exposure to amyloid pathology in AD. The alterations are specific to human neurons and contrast with the mild effects exhibited in mouse neurons. A core transcriptional program in the human neurons is characterized by strong upregulation of MEG3, a neuron-specific long noncoding RNA. MEG3 is also strongly upregulated in neurons from AD patients in situ. MEG3 expression alone is sufficient to induce necroptosis in human neurons in vitro. Orally administered small molecule receptor-interacting protein (RIP) kinase -1 and -3 inhibitors rescued the neuronal cell loss in this novel AD model. Thus, xenografted human neurons are uniquely sensitive to amyloid pathology, recapitulate all the defining neuropathological features of AD, and ultimately die by necroptosis.


title: Prefrontally Modulated Vagal Tone Inhibits Inflammatory Responses to Prevent Telomere Damage in Healthy Participants

abstract:
BackgroundAccumulated senescent cells are proposed to be one of the main drivers of age-related pathology through disruption of tissue structure and function. We recently proposed the Neuro-Immuno-Senescence Integrative Model (NISIM; Ask et al., 2018) which relates prefrontally modulated vagal tone and subsequent balance between vagal and sympathetic input to the spleen to inflammatory responses leading to generation of reactive oxygen species and oxidative telomere damage. The NISIM is based on converging evidence and argues for the existence of a prefrontal cortex-autonomic nervous system-spleen (PAS) axis, suggesting that the inflammation that induces reactive oxygen species-generation is downstream of this axis.

AimIn this study, we aim to assess inflammation as a mediator in the relationship between prefrontally modulated vagal tone and leukocyte telomere length to test the hypothesis that PAS axis dysregulation accelerates cellular aging. We also assess the relationship between a recently proposed index of vagal immunomodulation (vagal tone/inflammation ratio; NIM index; Gidron et al., 2018) and telomere length, and compare results between the NIM index and vagal tone as predictors of telomere length.

MethodsThis study uses participant data from a large nationally representative longitudinal study since 1974 with a total of 45,000 Norwegian residents so far. A sub-sample of 1372 participants from which vagal tone, C Reactive Protein, and leukocyte telomere length could be obtained were included in the study. Relationships were analyzed with hierarchical multiple linear regression using either vagal tone and C Reactive Protein or the NIM index to predict telomere length. Sleeping problems, tobacco use status, alcohol use status, time since last meal, and symptoms of depression were included as control variables.

ResultsIn the mediation analysis, vagal tone was a significant positive predictor of telomere length, while C Reactive Protein was a significant negative predictor of telomere length. This relationship remained significant when individually controlling for some but not all confounding variables. The NIM index was a significant positive predictor of telomere length. This relationship remained significant when controlling for all confounding variables except one. In a reduced dataset excluding all participants where confounders were present, the NIM index remained a significant predictor of telomere length.

ConclusionThis is the first study suggesting that PAS axis activity is associated with telomere length thus supporting the NISIM. Results indicate that the NIM index is a more sensitive indicator of PAS axis activity than vagal tone and C Reactive Protein in isolation. Clinical relevance and suggestions for future research are discussed.


title: Description of Life History & Reproductive Size Threshold in Three High Elevation Puya (Bromeliaceae)

abstract: AO_SCPLOWBSTRACTC_SCPLOWThe Andes are a hotspot for biodiversity and high species endemism for both plants and animals. The genus Puya (Bromeliaceae) lives throughout the Andes, including puna and the paramo ecosystems above 3500m. Here, we studied the life history and reproductive size thresholds in three species of Puya (P. raimondii, P. cryptantha and P. goudotiana): P. raimondii in the Bolivian puna, and P. cryptantha and P. goudotiana in the Colombian paramo. We collected data on threshold size at flowering and clonal reproduction. All three species were found to have a consistent minimum size at flowering, while only P. cryptantha demonstrated a minimum size for clonal reproduction. No such evidence was found for P. goudotiana. We also found a positive correlation between leaf length and fruit number for P. goudotiana and P. cryptantha. Our data supported that P. raimondii is fully semelparous and indicated that P. goudotiana and P. cryptantha may be semi-semelparous.

category: ecology


title: Hyperflexion is unlikely to be the primary cervical spine injury mechanism in accidental head-on rugby tackling

abstract: In Rugby a high proportion of catastrophic cervical spine injuries occur during tackling. In the injury prevention literature, there is still an open debate on the injury mechanisms related to such injuries, with hyperflexion and buckling being under scrutiny. The aims of this study were to determine the primary cervical spine injury mechanism during head-on rugby tackling, and evaluate the effect of tackling technique on cervical spine intervertebral loading. We conducted an in silico study to examine the dynamic response of the cervical spine under loading conditions representative of accidental head-on rugby tackles by using a subject-specific musculoskeletal model of a rugby player. The computer simulations were driven by experimental in vivo data of an academy rugby player tackling a punchbag, and in vitro data of head-first impacts using a dummy head. Results showed that: i) the earlier generation of high compression and anterior shear loads with low values of flexion moments provides evidence that hyperflexion is unlikely to be the primary injury mechanism in the sub-axial cervical spine (C3-C7) during central and posterior head impact locations; ii) a higher degree of neck flexion at impact poses the cervical spine in a more hazardous position. These findings provide objective evidence to inform injury prevention strategies or rugby law changes, with the final view of improving the safety of the game of rugby.


title: Acute and chronic toxicity of imidacloprid in the pollinator fly, Eristalis tenax

abstract: Imidacloprid is a neonicotinoid neurotoxin that remains the most used insecticide worldwide. It persists in the environment long after the initial application resulting in chronic exposure to non-target insects. To accurately map the dose-dependent effects of these exposures across taxa, toxicological assays need to assess various modes of exposure across relevant indicator species. However, due to the difficulty of these experiments, contact bioassays are frequently used to quantify dose, and dipterans remain underrepresented. Here, we developed a novel naturalistic feeding bioassay to precisely measure imidacloprid ingestion and its toxicity for acute and chronic exposures in a dipteran pollinator, Eristalis tenax. Flies which ingested imidacloprid dosages lower than 12.1 ng/mg all showed consistent intake volumes and learned improved feeding efficiency over successive feeding sessions. In contrast, at doses of 12.1 ng/mg and higher flies had a rapid onset of severe locomotive impairment which prevented them from completing the feeding task. Neither probability of survival nor severe locomotive impairment were significantly higher than the control group until doses of 1.43 ng/mg or higher were reached. We were unable to measure a median lethal dose for acute exposure (72 hours) due to flies possessing a relatively high tolerance for imidacloprid. However, with chronic exposure (18 days), mortality went up and an LD50 of 0.41 ng/mg was estimated. Severe locomotive impairment tended to occur earlier and at lower dosages than lethality, with ED50s of 0.17 ng/mg and 7.82 ng/mg for acute and chronic exposure, respectively. Although the adult Eristalis is a honeybee mimic, it possesses a much higher tolerance to this toxin than its model. The similarity in the LD50 to other dipterans such as the fruitfly and the housefly suggests that there may be a phylogenetic component to pesticide tolerance that needs to be further investigated. The absence of obvious adverse effects at sublethal dosages also underscores a need to develop better tools for quantifying animal behaviour to evaluate the impact of insecticides on foraging efficiency in economically important species.

category: pharmacology and toxicology


title: Cardiac and gastric interoception have distinct neural substrates

abstract: The functional roles of interoception or senses that arise from within the body have been thought to guide agents in adaptive behavior by informing various bodily states like heart rate or energy status. However, direct evidence that different types of interoception are processed or represented differently in the human brain is still lacking. Hence, we directly compared the neural activation for cardiac (awareness related to heartbeats) and gastric (awareness related to the stomach) interoception in the same population (N = 31). Participants were asked to focus on their heart and stomach sensations in a magnetic resonance imaging scanner. The results showed that neural activation underlying gastric interoception encompassed the sensorimotor and reward-related regions when compared with cardiac interoception, including the occipitotemporal visual cortices, bilateral primary motor cortex, primary somatosensory cortex, left orbitofrontal cortex, and bilateral hippocampal regions. Conversely, cardiac interoception selectively activated the right frontal operculum/insula, in contrast to gastric interoception. Moreover, we found that the left dorsal middle insula encoded cardiac and gastric interoception differently, but the posterior insula was unaffected by the multivoxel pattern analysis. Our results present novel data demonstrating that cardiac and gastric interoception have distinct neural substrates particularly relevant to the functions for each interoception.


title: A Methodological Framework to Assess the Accuracy of Virtual Reality Hand-Tracking Systems: A case study with the Oculus Quest 2

abstract: Optical marker-less hand-tracking systems incorporated into virtual reality (VR) headsets are transforming the ability to assess motor skills, including hand movements, in VR. This promises to have far-reaching implications for the increased applicability of VR across scientific, industrial and clinical settings. However, so far, there is little data regarding the accuracy, delay and overall performance of these types of hand-tracking systems. Here we present a novel methodological framework which can be easily applied to measure these systems absolute positional error, temporal delay and finger joint-angle accuracy. We used this framework to evaluate the Meta Quest 2 hand-tracking system. Our results showed an average fingertip positional error of 1.1cm, an average finger joint angle error of 9.6o and an average temporal delay of 38.0ms. Finally, a novel approach was developed to correct for these positional errors based on a lens distortion model. This methodological framework provides a powerful tool to ensure the reliability and validity of data originating from VR-based, marker-less hand-tracking systems.


title: Unsupervised physiological noise correction of fMRI data using phase and magnitude information (PREPAIR)

abstract: Of the sources of noise which affect BOLD fMRI, respiration and cardiac fluctuations are responsible for the largest part of the variance, particularly at high and ultra-high field. Existing approaches to removing physiological noise either use external recordings, which can be unwieldy and unreliable, or attempt to identify physiological noise from the magnitude fMRI data. Data-driven approaches are limited by sensitivity, temporal aliasing and the need for user interaction. In the light of the sensitivity of the phase of the MR signal to local changes in the field stemming from physiological processes, we have developed an unsupervised physiological noise correction method which uses the information carried in both the phase and the magnitude of EPI data. Our technique, Physiological Regressor Estimation from Phase and mAgnItude, sub-tR (PREPAIR) derives time series signals which are sampled at the slice TR from both phase and magnitude images. It allows physiological noise to be captured without aliasing, and efficiently removes other sources of signal fluctuations which are not related to physiology, prior to regressor estimation. We demonstrate that the physiological signal time courses identified with PREPAIR not only agree well with those from external devices, but also retrieve challenging cardiac dynamics. The removal of physiological noise was as effective as that achieved with the most commonly used approach based on external recordings, RETROICOR. In comparison with widely used physiological noise correction tools which do not use external signals, PESTICA and FIX, PREPAIR removed more respiratory and cardiac noise and achieved a larger increase in tSNR at both 3 T and 7 T.


title: Hybridization and a mixture of small and large-effect loci facilitate adaptive radiation

abstract: Adaptive radiations represent some of the most remarkable explosions of diversification across the tree of life. However, the constraints to rapid diversification and how they are sometimes overcome, particularly the relative roles of genetic architecture and hybridization, remain unclear. Here, we address these questions in the Alpine whitefish radiation, using a whole-genome dataset that includes multiple individuals of each of the 22 species belonging to six ecologically distinct ecomorph classes across several lake-systems. We reveal that repeated ecological and morphological diversification along a common environmental axis is associated with both genome-wide allele frequency shifts and a specific, larger effect, locus, associated with the gene edar. Additionally, we highlight the role of introgression between species from different lake-systems in facilitating the evolution and persistence of species with unique phenotypic combinations and ecology. These results highlight the role of both genome architecture and secondary contact with hybridization in fuelling adaptive radiation.


title: Network communication models narrow the gap between the modular organization of structural and functional brain networks

abstract: Structural and functional brain networks are modular. Canonical functional systems, such as the default mode network, are well-known modules of the human brain and have been implicated in a large number of cognitive, behavioral and clinical processes. However, modules delineated in structural brain networks inferred from tractography generally do not recapitulate canonical functional systems. Neuroimaging evidence suggests that functional connectivity between regions in the same systems is not always underpinned by anatomical connections. As such, direct structural connectivity alone would be insufficient to characterize the functional modular organization of the brain. Here, we demonstrate that augmenting structural brain networks with models of indirect (polysynaptic) communication unveils a modular network architecture that more closely resembles the brains established functional systems. We find that diffusion models of polysynaptic connectivity, particularly communicability, narrow the gap between the modular organization of structural and functional brain networks by 20-60%, whereas routing models based on single efficient paths do not improve mesoscopic structure-function correspondence. This suggests that functional modules emerge from the constraints imposed by local network structure that facilitates diffusive neural communication. Our work establishes the importance of modeling polysynaptic communication to understand the structural basis of functional systems.


title: With a pinch of salt: metagenomic insights into Namib Desert salt pan microbial mats and halites reveal functionally adapted and competitive communities

abstract:
Playas, saline-rich springs surrounded by halite evaporates (also called salt pans) have played an essential role in landscape erosion during the formation of the Namib Desert and are numerous in its central region. In this study, we used shotgun metagenomics to investigate the phylogenetic and functional capacities of the microbial communities in two salt pans (namely, Eisefeld and Hosabes) located in the Central Namib Desert. We studied the source and sink sediment mat communities of the saline streams, as well as those from two halites (crystallized structures on the stream margins). The microbial assemblages and potential functions were distinct in both niches. Independently from their localization (Eisfeld vs Hosabes and source vs sink), the sediment mat communities were dominated by members of the Alphaproteobacteria and Gammaproteobacteria classes, while halites were Archaea-dominated (Euryarchaea phylum) and also contained high abundances of the extremely halophilic bacterium Salinibacter sp. (phylum Bacteroidetes). Photoheterotrophy and chemoheterotrophy were the principal lifestyles in both niches, with halite communities having a reduced diversity of metabolic pathways. Intense microbial-virus interactions in both niches were implied by the widespread detection of CRISPR-Cas defense systems. We identified a putatively novel clade of type II CRISPR-Cas systems, as well as novel candidate viral lineages of the order Caudovirales and of Euryarchaea-infecting haloviruses. Putative gene transfer agent-like sequences within the Alphaproteobacteria were identified in the sediment mat communities. These horizontal gene transfer elements have the potential to drive genome plasticity and evolution of the Alphaproteobacteria in the Namib Desert salt pan microbiomes.

ImportanceThe hyperarid Namib Desert has is one of the oldest deserts on Earth. It contains multiple clusters of playas which are saline-rich springs surrounded by halite evaporites. Playas are of great ecological importance and the indigenous microorganisms that inhabit them are potentially involved in precipitation of minerals such as carbonates and sulfates. While there has been a considerable amount of research on the diversity and ecology of the microbiomes in the different edaphic niches of the Namib Desert, little is known about the microbial communities inhabiting its multiple playas. In this work, we provide a comprehensive taxonomic and functional characterization of the microbial and viral communities of sediment mats and halites from two salt pans in the central region of the Namib Desert, contributing towards the understanding of the ecology of this biome.

category: microbiology


title: How to resolve conflicting conservation objectives: A decision support tool for the global selection of multi-purpose protected areas

abstract: The establishment and maintenance of protected areas(PAs) is viewed as a key action in delivering post-2020 biodiversity targets. PAs often need to meet a multitude of objectives, ranging from biodiversity protection to ecosystem service provision and climate change mitigation. As available land and conservation funding are limited, optimizing resources by selecting the most beneficial PAs is vital. Here we present a decision support tool that enables a flexible approach to PA selection on a global scale, allowing different conservation objectives to be weighted and prioritized according to user-specified preferences. We apply the tool across 1347 terrestrial PAs and highlight frequent trade-offs among different objectives, e.g., between biodiversity protection and ecosystem integrity. These results indicate that decision makers must usually decide among conflicting objectives. To assist this our decision support tool provides an explicitly value-based approach that can help resolve such conflicts by considering divergent societal and political demands and values.


title: Imaging-based coculture model for high-throughput compound screening in hematological cancers

abstract: The bone marrow microenvironment modulates treatment response in blood cancers but a systematic assessment of anticancer drug effects in the context of this protective niche has been missing. To fill this gap, we established an ex-vivo model that enables high-throughput compound screening in leukemia-stroma coculture. We applied 50 compounds to 108 patient samples with hematological malignancies in monoculture and coculture with bone marrow stromal cells and measured cellular phenotypes via automated confocal microscopy. Stromal coculture conferred resistance to 52% of compounds in chronic lymphocytic leukemia (CLL) and to 36% of compounds in acute myeloid leukemia (AML). Although a partial loss in efficacy was observed for a considerable number of investigated drugs, responses to many of the drugs were comparable between mono- and cocultures. This suggests that large-scale monoculture drug perturbation studies might be sufficient for the purpose of first-line screening. However, our study also uncovered a substantial heterogeneity of stromal protection across the probed samples and compounds, which highlights the importance of validating the findings of monoculture screens in stroma coculture models.


title: An increase in serial sarcomere number induced via weighted downhill running improves work loop performance in the rat soleus

abstract: Increased serial sarcomere number (SSN) has been observed in rats via downhill running training due to the emphasis on active lengthening contractions; however, little is known about the influence on dynamic contractile function. Therefore, we employed 4 weeks of weighted downhill running training in rats, then assessed soleus SSN and work loop performance. We hypothesized trained rats would produce greater net work output during faster, higher-strain work loops due to a greater SSN. Thirty-one Sprague-Dawley rats were assigned to a control or training group. Weight was added during downhill running via a custom-made vest, progressing from 5-15% body mass. Following sacrifice, the soleus was dissected, and a force-length relationship was constructed. Work loops (active shortening followed by passive lengthening) were then performed about optimal muscle length (LO) at 1.5-3-Hz cycle frequencies and 1-7-mm strains to assess net work output. Muscles were then fixed in formalin at LO. Fascicle lengths and sarcomere lengths were measured and used to calculate SSN. Intramuscular collagen content and crosslinking were quantified via a hydroxyproline content and pepsin-solubility assay. Trained rats had longer fascicle lengths (+13%), greater SSN (+8%), greater specific active forces (+50%), and lower passive forces (-45-62%) than controls (P<0.05). There were no differences in collagen parameters (P>0.05). Net work output was greater (+101-424%) in trained than control rats for the 1.5-Hz loops at 1, 3, and 5-mm strains (P<0.05) and showed relationships with fascicle length (R2=0.14-0.24, P<0.05). These results suggest training-induced longitudinal muscle growth may improve dynamic performance.


title: Sample preparation and warping accuracy for correlative multimodal imaging in the mouse olfactory bulb using 2-photon, synchrotron X-ray and volume electron microscopy

abstract: Integrating physiology with structural insights of the same neuronal circuit provides a unique approach to understanding how the mammalian brain computes information. However, combining the techniques that provide both streams of data represents an experimental challenge. When studying glomerular column circuits in the mouse olfactory bulb, this approach involves e.g. recording the neuronal activity with in vivo 2-photon (2P) calcium imaging, retrieving the circuit structure with synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT) and/or serial block-face electron microscopy (SBEM) and correlating these datasets. Sample preparation and dataset correlation are two key bottlenecks in this correlative workflow. Here, we first quantify the occurrence of different artefacts when staining tissue slices with heavy metals to generate X-ray or electron contrast. We report improvements in the staining procedure, ultimately achieving perfect staining in [~]67% of the 0.6 mm thick olfactory bulb slices that were previously imaged in vivo with 2P. Secondly, we characterise the accuracy of the spatial correlation between functional and structural datasets. We demonstrate that direct, single-cell precise correlation between in vivo 2P and SXRT tissue volumes is possible and as reliable as correlating between 2P and SBEM. Altogether, these results pave the way for experiments that require retrieving physiology, circuit structure and synaptic signatures in targeted regions. These correlative function-structure studies will bring a more complete understanding of mammalian olfactory processing across length scales and time.


title: Measuring brain beats: cardiac-aligned fast fMRI signals

abstract: Blood and cerebrospinal fluid (CSF) pulse and flow throughout the brain, driven by the cardiac cycle. These fluid dynamics, which are essential to healthy brain function, are characterized by several noninvasive magnetic resonance imaging (MRI) methods. Recent developments in fast MRI, specifically simultaneous multislice (SMS) acquisition methods, provide a new opportunity to rapidly and broadly assess cardiac-driven flow, including CSF spaces, surface vessels and parenchymal vessels. We use these techniques to assess blood and CSF flow dynamics in brief (3.5 minute) scans on a conventional 3T MRI scanner. Cardiac pulses are measured with a photoplethysmograph (PPG) on the index finger, along with fMRI signals in the brain. We retrospectively analyze the fMRI signals gated to the heart beat. Highly reliable cardiac-gated fMRI temporal signals are observed in CSF and blood on the timescale of one heartbeat (test-retest reliability within subjects R2>0.50). Cardiac pulsations with a local minimum following systole are observed in blood vessels, with earlier extrema in the carotid and basilar arteries and in branches of the anterior, posterior and middle cerebral arteries and extrema [~]200 ms later in the superior sagittal, transverse and straight sinuses. CSF spaces in the ventricles and subarachnoid space showed cardiac pulsations with a local maximum following systole instead. Similar responses are observed, with less temporal detail, in slower resting state scans with slice timing retrospectively aligned to the cardiac pulse in the same manner. The SMS measurements rapidly, noninvasively and reliably sample brain-wide fMRI signal pulsations aligned to the heartbeat. The measurements estimate the amplitude and phase of cardiac driven fMRI pulsations in the CSF relative to those in the arteries, which is thought to be an estimate of the local intracranial impedance. Cardiac aligned fMRI signals can provide new insights about fluid dynamics or diagnostics for diseases where these dynamics are important.


title: Improved Heat Tolerance in Heat-acclimated Mice: the Probable Role of the PD-L1 Pathway

abstract: Heat stroke is a life-threatening illness and is related to systematic inflammation-induced multiple organ dysfunction. Available evidence indicates that the severity of the systematic inflammatory response in heat stroke may be related to the changes in immune regulation brought by heat acclimation. However, the mechanisms of heat acclimation are still unclear. Here, we assessed the differences in immunocyte subsets in the spleen and lymph nodes of heat-acclimated and unacclimated mice. A higher frequency of CD4+Foxp3+ Tregs was observed in heat-acclimated mice. Our results indicated that the improved heat tolerance exhibited during acute heat stress exposure was related to an increased number of Tregs. In heat-acclimated mice, an increase in the number of Tregs was able to mitigate the recruitment of neutrophils, inhibit the activation of neutrophils, and suppress the severity of acute inflammation. Increased differentiation and development of Tregs in peripheral immune organs in heat-acclimated mice might stem from enhanced expression of Foxp3 and PD-L1. Our results strongly suggest that the regulatory function of increased Tregs on neutrophils may be regulated through the PD-1/PD-L1 pathway. The anti-inflammatory effects of Tregs have never been studied in the context of heat stress-induced systemic inflammation. Thus, our results on immunoregulation involving Tregs in heat-acclimated mice might be significant for devising a potential treatment for systemic inflammatory response syndrome and heatstroke.

category: immunology